WO2019048881A1 - Oligonucleotides comprising triazole and (phosphor)amidate internucleoside linkages, method for the preparation and uses thereof - Google Patents

Oligonucleotides comprising triazole and (phosphor)amidate internucleoside linkages, method for the preparation and uses thereof Download PDF

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Publication number
WO2019048881A1
WO2019048881A1 PCT/GB2018/052555 GB2018052555W WO2019048881A1 WO 2019048881 A1 WO2019048881 A1 WO 2019048881A1 GB 2018052555 W GB2018052555 W GB 2018052555W WO 2019048881 A1 WO2019048881 A1 WO 2019048881A1
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oligonucleotide
formula
nucleoside
alkyl
independently selected
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PCT/GB2018/052555
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French (fr)
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Tom Brown
Afaf Helmy El-Sagheer
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Oxford University Innovation Limited
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids

Definitions

  • the present invention relates to a process for preparing oligonucleotides or oligonucleotide analogues.
  • the present invention also relates to the oligonucleotides and oligonucleotide analogues formed from said process and to use of these oligonucleotides and oligonucleotide analogues in the synthesis of genes, as therapeutics in treatment of certain diseases and disorders (e.g. cancer) and as templates in polymerase chain reactions, DNA replication processes, RNA transcription processes and/or translation processes.
  • PCR DNA polymerases cannot discriminate between the canonical deoxyribonucleoside triphosphates (dNTPs) and modified versions. This is because any modified dNTP must possess the same fundamental Watson-Crick base pairing properties as its natural counterpart in order to be incorporated into DNA by polymerase enzymes. Consequently, the natural and unnatural dNTPs compete in an uncontrollable manner.
  • An obvious solution to this problem is to assemble DNA by ligation of pre-synthesized chemically modified oligonucleotides. This would open up new areas of biology, allowing a vast array of modifications to be incorporated into genomic DNA.
  • Ligation can be carried out enzymatically, 3 but chemical ligation offers an attractive alternative. 4 It is compatible with large scale applications, radical modifications to the sugars and nucleobases, templated or non-templated reactions, and can be carried out in conditions under which ligase enzymes would not remain functional, including automated nucleic acid assembly. Moreover, chemical ligation is not restricted to the natural phosphodiester backbone of DNA; other backbones can be produced, some of which have been found to be biocompatible.
  • a process for preparing a target oligonucleotide or oligonucleotide analogue comprising two or more different phosphodiester mimic inter-nucleoside linkages, as defined herein.
  • an oligonucleotide or oligonucleotide analogue as defined herein.
  • a use of an oligonucleotide or oligonucleotide, as defined herein, in the synthesis of genes in the synthesis of genes.
  • a use of an oligonucleotide or oligonucleotide, as defined herein, as a template in the synthesis of genes is provided.
  • oligonucleotide or oligonucleotide as defined herein, as:
  • interference RNA e.g. siRNA
  • an RNA component of a CRISPR-Cas system e.g. crRNA, tracrRNA or gRNA
  • the disease or disorder is cancer, a genetic disorder or an infection.
  • oligonucleotide or oligonucleotide as defined herein, as:
  • PCR polymerase chain reaction
  • alkyl includes both straight and branched chain alkyl groups. References to individual alkyl groups such as “propyl” are specific for the straight chain version only and references to individual branched chain alkyl groups such as “isopropyl” are specific for the branched chain version only.
  • (1-6C)alkyl includes (1-4C)alkyl, (1-3C)alkyl, propyl, isopropyl and f-butyl.
  • (m-nC) or "(m-nC) group” used alone or as a prefix, refers to any group having m to n carbon atoms.
  • halo refers to fluoro, chloro, bromo and iodo.
  • oligonucleotide or oligonucleotide analogue of the invention means those oligonucleotides or oligonucleotide analogues which are disclosed herein, both generically and specifically.
  • oligonucleotide refers to a polynucleotide strand. It will therefore be understood that the term oligonucleotide used herein covers both "short" polynucleotide strands comprising between 2 and 500 nucleotide residues and "long” polynucleotide strands comprising greater than 500 nucleotide residues. It will also be appreciated by those skilled in the art that an oligonucleotide has a 5' and a 3' end and comprises a sequence of nucleosides linked together by inter-nucleoside linkages.
  • oligonucleotide analogue and “nucleotide analogue” refer to any modified synthetic analogues of oligonucleotides or nucleotides respectively that are known in the art.
  • oligonucleotide analogues include peptide nucleic acids (PNAs), morpholino oligonucleotides, phosphorothioate oligonucleotides, phosphorodithioate oligonucleotides, alkylphosphonate oligonucleotides, acylphosphonate oligonucleotides and phosphoramidate oligonucleotides.
  • PNAs peptide nucleic acids
  • morpholino oligonucleotides include morpholino oligonucleotides, phosphorothioate oligonucleotides, phosphorodithioate oligonucleotides, alkylphosphonate oligonucle
  • nucleobase analogue refers to any analogues of nucleobases known in the art.
  • the skilled person will appreciate there to be numerous natural and synthetic nucleobase analogues available in the art which could be employed in the present invention. As such, the skilled person will readily be able to identify suitable nucleobase analogues for use in the present invention.
  • Commonly available nucleobase analogues are commercially available from a number of sources (for example, see the Glen Research catalogue (http://www.glenresearch.com/Catalog/contents.php). It will also be appreciated that the term “nucleobase analogue” covers: universal/degenerate bases (e.g.
  • 3-nitropyrrole, 5-nitroindole and hypoxanthine fluorescent bases (e.g. tricyclic cytosine analogues (tCO, tCS) and 2- aminopurine); base analogues bearing reactive groups selected from alkynes, thiols or amines; and base analogues that can crosslink oligonucleotides to DNA, RNA or proteins (e.g. 5-bromouracil or 3-cyanovinyl carbazole).
  • fluorescent bases e.g. tricyclic cytosine analogues (tCO, tCS) and 2- aminopurine
  • base analogues bearing reactive groups selected from alkynes, thiols or amines e.g. 5-bromouracil or 3-cyanovinyl carbazole.
  • the nucleobase or nucleobase analogue is attached to a sugar moiety (typically ribose or deoxyribose) or a ribose or deoxyribose mimic, for example a chemically modified sugar derivative (e.g. a chemically modified ribose or deoxyribose) or a cyclic group that functions as a synthetic mimic of a ribose or deoxyribose sugar moiety (e.g. the morpholino ring present in morpholino oligonucleotides).
  • a sugar moiety typically ribose or deoxyribose
  • a ribose or deoxyribose mimic for example a chemically modified sugar derivative (e.g. a chemically modified ribose or deoxyribose) or a cyclic group that functions as a synthetic mimic of a ribose or deoxyribose sugar moiety (e.g. the morph
  • nucleoside is used herein to refer to a moiety composed of a sugar / a ribose or deoxyribose mimic bound to a nucleobase/nucleobase analogue.
  • nucleoside as used herein excludes the inter-nucleoside linkage that connects adjacent nucleosides together.
  • An "inter-nucleoside linkage” is a linking group that connects the rings of the sugar / ribose or deoxyribose mimic of adjacent nucleosides.
  • locked nucleic acid LIMA or locked nucleoside
  • nucleic acids or nucleosides comprising a ribose or deoxyribose moiety in which the conformation of the ribose or deoxyribose ring is fixed or locked in a specific conformation, typically by a bridging group.
  • the bridging group connects the 2' and 4' carbon atoms of the ribose or deoxyribose rings and locks the ribose or deoxyribose in the 3'-endo conformation (which is often found in A-form duplexes).
  • Examples of locked nucleic acid/nucleoside structures are well known in the art and are commercially available.
  • a process for preparing a target oligonucleotide or oligonucleotide analogue comprising two or more different phosphodiester mimic inter-nucleoside linkages comprising the steps of:
  • R 1 a , R 1 b , R 1 c , R 1 d , R 1 e , R 1 f , R 1 9 and R 1 h are each independently selected from hydrogen or (1 -4C)alkyl, wherein each (1 -4C)alkyl is optionally substituted with one or more NH2, OH or SH;
  • V and W are independently selected from O, S or NR X , wherein R x is selected from hydrogen or (1 -4C)alkyl;
  • x, xi , z and zi are integers independently selected from 0 to 2; and y and yi are integers independently selected from 0 to 1 ;
  • ⁇ f independently denote the points of attachment to the target oligonucleotide or oligonucleotide analogue
  • R 2a , R 2b , R 2c , R 2d , R 3a , R 3b , R 3c and R 3d are independently selected from hydrogen or (1-4C)alkyl, wherein each (1-4C)alkyl is optionally substituted with one or more NH2, OH or SH
  • R 2e and R 3e are independently selected from hydrogen or (1-4C)alkyl
  • Vi, Wi and W2 are independently selected from O, S or NR Z , wherein R z is selected from hydrogen or (1-4C)alkyl;
  • Q is selected from S or O
  • n, rii , m and m 1 are integers independently selected from 0 to 2; and p, pi and P2 are integers independently selected from 0 to 1 ;
  • steps A) and B) above are conducted in either order;
  • the inventors have discovered a new process for preparing oligonucleotides or oligonucleotide analogues comprising two or more different phosphodiester mimic inter- nucleoside linkages.
  • the inventors In exploiting orthogonal chemistries to prepare the two or more different phosphodiester mimic inter-nucleoside linkages, the inventors have provided a cheap and highly efficient process for preparing oligonucleotides and analogues thereof comprising a vast array of modifications.
  • the inventors have also discovered that the two or more different phosphodiester mimic inter-nucleoside linkages used in the present process are fully compatible with and readable by DNA and RNA polymerases, which makes the oligonucleotides and oligonucleotide analogues prepared by the present process of particular use in a number of biological applications.
  • the process of the present invention also comprises the preparation of long polynucleotides (e.g. genes), wherein the long polynucleotide comprises greater than 500 nucleotide residues.
  • the process of the present invention is conducted in the presence of a template.
  • any suitable template may be used.
  • a person skilled in the art will be able to select a suitable template having the correct size and nucleoside sequence for hybridisation with the oligonucleotides and oligonucleotide analogues of the present process that are to be ligated together.
  • the template may comprise both an oligonucleotide and a synthetic oligonucleotide analogue, such as, for example, a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • the template is a single stranded oligonucleotide or oligonucleotide analogue that the oligonucleotides or oligonucleotide analogues to be ligated bind to, such that, functional groups on the termini of adjacent oligonucleotides or oligonucleotide analogues are available to be ligated together to form an inter-nucleoside linkage of Formula I, II and III as defined herein.
  • the process of the present invention is conducted in the absence of a template.
  • Processes conducted in the absence of a template will be understood to encompass reactions such as, for example, solution phase reactions and/or solid supported reactions.
  • At least one of the oligonucleotides or oligonucleotide analogues to be ligated is attached to a solid support.
  • the solid support is selected from controlled pore glass (CPG), silica, hydroxylated methacrylic polymer beads (e.g. Toyopearl® beads), grafted copolymers comprising a crosslinked polystyrene matrix onto which polyethylene glycol is grafted (e.g. Tenagel®) or microporous polystyrene (MPPS).
  • CPG controlled pore glass
  • MPPS microporous polystyrene
  • the solid support is selected from controlled pore glass (CPG) or microporous polystyrene (MPPS).
  • the solid support is a controlled pore glass (CPG) support.
  • One of the advantages of the present invention is that it allows multiple (i.e. three or more) oligonucleotides or oligonucleotide analogous to ligated together sequentially.
  • the inventors have advantageously discovered that in exploiting the orthogonal chemistries used to prepare the one or more phosphodiester backbone mimic inter-nucleoside linkages of Formulae I, II or III, oligonucleotides and oligonucleotide analogues can be prepared easily and very efficiently, without the need for any functional group interconversions.
  • target oligonucleotides or oligonucleotide analogues prepared by the present process may comprise any number of phosphodiester backbone mimic inter-nucleoside linkages of Formula I and any number of phosphodiester backbone mimic inter-nucleoside linkages of Formula II or Formula III.
  • the target oligonucleotide or oligonucleotide analogue comprises one or more phosphodiester backbone mimic inter- nucleoside linkages of Formula I and one or more phosphodiester backbone mimic inter- nucleoside linkages of Formula II.
  • the target oligonucleotide or oligonucleotide analogue comprises one phosphodiester backbone mimic inter-nucleoside linkage of Formula I and one phosphodiester backbone mimic inter-nucleoside linkage of Formula II.
  • the target oligonucleotide or oligonucleotide analogue comprises one or more phosphodiester backbone mimic inter- nucleoside linkages of Formula I and one or more phosphodiester backbone mimic inter- nucleoside linkages of Formula III.
  • the target oligonucleotide or oligonucleotide analogue comprises one phosphodiester backbone mimic inter-nucleoside linkage of Formula I and one phosphodiester backbone mimic inter-nucleoside linkage of Formula III.
  • the one or more phosphodiester backbone mimic inter- nucleoside linkages of Formula II or Formula III are formed before the one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula I.
  • step B) of the present process is conducted before step A).
  • the target oligonucleotide prepared by the present process may also comprises at least one locked nucleoside.
  • Locked nucleosides are well known in the art and include nucleic acids and/or nucleosides comprising a ribose or deoxyribose moiety in which the conformation of the ribose or deoxyribose ring is fixed or "locked" in a specific conformation, typically by a bridging group.
  • a non-limiting list of suitable locked nucleosides which may be used in the present invention are described in K. Singh, S. and J. Wengel (1998).
  • the at least one locked nucleoside may be positioned at either the 3' or 5' end of an inter-nucleoside linkage of Formula I, II or III defined herein, or a locked nucleoside may be positioned at both the 3' and 5' end of an inter- nucleoside linkage of Formula I, II or III defined herein.
  • the at least one locked nucleoside is positioned at the 3' end of an inter-nucleoside linkage of Formula I, II or III defined herein.
  • the at least one locked nucleoside is positioned at the 5' end of an inter-nucleoside linkage of Formula I, II or III defined herein.
  • the target oligonucleotide or oligonucleotide analogue comprises at least two locked nucleosides, with at least one locked nucleoside positioned at the 3' end of an inter-nucleoside linkage of Formula I, II or III defined herein and at least one locked nucleoside position at 5' end of an inter-nucleoside linkage of Formula I, II or III defined herein.
  • target oligonucleotides or oligonucleotide analogues prepared by the process of the present invention may be isolated and purified using any suitable techniques known in the art.
  • the target oligonucleotides or oligonucleotide analogues formed by the process of the present invention are isolated and purified using column chromatography, such as, for example, using Sephadex® columns (i.e. cross-linked dextran gels).
  • step A) of the present process involves ligating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula la, shown below, at the point(s) of ligation:
  • V and W are independently selected from O, S or NR X , wherein R x is selected from hydrogen or (1-4C)alkyl;
  • x, xi , z and zi are integers independently selected from 0 to 2;
  • y and yi are integers independently selected from 0 to 1 ; with the proviso that the sum of x, xi , y, yi , z and zi is either 0, 1 , 2, 3, 4, 5 or 6.
  • step A) of the present process involves ligating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula lb, shown below, at the point(s) of ligation:
  • x, xi , z and zi are integers independently selected from 0 to 2;
  • y and yi are integers independently selected from 0 to 1 ;
  • step A) of the present process involves ligating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages at the point(s) of ligation selected from:
  • ⁇ denotes the point of attachment to a 5' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue.
  • step A) of the present process involves ligating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages at the point(s) of ligation selected from:
  • / a denotes the point of attachment to a 3' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue
  • ⁇ denotes the point of attachment to a 5' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue.
  • step A) of the present process involves ligating two or more oligonucleotides or oligonucleotide analogues together to form a phosphodiester backbone mimic inter-nucleoside linkage at the point of ligation of the formula:
  • / a denotes the point of attachment to a 3' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue
  • ⁇ denotes the point of attachment to a 5' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue.
  • the one or more phosphodiester backbone mimic inter- nucleoside linkages of Formulae I, la or lb may be prepared using any suitable technique known in the art. The person skilled in the art will be able to select suitable reaction conditions and reagents to prepare the one or more phosphodiester backbone mimic inter-nucleoside linkages of Formulae I, la or lb.
  • the one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula I are formed at the point(s) of ligation by reacting:
  • ⁇ a' denotes the point of attachment to the alkyne terminating oligonucleotide or oligonucleotide analogue
  • V is selected from O, S or NR X , wherein R x is selected from hydrogen or (1- 4C)alkyl; R 1 a , R 1 b , R 1 c and R 1 d are independently selected from hydrogen or (1 - 4C)alkyl, wherein each (1 -4C)alkyl is optionally substituted with one or more NH 2 , OH or SH;
  • x and z are integers independently selected from 0 to 2;
  • y is an integer selected from 0 to 1 ;
  • ' denotes the point of attachment to the azide terminating oligonucleotide or oligonucleotide analogue
  • W is selected from O, S or NR X , wherein R x is selected from hydrogen or (1 - 4C)alkyl;
  • R 1 e , R 1 f , R 19 and R 1 h are independently selected from hydrogen or (1 -4C)alkyl, wherein each (1 -4C)alkyl is optionally substituted with one or more NH2, OH or SH;
  • xi and zi are integers independently selected from 0 to 2; and yi is an integer selected from 0 to 1 ;
  • the alkyne terminal functional group of Formula A may be protected using a suitable protecting group.
  • suitable protecting groups include, for example, trialkylsilylacetylenes (e.g. trimethylsilylacetylene).
  • alkyne terminal functional group of Formula A it may be necessary to conduct the reaction between the one or more alkyne terminating oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula A and the one or more azide terminating oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula B in the presence of a deprotecting agent.
  • Suitable deprotecting agents will be apparent to those skilled in the art.
  • the reaction between the one or more alkyne terminating oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula A and the one or more azide terminating oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula B may optionally be carried out in the presence of tetrabutylammonium fluoride (TBAF) as the deprotecting agent.
  • TBAF tetrabutylammonium fluoride
  • the azido terminal functional group of Formula B may be masked in the form of an azide precursor.
  • suitable azide precursors will be known to the person skilled in the art.
  • Non-limiting examples of azide precursors include alkyl halides, tosylates or mesylates.
  • the azido terminal functional group of Formula B is delivered in the form of an azide precursor, it will be understood that it will be necessary to conduct the reaction between the one or more alkyne terminating oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula A and the one or more azide terminating oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula B in the presence of a suitable azide source (e.g. NaNs), to firstly convert the azide precursor to the terminal functional group of Formula B.
  • a suitable azide source e.g. NaNs
  • the sum of integers x, xi , y, yi , z and zi is either 0, 1 , 2, 3, 4 or 5.
  • the sum of integers x, xi , y, yi , z and zi is either 0, 1 , 2, 3 or 4. More suitably, the sum of integers x, xi , y, yi , z and zi is either 1 , 2, 3 or 4.
  • the sum of integers x, xi , y, yi , z and zi is either 1 , 2 or 3.
  • V is selected from O or NR X , wherein R x is selected from hydrogen or (1-4C)alkyl.
  • V is selected from O or NR X , wherein R x is selected from hydrogen or methyl.
  • V is O.
  • y is 1. In another embodiment, y is 0.
  • W is selected from O or NR X , wherein R x is selected from hydrogen or (1-4C)alkyl.
  • W is selected from O or NR X , wherein R x is selected from hydrogen or methyl.
  • W is O.
  • yi is 1. In other embodiments, yi is 0.
  • R 1a , R 1 b , R 1c , R 1d , R 1e , R 1f , R 19 and R 1 h are independently selected from hydrogen or (1-4C)alkyl.
  • R 1a , R 1 b , R 1c , R 1d , R 1 e , R 1f , R 19 and R 1 h are independently selected from hydrogen or methyl.
  • R 1a , R 1 b , R 1c , R 1d , R 1e , R 1f , R 19 and R 1 h are hydrogen.
  • x, xi , z and zi are integers independently selected from 0 to 1.
  • ⁇ a' denotes the point of attachment to a 3' carbon of a
  • ⁇ v denotes the point of attachment to a 5' carbon of a nucleoside of the azide terminating oligonucleotide or oligonucleotide analogue.
  • step A) of the process may be conducted using any suitable reaction conditions.
  • reaction conditions used in the step A) of the present process will vary according to the specific oligonucleotide, oligonucleotide analogue and/or functional groups of Formula A and B that are used.
  • suitable reaction conditions e.g. temperature, pressures, reaction times, concentration etc.
  • the reaction between the one or more alkyne terminating oligonucleotides or oligonucleotide analogues of step A1) and the one or more azide terminating oligonucleotides or oligonucleotide analogues of step A2) is conducted in the presence of a catalyst.
  • the catalyst is a copper (I) species.
  • suitable catalysts include copper iodide (Cul), copper bromide (CuBr) or copper iodide-triethyl phosphite (Cul.P(OEt) 3 ).
  • the catalyst e.g. the copper (I) species
  • the catalyst may be formed in situ upon adding a pre-catalyst complex and reducing agent to the reaction conditions. Accordingly, in an embodiment, the catalyst may be added in the form of a pre-catalyst together with a reducing agent.
  • suitable pre-catalysts include copper sulfate (CuSCU), copper chloride (CuC ), copper bromide (CuBr2), copper formate (Cu(OC(0)H)2), copper hydroxide (CuOhb) and copper nitrate (Cu(NOs)2).
  • a non-limiting example of a suitable reducing agent is sodium ascorbate.
  • the pre-catalyst is copper sulfate and the reducing agent is sodium ascorbate.
  • the step of reacting together the alkyne terminating oligonucleotide or oligonucleotide analogue comprising a terminal functional group of Formula A with the azide terminating oligonucleotide or oligonucleotide analogue comprising a terminal functional group of Formula B may be repeated sequentially more than once, for example, more than twice, more than three times, more than four times or more than five times, to form an oligonucleotide or oligonucleotide analogue comprising the one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula I.
  • step B) of the present process involves ligating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula lla, shown below, at the point(s) of ligation:
  • / c and d independently denote the points of attachment to the target oligonucleotide or oligonucleotide analogue
  • R 2a , R 2b , R 2c , R 2d , R 2e and R z are independently selected from hydrogen or (1- 4C)alkyl;
  • n and ni are integers independently selected from 0 to 2;
  • q is an interger from 0 to 1 ;
  • n + ni + p 2, 3 or 4.
  • step B) of the present process involves ligating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula lib, shown below, at the point(s) of ligation:
  • / c and ⁇ d independently denote the points of attachment to the target oligonucleotide or oligonucleotide analogue
  • R 2a , R 2b , R 2c , R 2d and R 2e are independently selected from hydrogen or (1- 4C)alkyl;
  • n and ni are integers independently selected from 0 to 2, with the proviso that
  • step B) of the present process involves ligating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages at the point(s) of ligation selected from:
  • step B) of the present process involves ligating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages at the point(s) of ligation selected from:
  • step B) of the present process involves ligating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages at the point(s) of ligation selected from:
  • c denotes the point of attachment to a 3' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue
  • ⁇ d denotes the point of attachment to a 5' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue.
  • the one or more phosphodiester backbone mimic inter- nucleoside linkages of Formulae II, lla or lib may be prepared using any suitable technique known in the art.
  • the person skilled in the art will be able to select suitable reaction conditions and reagents to prepared the one or more phosphodiester backbone mimic inter-nucleoside linkages of Formulae II, lla or lib.
  • a suitable protecting group for an amino or alkylamino group is, for example, an acyl group, for example an alkanoyl group such as acetyl, an alkoxycarbonyl group, for example a methoxycarbonyl, ethoxycarbonyl or t-butoxycarbonyl group, an arylmethoxycarbonyl group, for example benzyloxycarbonyl, or an aroyl group, for example benzoyl.
  • the deprotection conditions for the above protecting groups necessarily vary with the choice of protecting group.
  • an acyl group such as an alkanoyl or alkoxycarbonyl group or an aroyl group may be removed by, for example, hydrolysis with a suitable base such as an alkali metal hydroxide, for example lithium or sodium hydroxide.
  • a suitable base such as an alkali metal hydroxide, for example lithium or sodium hydroxide.
  • an acyl group such as a te/f-butoxycarbonyl group may be removed, for example, by treatment with a suitable acid as hydrochloric, sulfuric or phosphoric acid or tnfluoroacetic acid and an arylmethoxycarbonyl group such as a benzyloxycarbonyl group may be removed, for example, by hydrogenation over a catalyst such as palladium-on-carbon, or by treatment with a Lewis acid for example boron tris(trifluoroacetate).
  • a suitable alternative protecting group for a primary amino group is, for example, a phthaloyl group which may be removed by treatment with an alkylamine, for example dimethylaminopropylamine, or with hydrazine.
  • a suitable protecting group for a carboxy group is, for example, an esterifying group, for example a methyl or an ethyl group which may be removed, for example, by hydrolysis with a base such as sodium hydroxide, or for example a t-butyl group which may be removed, for example, by treatment with an acid, for example an organic acid such as tnfluoroacetic acid, or for example a benzyl group which may be removed, for example, by hydrogenation over a catalyst such as palladium-on-carbon.
  • a base such as sodium hydroxide
  • a t-butyl group which may be removed, for example, by treatment with an acid, for example an organic acid such as tnfluoroacetic acid, or for example a benzyl group which may be removed, for example, by hydrogenation over a catalyst such as palladium-on-carbon.
  • the one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula II are formed by reacting:
  • / c denotes the point of attachment to the oligonucleotide or oligonucleotide analogue comprising a terminal functional group of Formula C;
  • X is a leaving group selected from halo, OS02R x1 , (1-2C)haloalkyl, (1- 2C)haloalkoxy, OR" 2 , heteroaryl, wherein R x1 and R* 2 are independently selected from H, (1-6C)alkyl, (1-6C)alkanoyl, cycloalkyl, heteroalkyl, aryl, heteroaryl, (1-2C)haloalkyl, and wherein each of (1-6C)alkyl, cycloalkyl, heteroalkyl, aryl, heteroaryl are optionally further substituted with one or more groups selected from (1-4C)alkyl, halo, cyano, nitro or (1-2C)haloalkyl;
  • Vi is selected from O, S or NR Z , wherein R z is selected from hydrogen or (1- 4C)alkyl;
  • Q is selected from O or S
  • R 2a and R 2b are independently selected from hydrogen or (1-4C)alkyl, wherein each (1-4C)alkyl is optionally substituted with one or more NH2, OH or SH; n is an integer selected from 0 to 2; and
  • p is an integer selected from 0 to 1 ;
  • oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula D, shown below:
  • ⁇ d' denotes the point of attachment to the oligonucleotide or oligonucleotide analogue comprising a terminal functional group of Formula D;
  • R 2c and R 2d are independently selected from hydrogen or (1-4C)alkyl
  • Wi is selected from O or NH
  • X a is selected from NR e or SH, wherein R e is selected from hydrogen or (1- 4C)alkyl;
  • ni is an integer selected from 0 to 2;
  • pi is an integer selected from 0 or 1 ;
  • reaction is optionally conducted in the presence of one or more of the following:
  • the sum of integers n, ni , p is equal to 2, 3 or 4.
  • the sum of integers n, ni , p is equal to 2 or 3.
  • the sum of integers n, ni , p is equal to 2.
  • X is selected from halo, OS0 2 R x1 , (1-2C)haloalkyl, (1- 2C)haloalkoxy, OR" 2 , 5-membered heteroaryl, wherein R x1 and R* 2 are independently selected from H, (1-6C)alkyl, (1-6C)alkonyl, aryl or (1-2C)haloalkyl, and wherein each of (1-6C)alkyl or aryl is optionally further substituted with one or more groups selected from (1-4C)alkyl, halo, cyano, nitro or (1-2C)haloalkyl.
  • X is selected from halo, OS0 2 R x1 , (1-2C)haloalkyl, (1-2C)haloalkoxy, OR" 2 , triazolyl, wherein R x1 and R" 2 are independently selected from H, (1- 6C)alkyl, (1-6C)alkonyl, phenyl or (1-2C)haloalkyl. More suitably, X is selected from halo, (1- 2C)haloalkyl or OR" 2 , wherein R* 2 is selected from H, (1-6C)alkyl or a (1-6C)alkonyl. Even more suitably, X is selected from OR" 2 , wherein R" 2 is selected from H or (1-6C)alkyl. Most suitably, X is OH.
  • Vi is selected from O or NR Z , wherein R z is selected from hydrogen or (1-4C)alkyl.
  • R z is selected from hydrogen or (1-4C)alkyl.
  • Vi is selected from O or N z , wherein R z is selected from hydrogen or methyl.
  • Vi is O.
  • Q is O.
  • Wi is O.
  • n and ni are integers selected from 0 or 1.
  • the sum of integers n, ni , p and pi is equal to 2, 3 or 4.
  • the sum of integers n, ni , p and pi is equal to 2 or 3.
  • the sum of integers n, ni , p and pi is equal to 2.
  • R 2a , R 2b , R 2c and R 2d are independently selected from hydrogen or (1-4C)alkyl.
  • R 2a , R 2b , R 2c and R 2d are independently selected from hydrogen or methyl.
  • R 2a , R 2b , R 2c and R 2d are hydrogen.
  • pi is 0.
  • p is 0.
  • X a is NR e , wherein R e is selected from hydrogen or (1- 4C)alkyl.
  • R e is selected from hydrogen or (1- 4C)alkyl.
  • X a is NH 2 .
  • the reaction between the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula C and the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula D is conducted at a temperature of between 0 °C and 150 °C.
  • the reaction between the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula C and the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula D is conducted at a temperature of between 0 °C and 100 °C.
  • the reaction between the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula C and the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula D is conducted at a temperature of between 0 °C and 75 °C.
  • the reaction between the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula C and the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula D is carried out in a polar solvent.
  • the polar solvent may be used to solubilise the oligonucleotides comprising functional groups of Formulae C and D and thereby facilitate reaction therebetween. Accordingly, it will be understood that the polar solvent selected will depend on the specific oligonucleotides selected.
  • Suitable polar solvents may include, but are not limited to, water, an aqueous buffered solution (e.g. a solution of sodium phosphate or sodium carbonate), DMF, DMSO, acetonitrile, tetrahydrofuran (THF) and mixtures thereof with aqueous salt solutions.
  • the reaction between the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula C and the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula D is carried out in an aqueous medium at a pH within the range of 5 to 9.
  • the reaction between the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula C and the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula D is carried out at a pH within the range of 6 to 8.
  • the reaction between the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula C and the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula D is carried out at a pH within the range of 6.5 to 7.5.
  • a suitable buffer is present to maintain the reaction medium within the pH range 5 to 9. In a further embodiment, the buffer maintains the reaction medium within the pH range 6 to 8. In another embodiment, the buffer maintains the reaction medium within the pH range 6.5 to 7.5.
  • the buffer is selected from the group comprising: phosphate, acetate, borate, citrate, sulfonic acid, ascorbate, linolenate, carbonate and bicarbonate based buffers.
  • the buffer is selected from the group comprising: phosphate, acetate, carbonate and bicarbonate based buffers.
  • the buffer is sodium phosphate or sodium carbonate.
  • the reaction between the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula C and the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula D is conducted in the presence of a salt (e.g. NaCI).
  • a salt e.g. NaCI
  • Any suitable concentration of salt may be used.
  • the salt is present in a concentration of between 20 mM and 500 mM. More suitably, the salt is present in a concentration between 50 mM and 300 mM. Yet more suitably, the salt is present in a concentration between 100 mM and 250 mM.
  • the reaction between the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula C and the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula D is carried out in the presence of a catalyst.
  • a catalysts may be any suitable reagent that helps to promote the rate of the reaction between the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula C and the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula D.
  • the catalyst is an acid and/or a base. Most suitably, the catalyst is a base.
  • suitable bases include NaOH, trimethylamine, diisopropylethylamine and N-methylmorpholine.
  • the reaction between the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula C and the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula D is carried out in the presence of one or more peptide coupling agents.
  • Any suitable peptide coupling reagent capable of enhancing the reaction between the functional group of Formula C and the functional group of Formula D may be used. It will be understood that the peptide coupling agent is preferably present when X is OH (i.e. the functional group of Formula C comprises a carboxy group).
  • the peptide coupling reagent is a carbodiimide-based coupling reagent.
  • the peptide coupling reagent is selected from 1- [Bis(dimethylamino)methylene]-1 H-1 ,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU), 2-(1 H-benzotriazol-1-yl)-1 ,1 ,3,3-tetramethyluronium hexafluorophosphate (HBTU), (Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), 4-(4,6-Dimethoxy-1 ,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM), N-Ethoxycarbonyl-2-ethoxy-1 ,2-dihydroquinoline (EEDQ), ⁇ ,
  • the coupling reagent is selected from ⁇ , ⁇ '-dicyclohexylcarbodiimide (DCC), ⁇ , ⁇ '-diisopropylcarbodiimide (DIC) or 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDCI).
  • the coupling reagent is 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDCI).
  • Additional activating agents such as, for example, hydroxybenzotriazole (HOBt), N-hydroxy 2-phenyl benzimidazole (HOBI), 1-hydroxy-7-azabenzotriazole (HOAt), N- hydroxysuccinimide (NHS), N-hydroxysulfosuccinimide (Sulfo-NHS), 4-dimethylaminopyridine (DMAP) and ethyl cyano(hydroxyimino)acetate (Oxyma Pure ® ) may also be used together with the peptide coupling reagent defined hereinabove, to further enhance reactivity between the functional group of Formula C and the functional group of Formula D.
  • HOBt hydroxybenzotriazole
  • HOBI N-hydroxy 2-phenyl benzimidazole
  • HOAt 1-hydroxy-7-azabenzotriazole
  • NHS N- hydroxysuccinimide
  • Sulfo-NHS N-hydroxysulfosuccinimide
  • DMAP 4-
  • the activating agent is N-hydroxysuccinimde (NHS), N- hydroxysulfosuccinimide (Sulfo-NHS) or ethyl cyano(hydroxyimino)acetate (Oxyma Pure ® ).
  • the activating agent is N-hydroxysuccinimde (NHS).
  • the reaction between the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula C and the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula D is carried out in the presence of both a peptide coupling agent (e.g. EDCI) and an activating agent (e.g. NHS).
  • a peptide coupling agent e.g. EDCI
  • activating agent e.g. NHS
  • the ratio of peptide coupling agent (e.g. EDCI) to activating agent (e.g. NHS) is from between 10: 1 to 1 : 1. More suitably, the ratio of peptide coupling agent (e.g. EDCI) to activating agent (e.g. NHS) is from between 6: 1 to 1 :1.
  • the step of reacting together the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula C and the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula D may be repeated sequentially more than once, for example, more than twice, more than three times, more than four times or more than five times, to form an oligonucleotide or oligonucleotide analogue comprising the one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula II.
  • step B) of the present process involves ligating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula Ilia, shown below, at the point(s) of ligation:
  • e and / f independently denote the points of attachment to the target oligonucleotide or oligonucleotide analogue
  • W2 is selected from O or NH
  • n and m 1 are integers independently selected from 0 to 2; and P2 is an integer selected from 0 or 1 ;
  • step B) of the present process involves ligating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula ll lb, shown below, at the point(s) of ligation:
  • e and / f independently denote the points of attachment to the target oligonucleotide or oligonucleotide analogue
  • R 3a , R 3b , R 3c and R 3d are independently selected from hydrogen or (1 -4C)alkyl
  • n and m 1 are integers independently selected from 0 to 2;
  • step B) of the present process involves ligating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula lllc, shown below, at the point(s) of ligation:
  • e and / f independently denote the points of attachment to the target oligonucleotide or oligonucleotide analogue
  • n and m ⁇ are integers independently selected from 0 to 2;
  • step B) of the present process involves ligating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages at the point(s) of ligation of the formula:
  • e denotes the point of attachment to a 3' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue
  • f denotes the point of attachment to a 5' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue
  • the one or more phosphodiester backbone mimic inter- nucleoside linkages of Formulae I II , Il ia, 1 Mb or ll lc may be prepared using any suitable technique known in the art. The person skilled in the art will be able to select suitable reaction conditions and reagents to prepared the one or more phosphodiester backbone mimic inter- nucleoside linkages of Formulae II I, I lia, 1 Mb or l llc.
  • the one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula II I are formed by reacting:
  • ⁇ e' denotes the point of attachment to the oligonucleotide or oligonucleotide analogue comprising a terminal functional group of Formula E;
  • R 3a and R 3b are independently selected from hydrogen or (1 -4C)alkyl, wherein each (1 -4C)alkyl is optionally substituted with one or more NH2, OH or SH; and
  • n is an integer selected from 0 to 2;
  • oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula F shown below:
  • f denotes the point of attachment to the oligonucleotide or oligonucleotide analogue comprising a terminal functional group of Formula F;
  • R 3c and R 3d are independently selected from hydrogen or (1 -4C)alkyl, wherein each (1 -4C)alkyl is optionally substituted with one or more NH2, OH or SH;
  • R 3e is selected from and hydrogen or (1 -4C)alkyl
  • W2 is selected from O, S or NR Z , wherein R z is selected from hydrogen or (1 - 4C)alkyl
  • mi is an integer selected from 0 to 2;
  • P2 is an integer selected from 0 or 1 ;
  • reaction is optionally conducted in the presence of one or more of the following:
  • s e denotes the point of attachment to a 3' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue
  • ⁇ f denotes the point of attachment to a 5' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue
  • R 3a , R 3b , R 3c and R 3d are independently selected from hydrogen or (1 -4C)alkyl.
  • R 3a , R 3b , R 3c and R 3d are independently selected from hydrogen or methyl.
  • R 3a , R 3b , R 3c and R 3d are hydrogen.
  • R 3e is selected from hydrogen or methyl.
  • R 3e is hydrogen.
  • W2 is selected from O or NH.
  • P2 is 0.
  • n and mi are integer independently selected from 0 or 1 .
  • m and mi are 0.
  • the sum of integers m, mi and P2 is equal to 0, 1 , 2, 3 or 4.
  • the sum of integers m, mi and p2 is equal to 0, 1 , 2 or 3. More suitably, the sum of integers m, mi and P2 is equal to 0, 1 or 2. Most suitably, the sum of integers m, mi and P2 is equal to 0 or 1 .
  • the reaction between the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula E and the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula F is conducted in the presence of one or more peptide coupling reagents.
  • Suitable peptide coupling reagents are analogous to those described in paragraphs [0098] and [0099] hereinabove.
  • Additional activating may also be used together with the peptide coupling reagent defined hereinabove, to further enhance reactivity between the functional group of Formula E and the functional group of Formula F.
  • Suitable activating agents are analogous to those described in paragraphs [00100] and [00101] hereinabove.
  • the step of reacting together the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula E and the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula F may be repeated sequentially more than once, for example, more than twice, more than three times, more than four times or more than five times, to form an oligonucleotide or oligonucleotide analogue comprising the one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula III.
  • the one or more alkyne terminating oligonucleotides comprising a terminal functional group of Formula A, or the one or more azide terminating oligonucleotides comprising a terminal functional group of Formula B further comprise a terminal functional group selected from Formula C, Formula D, Formula E or Formula F, as defined hereinabove.
  • the one or more alkyne terminating oligonucleotides comprising a terminal functional group of Formula A further comprise a terminal functional group selected from Formula D or Formula F, as defined hereinabove.
  • the one or more azide terminating oligonucleotides comprising a terminal functional group of Formula B further comprise a terminal functional group selected from Formula C or Formula E, as defined hereinabove.
  • the one or more alkyne terminating oligonucleotides comprising a terminal functional group of Formula A further comprise a terminal functional group selected from Formula C or Formula E, as defined hereinabove.
  • the one or more azide terminating oligonucleotides comprising a terminal functional group of Formula B further comprise a terminal functional group selected from Formula D or Formula F, as defined hereinabove.
  • a process for preparing a target oligonucleotide or oligonucleotide analogue comprising two or more different phosphodiester mimic inter-nucleoside linkages comprising the steps of:
  • V and W are independently selected from O, S or NR X , wherein R x is selected from hydrogen or (1 -4C)alkyl;
  • x, xi , z and zi are integers independently selected from 0 to 2; and y and yi are integers independently selected from 0 to 1 ;
  • ⁇ f independently denote the points of attachment to the target oligonucleotide or oligonucleotide analogue
  • R 2a , R 2b , R 2c , R 2d , R 2e , R 3a , R 3b , R 3c and R 3d are independently selected from hydrogen or (1-4C)alkyl
  • W2 is selected from O or NH
  • n, rii, m and m 1 are integers independently selected from 0 to 2; and p and P2 are integers independently selected from 0 to 1 ;
  • steps A) and B) above are conducted in either order;
  • x, xi , z and zi are integers independently selected from 0 to 2; and y and yi are integers independently selected from 0 to 1 ;
  • / c d e and / f independently denote the points of attachment to the target oligonucleotide or oligonucleotide analogue
  • R 2a , R 2b , R 2c , R 2d and R 2e are independently selected from hydrogen or (1 - 4C)alkyl, n, ⁇ , m and m ⁇ are integers independently selected from 0 to 2; and and wherein steps A) and B) above are conducted in either order;
  • / a denotes the point of attachment to a 3' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue
  • ⁇ denotes the point of attachment to a 5' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue
  • ligating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages at the point(s) of ligation selected from:
  • / c denotes the point of attachment to a 3' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue
  • ⁇ d denotes the point of attachment to a 5' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue
  • e denotes the point of attachment to a 3' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue
  • f denotes the point of attachment to a 5' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue
  • steps A) and B) above are conducted in either order.
  • a catalyst e.g. a copper (I) species
  • ⁇ a' denotes the point of attachment to the alkyne terminating oligonucleotide or oligonucleotide analogue
  • ' denotes the point of attachment to the azide terminating oligonucleotide or oligonucleotide analogue
  • V and W are independently selected from O, S or NR X , wherein R x is selected from hydrogen or (1 -4C)alkyl;
  • x, xi , z and zi are integers independently selected from 0 to 2; and y and yi are integers independently selected from 0 to 1 ; and
  • ⁇ f independently denote the points of attachment to the target oligonucleotide or oligonucleotide analogue
  • / c denotes the point of attachment to the oligonucleotide or oligonucleotide analogue comprising a terminal functional group of Formula C1 ;
  • ⁇ d' denotes the point of attachment to the oligonucleotide or oligonucleotide analogue comprising a terminal functional group of Formula D1 ;
  • ⁇ e' denotes the point of attachment to the oligonucleotide or oligonucleotide analogue comprising a terminal functional group of Formula E1
  • f denotes the point of attachment to the oligonucleotide or oligonucleotide analogue comprising a terminal functional group of Formula F1 ;
  • X is selected from OR" 2 , wherein R" 2 is selected from hydrogen or (1 - 6C)alkyl;
  • R 2a , R 2b , R 2c , R 2d , R 2e , R 3a , R 3b , R 3c and R 3d are independently selected from hydrogen or (1 -4C)alkyl
  • n, ni , m and m ⁇ are integers independently selected from 0 to 2; and p is an integer selected from 0 to 1 ;
  • steps A) and B) above are conducted in either order; with the proviso that:
  • ⁇ a' denotes the point of attachment to the alkyne terminating oligonucleotide or oligonucleotide analogue
  • ' denotes the point of attachment to the azide terminating oligonucleotide or oligonucleotide analogue
  • x, xi , z and zi are integers independently selected from 0 to 2; and y and yi are integers independently selected from 0 to 1 ; and
  • Formula ll lc wherein the one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula lib are formed at the point(s) of ligation by reacting:
  • oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula D2, shown below:
  • oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula F2 shown below:
  • Formula F2 optionally, in the presence of one or more peptide coupling reagents (e.g. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide);
  • one or more peptide coupling reagents e.g. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
  • ⁇ f independently denote the points of attachment to the target oligonucleotide or oligonucleotide analogue
  • / c denotes the point of attachment to the oligonucleotide or oligonucleotide analogue comprising a terminal functional group of Formula C2;
  • ⁇ d' denotes the point of attachment to the oligonucleotide or oligonucleotide analogue comprising a terminal functional group of Formula D2;
  • ⁇ e' denotes the point of attachment to the oligonucleotide or oligonucleotide analogue comprising a terminal functional group of Formula E2;
  • ⁇ f denotes the point of attachment to the oligonucleotide or oligonucleotide analogue comprising a terminal functional group of Formula F2;
  • X is OH
  • R 2a , R 2b , R 2c and R 2d are independently selected from hydrogen or methyl
  • n, rii , m and m ⁇ are integers independently selected from 0 to 2;
  • steps A) and B) above are conducted in either order; with the proviso that:
  • an oligonucleotide or oligonucleotide analogue comprising:
  • R 1 a , R 1 b , R 1 c , R 1 d , R 1 e , R 1 f , R 1 9 and R 1 h are each independently selected from hydrogen or (1 -4C)alkyl, wherein each (1 -4C)alkyl is optionally substituted with one or more NH2, OH or SH;
  • V and W are independently selected from O, S or NR X , wherein R x is selected from hydrogen or (1 -4C)alkyl;
  • x, xi, z and zi are integers independently selected from 0 to 2; and y and yi are integers independently selected from 0 to 1 ;
  • ⁇ f independently denote the points of attachment to the target oligonucleotide or oligonucleotide analogue
  • R 2a , R 2b , R 2c , R 2d , R 3a , R 3b , R 3c and R 3d are independently selected from hydrogen or (1-4C)alkyl, wherein each (1-4C)alkyl is optionally substituted with one or more NH2, OH or SH
  • R 2e and R 3e are independently selected from hydrogen or (1-4C)alkyl
  • Vi, Wi and W2 are independently selected from O, S or NR Z , wherein R z is selected from hydrogen or (1-4C)alkyl;
  • Q is selected from S or O
  • n, ni, m and mi are integers independently selected from 0 to 2; and p, pi and P2 are integers independently selected from 0 to 1 ;
  • the oligonucleotide or oligonucleotide analogue comprises one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula I and one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula II.
  • the oligonucleotide or oligonucleotide analogue comprises one phosphodiester backbone mimic inter-nucleoside linkage of Formula I and one phosphodiester backbone mimic inter- nucleoside linkage of Formula II.
  • the oligonucleotide or oligonucleotide analogue comprises one or more phosphodiester backbone mimic inter-nucleoside linkage of Formula I and one or more phosphodiester backbone mimic inter-nucleoside linkage of Formula III.
  • the oligonucleotide or oligonucleotide analogue comprises one phosphodiester backbone mimic inter-nucleoside linkages of Formula I and one phosphodiester backbone mimic inter- nucleoside linkages of Formula III.
  • R 1a , R 1 b , R 1c , R 1d , R 2a , R 2b , R 2c , R 2d , R 3a , R 3b , R 3c and R 3d are independently selected from hydrogen or (1-4C)alkyl.
  • R 1a , R 1 b , R 1c , R 1d , R 2a , R 2b , R 2c , R 2d , R 3a , R 3b , R 3c and R 3d are independently selected from hydrogen or methyl.
  • R 1a , R 1 b , R 1c , R 1d , R 2a , R 2b , R 2c , R 2d , R 3a , R 3b , R 3c and R 3d are hydrogen.
  • V and W are independently selected from O or NR X , wherein R x is selected from hydrogen or (1-4C)alkyl.
  • V and W are independently selected from O or NR X , wherein R x is selected from hydrogen or methyl. More suitably, V and W are independently selected from O or NH2. Most suitably, V and W are both O.
  • the sum of integers x, xi , y, yi , z and zi is either 0, 1 , 2, 3, 4 or 5.
  • the sum of integers x, xi , y, yi , z and zi is either 0, 1 , 2, 3 or 4. More suitably, the sum of integers x, xi , y, yi , z and zi is either 1 , 2, 3 or 4.
  • the sum of integers x, xi , y, yi , z and zi is either 1 , 2 or 3.
  • Q is oxygen
  • pi is 0.
  • p is 0.
  • Vi and Wi are independently selected from O or NR Z , wherein R z is selected from hydrogen or (1-4C)alkyl.
  • R z is selected from hydrogen or (1-4C)alkyl.
  • Vi and Wi are independently selected from O or NR Z , wherein R z is selected from hydrogen or methyl. More suitably, Vi and Wi are independently selected from O or NH. Most suitably, Vi and Wi are both oxygen.
  • the sum of integers n, ni , p is equal to 2, 3 or 4.
  • the sum of integers n, ni , p is equal to 2 or 3.
  • the sum of integers n, ni , p is equal to 2.
  • the sum of integers m and mi is equal to 0, 1 , 2, 3 or 4.
  • the sum of integers m and mi is equal to 0, 1 , 2 or 3. More suitably, the sum of integers m and mi is equal to 0, 1 or 2. Most suitably, the sum of integers m and mi is equal to 0 or 1.
  • s e denotes the point of attachment to a 3' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue and ⁇ f denotes the point of attachment to a 5' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue.
  • the one or more phosphodiester backbone mimic inter- nucleoside linkages of Formula I are selected from one of the following:
  • Zi and ⁇ 2 are independently selected from O or NH; a denotes the point of attachment to a 3' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue;
  • ⁇ denotes the point of attachment to a 5' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue.
  • the one or more phosphodiester backbone mimic inter- nucleoside linkages of Formula I I are selected from one of the following:
  • / c denotes the point of attachment to a 3' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue
  • ⁇ d denotes the point of attachment to a 5' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue.
  • the one or more phosphodiester backbone mimic inter- nucleoside linkages of Formula II I is of the following formula:
  • e denotes the point of attachment to a 3' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue
  • f denotes the point of attachment to a 5' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue.
  • an oligonucleotide or oligonucleotide analogue comprising:
  • x, xi , z and zi are integers independently selected from 0 to 2; and y and yi are integers independently selected from 0 to 1 ;
  • ⁇ f independently denote the points of attachment to the target oligonucleotide or oligonucleotide analogue
  • R 2a , R 2b , R 2c and R 2d are independently selected from hydrogen or (1- 4C)alkyl
  • n, ni , m and m ⁇ are integers independently selected from 0 to 2; and with the proviso that:
  • an oligonucleotide or oligonucleotide analogue comprising:
  • Zi and ⁇ 2 are independently selected from O or NH;
  • / a denotes the point of attachment to a 3' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue
  • ⁇ denotes the point of attachment to a 5' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue
  • c denotes the point of attachment to a 3' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue
  • ⁇ d denotes the point of attachment to a 5' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue
  • e denotes the point of attachment to a 3' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue
  • f denotes the point of attachment to a 5' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue.
  • oligonucleotides may be used therapeutically for the treatment of various diseases and disorders, such as, for example, cancer, genetic disorders and infection.
  • the present invention provides a use of an oligonucleotide or oligonucleotide analogue, as defined herein, in the treatment of a disease or disorder.
  • the disease or disorder is cancer.
  • the disease or disorder is a genetic disorder.
  • the disease or disorder is an infection.
  • a method for the treatment of a disease or disorder comprising administering a therapeutically effective amount of an oligonucleotide or oligonucleotide analogue, as defined herein, or a pharmaceutically acceptable salt or solvate thereof.
  • the disease or disorder is cancer.
  • the disease or disorder is a genetic disorder.
  • the disease or disorder is an infection.
  • oligonucleotide or oligonucleotide as defined herein, in the synthesis of genes.
  • oligonucleotide or oligonucleotide, as defined herein as a template in the synthesis of genes.
  • oligonucleotide or oligonucleotide as defined herein, as:
  • interference RNA e.g. siRNA
  • an RNA component of a CRISPR-Cas system e.g. crRNA, tracrRNA or
  • oligonucleotide or oligonucleotide analogue as defined herein, as:
  • PCR polymerase chain reaction
  • a method for amplifying an oligonucleotide or oligonucleotide analogue sequence comprising the steps of:
  • step 2 2) carrying out a polymerase chain reaction (PCR) using the oligonucleotide or oligonucleotide analogue of step 1 as a template.
  • PCR polymerase chain reaction
  • a method for replicating an oligonucleotide or oligonucleotide analogue sequence comprising the steps of:
  • step 2 2) carrying out a replication reaction using the oligonucleotide or oligonucleotide analogue of step 1 as a template.
  • RNA sequence comprising the steps of:
  • step 2 2) transcribing the oligonucleotide or oligonucleotide analogue of step 1 to form a
  • RNA transcript ribonucleic acid
  • a method for producing a deoxyribonucleic acid (DNA) sequence comprising the steps of:
  • step 2 2) reverse-transcribing the oligonucleotide or oligonucleotide analogue of step 1 to form a complementary deoxyribonucleic acid (cDNA) sequence.
  • cDNA complementary deoxyribonucleic acid
  • a method for preparing a protein or peptide comprising the steps of: 1) providing an oligonucleotide or oligonucleotide ananlogue as defined herein; and
  • step 2 2) translating the oligonucleotide or oligonucleotide analogue of step 1 to form the protein or peptide.
  • Class 1 systems have a multi-subunit crRNA-effector complex such as Cascade- Cas3, whereas Class 2 systems have a crRNA-effector complex having a single Cas protein, such as Cas9, Cas12 (previously referred to as Cpfl) and Cas 13a (previously referred to as C2c2).
  • Cpfl Cas9
  • Cas12 previously referred to as Cpfl
  • Cas 13a previously referred to as C2c2
  • Type II systems there is a second RNA component tracrRNA which hybridises to crRNA to form a crRNA:tracr RNA duplex, these two RNA components may be linked to form single guide RNA.
  • RNA components in such CRISPR-Cas systems may be adapted to be an oligonucleotide in accordance with the invention or a dinucleotide of the invention may be comprised within an RNA components of a CRISPR-Cas system.
  • RNA component e.g., to guide the crRNA:effector complex to a target site.
  • Standard methods are known in the art for testing whether oligonucleotides of the invention when used as such CRISPR RNA components retain the desired function (e.g. by comparing the desired function to that of a control CRISPR RNA component which has the same nucleosides without any-triazole linker moieties between nucleosides or locked nucleosides).
  • CRISPR RNA components or "RNA component of a CRISPR-Cas system” is used herein, as in most CRISPR-Cas systems, the nucleic acid sequences which guide the effector protein(s) to a desired target sequence are RNA components.
  • CRISPR hybrid DNA/RNA polynucleotides which can also function to guide effector protein(s) to a desired target site in a DNA or RNA sequence are also known in the art - see for example Rueda et al. (Mapping the sugar dependency for rational generation of a DNA-RNA hybrid- guided Cas9 endonuclease, Nature Communications 8, Article Number: 1610 (2017)).
  • reference to CRISPR RNA components herein may also encompass hybrid RNA/DNA components such as crDNA/RNA, tracrDNA/RNA or gDNA/RNA.
  • the oligonucleotides of the invention may have particular utility in in vivo gene therapy applications.
  • one way of carrying out in vivo therapy using a Type II CRISPR-Cas system involves delivering the Cas9 and tracrRNA via a virus, which can assemble inactive complexes inside of cells.
  • the crRNA can then be administered later to assemble and selectively activate CRISPR/Cas9 complexes, which would then go on to target and edit specific sites in the human genome, such as disease relevant genes (Gagnon and Corey, Proc. Natl. Acad. Sci. USA 1 12: 15536-15537, 2015; Rahdar, et al, Proc. Natl. Acad. Sci.
  • crRNA:effector complexes i.e. CRISPR-Cas complexes, such as CRISPR/Cas9
  • CRISPR-Cas complexes such as CRISPR/Cas9
  • Oligonucleotides of the invention when used as crRNAs may improve this approach by offering stability against degradation.
  • the oligonucleotides of the invention when used as CRISPR RNA components can advantageously be used for the various applications of CRISPR-Cas systems known in the art including: gene-editing, gene activation (CRISPRa) or gene interference (CRISPRi), base-editing, multiplex engineering (CRISPRm), DNA amplification, diagnostics (e.g. SKERLOCK or DETECTR), cell recording (e.g. CAMERA), typing bacteria, antimicrobial applications, synthesising new chemicals etc..
  • CRISPR-Cas systems known in the art including: gene-editing, gene activation (CRISPRa) or gene interference (CRISPRi), base-editing, multiplex engineering (CRISPRm), DNA amplification, diagnostics (e.g. SKERLOCK or DETECTR), cell recording (e.g. CAMERA), typing bacteria, antimicrobial applications, synthesising new chemicals etc.
  • the oligonucleotides of the invention can be used as RNA components such as the "sacrificial RNA molecules" used to create a signal.
  • Figure 1 shows: A) a schematic representation of the templated simultaneous orthogonal phosphoramidate and CuAAC ligation reactions; and B) a schematic representation of the concept of single tube gene assembly by phosphoramidate ligation followed by transcription of modified DNA.
  • Figure 2 shows the 12% denaturing PAGE analysis of 3'-phosphate/5'-amine oligonucleotides ligation to give the phosphoramidate-containing product.
  • Lane 1 phosphoramidate reaction mixture (ODN 1 , 81-mer), lane 2; reference starting material ODN 3.
  • ODN 3 An excess of the amine oligonucleotide (ODN 3) was used, resulting in a residual lower band and complete consumption of the phosphate oligonucleotide.
  • Figure 3 shows the 12% denaturing PAGE analysis for optimisation of 3'-phosphate/5'-amine oligonucleotides ligation to give the phosphoramidate template (ODN 1 , 81-mer) top bands. Lanes 1-8; reaction mixture after 5, 10, 15, 30, 45, 60 120 and 360 min. An excess of the amine oligonucleotide was used resulting in a residual lower band and consumption of the phosphate oligonucleotide
  • Figure 4 shows the PCR amplification of the 81-mer phosphoramidate DNA template (ODN 1).
  • Lane 1 50 bp DNA ladder, lane 2; PCR using the phosphoramidate-containing template ODN 1 , lane 3; control PCR without a phosphoramidate linkage.
  • Figure 5 shows the 6% denaturing PAGE analysis of 3'-phosphate/5'-amine oligonucleotide ligation to give the product containing two phosphoramidate linkages (ODN 5, 303-mer).
  • Figure 6 shows the PCR amplification of the 303-mer DNA template ODN 5.
  • Lane 1 50 bp DNA ladder, lane 2; control PCR without phosphoramidate linkage, lane 3 and 4; PCR using the phosphoramidate-containing template ODN 5 (303-mer).
  • Figure 7 shows the sequence alignment of 20 Clones from PCR of ODN 5 (2x phosphoramidate linkages in 303-mer section of EGFP gene, in red with the ligation points in blue). All the sequences are identical indicating the biocompatibility of the phosphoramidate linkage. Only a few mutations were observed and these are far from the ligation points (see Table 1). The mutations could have occurred during sequencing or during oligonucleotide synthesis and purification.
  • Figure 8 shows: A) PCR amplification of the double stranded phosphoramidate EGFP gene (762-mer). Lane 1 ; 100 bp DNA ladder, lane 2; PCR using the double strand phosphoramidate EGFP gene (762-mer), lane 3; control PCR for individual oligos without ligation.
  • Figure 9 shows the data from cloning and sequencing of the PCR product from the phosphoramidate EGFP gene (762-mer) showing the faithful copying at the ligation points (shown in red in the sequence text) (A) and the water mark GTACA (B). All clones show the water mark which was inserted into the sequence of the synthesised EGFP gene as a unique signature to differentiate it from potential contaminant DNA.
  • Figure 10 shows the data from cloning and sequencing of the PCR product of the phosphoramidate EGFP gene (762-mer). The data show that the polymerase copied the gene faithfully including the bases around the phosphoramidate ligation points (shown in red in the inserted sequence text). Only one deletion mutation was found in this clone.
  • Figure 11 shows the transcription of 79-mer unmodified and phosphoramidate-containing DNA templates.
  • Lane 1 and 2 reaction using phosphoramidate template (ODN 27) and short coding strand (ODN 33) for 2 and 4 h respectively; lane 3; template ODN 31 lane 4 and 5, reaction using control template (ODN 31) and short coding strand (ODN 33) for 2 and 4 h respectively; Lane 6 and 7, reaction using phosphoramidate template (ODN 27) and long coding strand (ODN 32) for 2 and 4 h respectively; lane 8 and 9, reaction using control template (ODN 31) and long coding strand (ODN 32) for 2 and 4 h respectively. 15% polyacrylamide gel.
  • Figure 12 shows the ES- Mass spectra of A), the RNA transcripts from the phosphoramidate- containing template (ODN 27) and B), the normal template (ODN 31).
  • the transcripts have the expected 5'-triphosphate and an additional 3'-cytidine.
  • Required mass 17.236 KD. Found mass, 17.239 (transcript with 5'-triphosphate), 17.261 (transcript with 5'-triphosphate + Na + ) and 17.566 (transcript with 5'-triphosphate and 3'-cytidine).
  • Figure 13 shows the orthogonal phosphoramidate and CuAAC reactions for ligation of three oligonucleotides to make a 303-mer product.
  • Lane 1 ODN 6, Iane2; 2 x CuAAC reactions, lane 3; 2 x phosphoramidate reactions, lane 4; orthogonal phosphoramidate and CuAAC reactions.
  • Figure 14 shows the orthogonal phosphoramidate and CuAAC reactions for ligation of three oligonucleotides to make a fluorescent 331 -mer product.
  • Lane 1 starting material ODN 39, Iane2; orthogonal phosphoramidate and CuAAC reactions using ODN 39 (3'-phosphate, 5'- Cy3), ODN 38 (3'-propargyl and 5'-amine) and ODN 40 (5'-azide).
  • ODN 39 3'-phosphate, 5'- Cy3
  • ODN 38 (3'-propargyl and 5'-amine
  • ODN 40 (5'-azide).
  • Figure 15 shows a schematic representation of the orthogonal phosphoramidate and CuAAC reactions for ligation of three oligonucleotides to make a 303-mer product. Also shown is the gel electrophoresis trace. Lane 1 ; orthogonal phosphoramidate and CuAAC reactions using ODN 6 (3'-phosphate), ODN 38 (3'-propargyl and 5'-amine) and ODN 37 (5'-azide), Iane2; starting material ODN 6. Denaturing 8% polyacrylamide gel-electrophoresis.
  • Figure 16 shows a schematic representation of how the process of the present invention may be applied using solid supported chemistry.
  • Standard DNA phosphoramidites, solid supports, and additional reagents were purchased from Link Technologies Ltd and Applied Biosystems Ltd.
  • 5'- Monomethoxytritylamino-2'-deoxythymidine,3'-[(2-cyanoethyl)-(N,N-diisopropyl)]- phosphoramidite was purchased from Glen Research (Catalog Number: 10-1932-90).
  • oligonucleotides were synthesized on an Applied Biosystems 394 automated DNA/ RNA synthesizer using a standard 0.2 or 1.0 /ymole phosphoramidite cycle of acid- catalyzed detritylation, coupling, capping, and iodine oxidation. Stepwise coupling efficiencies and overall yields were determined by the automated trityl cation conductivity monitoring facility and in all cases were >98.0%.
  • oligonucleotides Purification of oligonucleotides was carried out by reversed-phase HPLC on a Gilson system using a Brownlee Aquapore column (C8, 8 mm x 250 mm, 300A pore) with a gradient of acetonitrile in triethylammonium bicarbonate (TEAB) increasing from 0% to 50% buffer B over 30 min with a flow rate of 4 mL/min (buffer A: 0.1 M triethylammonium bicarbonate, pH 7.0, buffer B: 0.1 M triethylammonium bicarbonate, pH 7.0 with 50% acetonitrile). Elution of oligonucleotides was monitored by ultraviolet absorption at 295 or 300 nm. After HPLC purification, oligonucleotides were freeze dried then dissolved in water without the need for desalting.
  • TEAB triethylammonium bicarbonate
  • oligonucleotides For long oligonucleotides, polyacrylamide gel electrophoresis was used for purification. Oligonucleotide bands were then visualized using a UV lamp and the desired bands excised, crushed and soaked in water overnight at 37 °C. After evaporation, samples were desalted using NAP-25 followed by NAP-10 columns (G.E. Healthcare Life Sciences). All oligonucleotides were characterised by electrospray mass spectrometry using a Bruker micrOTOF II focus ESI-TOF MS instrument in ESI " mode. Data were processed using MaxEnt.
  • GoTaq DNA polymerase was used to generate a PCR product from the 81 -mer template (ODN 1) which includes one phosphoramidate linkage.
  • ODN 1 81 -mer template
  • Reagents and conditions 4 ⁇ _ of 5x buffer (Promega green PCR buffer) was used in a total reaction volume of 20 ⁇ _ with 5 ng of the DNA template, 0.5 mM of each primer, 0.2 mM dNTP and 1.0 unit of GoTaq polymerase.
  • the reaction mixture was loaded onto a 2% agarose gel in 1xTBE buffer.
  • 5 X Promega green PCR buffer was provided with the enzyme (Promega GoTaq DNA polymerase), pH 8.5 containing 7.5 mM MgC to give a final Mg 2+ concentration of 1.5 mM.
  • the buffer contains Tris.HCI, KCI and two dyes (blue and yellow) that separate during electrophoresis to monitor the migration process.
  • Oligonucleotides ODN 6, ODN 7, ODN 8 with splints ODN 9 and ODN 10 were annealed by heating at 90 °C for 5 min then cooling slowly to room temperature.
  • a solution of 1-(2-hydroxyethyl) imidazole (1.0 M, 10 ⁇ _) (0.1 M final concentration) and EDC.HCI (6.0 M, 10 ⁇ _) (0.6 M final concentration) was added to the annealed oligonucleotides and the reaction mixture was kept at room temperature for 2 h. Reagents were removed using NAP-25 gel-filtration column and the ligated DNA was analysed by denaturing 6% polyacrylamide gel electrophoresis.
  • TC total clones
  • NMC non-mutant clones
  • MC mutant clones
  • IM insertion mutation
  • DM deletion mutation
  • SM substituted mutation
  • LPM ligation point mutation
  • TB total number of bases
  • TM total number of mutation.
  • EDC.HCI (30 mg) and a solution of 1-(2-hydroxyethyl) imidazole (1.0 M, 30 ⁇ _) were added to the annealed oligonucleotides and the reaction mixture was kept at room temperature for 2 h. Reagents were removed using NAP-25 gel-filtration column and the ligated DNA was analysed by denaturing 4% polyacrylamide gel electrophoresis. The band was cut and DNA was extracted then used in PCR.
  • a PCR product from the whole EGFP gene duplex was generated using GoTaq DNA polymerase under the same conditions explained above for PCR of 81-mer ODN 1.
  • the PCR product was purified by extraction from a 2% agarose gel ( Figure 8A) using a QIAquick Gel Extraction kit. It was then inserted into the vector pCR2.1. Cloning into the TOPO vector was done with a standard TOPO cloning protocol. Automated Sanger DNA sequencing was performed; and the data is shown in Figure 9 and Figure 10. This procedure was carried out by Eurofins GmbH.
  • Oligonucleotide bands were then visualized using a UV lamp and the desired bands excised, crushed and soaked in buffer (50 mM Tris-HCI, pH 7.5, 25 mM NaCI) overnight at 37 °C. After evaporation of the solvent, samples were desalted using two NAP-25 columns (G.E. Healthcare Life Sciences, cat. no. 17-0852-01). The expected product was confirmed by mass spectrometry of transcripts formed from phosphoramidate-containing and control strands using the long coding strand.
  • RNA transcripts were precipitated by adding sodium acetate (3 M, 50 ⁇ ) followed by isopropanol (150 ⁇ ). The mixture was left at -80 °C for 3 h then centrifuged at 4 °C and 13 RPM for 10 min. The RNA was dried then dissolved in 20 ⁇ water where 0.5 ⁇ was analysed by mass spectrometry. The crude transcripts gave the same (expected) mass for phosphoramidate and control templates.
  • a solution of Cu' click catalyst was prepared from fr/s-hydroxypropyltriazole (0.7 ⁇ in 0.2 M NaCI, 17.0 ⁇ _), and sodium ascorbate (1.0 ⁇ in 0.2 M NaCI, 2.0 ⁇ _) and CuS0 4 .5H 2 0 (0.1 ⁇ in 0.2 M NaCI, 1.0 ⁇ _) was added to the above annealed oligonucleotides. The mixture was kept at room temperature for 2 h before analysis by denaturing 4% polyacrylamide gel electrophoresis.
  • a solution of Cu' click catalyst was prepared from fr/s-hydroxypropyltriazole (0.35 ⁇ in 0.2 M NaCI, 17.0 ⁇ _), sodium ascorbate (1.0 ⁇ in 0.2 M NaCI, 1.0 ⁇ _) and CuS0 4 .5H 2 0 (0.1 ⁇ in 0.2 M NaCI, 1.0 ⁇ _) followed by EDC.HCI (10 mg) and a solution of 1-(2-hydroxyethyl) imidazole (1.0 M, 10 ⁇ _) were added to the annealed oligonucleotides and the reaction mixture was kept at room temperature for 2 h before being analysed by denaturing 8% polyacrylamide gel electrophoresis.
  • Figure 13 shows similar results for all three reactions indicating the orthogonality of CuAAC click and phosphoramidate ligations.
  • the orthogonal CuAAC and phosphoramidate reactions were repeated under the same conditions using fluorescently labelled oligonucleotide ODN 39, and gave a similar result as indicated by denaturing 8% polyacrylamide gel-electrophoresis ( Figure 14).
  • ODN 7 AAATTTATTTGTACTACTGGTAAATTGCCAGTTCCATGGCCAACCTT
  • ODN 8 I AAACAACATGACTTTTTCAAGTCTGCCATGCCAGAAGGTTATGTTCA
  • SeqI D 13 ⁇ ODN 11 GTTCTTTCTTGAACATAA PCR Primer 1 for 303-mer template j SeqI D 14 j ODN 12 ; AAGCTTT ATTAAAAT GTCTA PCR Primer 2 for 303-mer template SeqID 15 pTCGACGGTACCGCGGGCCCGGGATCCACCGGTCGCCACCATGGT
  • SeqID 16 pGGCCGCTTTACTTGTACAGCTCGTCCATGCCGAGAGTGATCCCGG
  • SeqID 17 gTCGACGGTACCGCGGGCCCGGGATCCACCGGTCGCCACCATGGT
  • ODN 27 TTCTCCCTATAGTGAGTCGTATTAGGACCAGCGT transcription template
  • SeqID 30 j ⁇ TCGCCCTTGCTCACCATGGTGGCGACTTCTCCCTATAGTGAGTCG
  • ODN 31 TTCTCCCTATAGTGAGTCGTATTAGGACCAGCGT control for transcription
  • SeqID 34 j ACGCTGGTCCTAATACGACTCACTATAGGGAGAAGTCGCCACCATG
  • SeqID 35 j ACGCTGGTCCTAATACGACTCACTATAGGGAGAAGTCGCC short
  • SeqID 36 j pppGGGAGAAGUCGCCACCAUGGUGAGCAAGGGCGAGGAGCUGU j
  • SeqID 37 j AAGCTTTATTAAAATGTCTAAAGGTGAAGAATTATTCACTGGTGTTG j
  • SeqID 39 JTTTCGGTTATGGTGTTCAATGTTTTGCTAGATACCCAGATCATATGA

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Abstract

The present invention relates to a process for preparing oligonucleotides or oligonucleotide analogues comprising triazole and (phosphor)amidate internucleoside linkages. The present invention also relates to the oligonucleotides and oligonucleotide analogues formed from said process and to the use of these oligonucleotides and oligonucleotide analogues in the synthesis of genes, as therapeutics in treatment of certain diseases and disorders (e.g. cancer) and as templates in polymerase chain reactions, DNA replication processes, RNA transcription processes and/or translation processes.

Description

OLIGONUCLEOTIDES COMPRISING TRIAZOLE AND (PHOSPHOR)AMIDATE INTERNUCLEOSIDE LINKAGES,
METHOD FOR THE PREPARATION AND USES THEREOF
INTRODUCTION
[0001] The present invention relates to a process for preparing oligonucleotides or oligonucleotide analogues. The present invention also relates to the oligonucleotides and oligonucleotide analogues formed from said process and to use of these oligonucleotides and oligonucleotide analogues in the synthesis of genes, as therapeutics in treatment of certain diseases and disorders (e.g. cancer) and as templates in polymerase chain reactions, DNA replication processes, RNA transcription processes and/or translation processes.
BACKGROUND OF THE INVENTION
[0002] Gene synthesis is an important and rapidly growing field. The most successful and by far most common methods are based on PCR amplification of synthetic oligonucleotide pools. This methodology is used routinely to produce large DNA constructs up to several kilobases (kb) in length, and has served the biological community well.1 However, it has limitations, foremost of which is its inability to produce DNA that contains modifications at specific pre-defined loci. Such modified DNA constructs, if available, would be useful in many applications including epigenetics.2
[0003] The limitations of PCR are due to the fact that DNA polymerases cannot discriminate between the canonical deoxyribonucleoside triphosphates (dNTPs) and modified versions. This is because any modified dNTP must possess the same fundamental Watson-Crick base pairing properties as its natural counterpart in order to be incorporated into DNA by polymerase enzymes. Consequently, the natural and unnatural dNTPs compete in an uncontrollable manner. An obvious solution to this problem is to assemble DNA by ligation of pre-synthesized chemically modified oligonucleotides. This would open up new areas of biology, allowing a vast array of modifications to be incorporated into genomic DNA.
[0004] Ligation can be carried out enzymatically,3 but chemical ligation offers an attractive alternative.4 It is compatible with large scale applications, radical modifications to the sugars and nucleobases, templated or non-templated reactions, and can be carried out in conditions under which ligase enzymes would not remain functional, including automated nucleic acid assembly. Moreover, chemical ligation is not restricted to the natural phosphodiester backbone of DNA; other backbones can be produced, some of which have been found to be biocompatible.5,6 The use of chemical ligation to produce modified backbones has other advantages; highly efficient chemical reactions can be chosen, orthogonal ligation chemistries can be used simultaneously for special applications (Figure 1A), and successful DNA ligation strategies, once developed, can be applied to the synthesis of long RNA strands7 with potential applications in gene editing.8
[0005] However, importantly, for any modified DNA ligation chemistry to be useful in biology, it should be compatible with the synthesis of functional genes, i.e. as well as being efficient, it must not give rise to mutations, and the modified linkage should be compatible with polymerase enzymes. Presently, very few DNA chemical ligation methodologies exist which produce oligonucleotides comprising phosphodiester mimic inter-nucleoside linkages that are compatible with polymerase enzymes (e.g. DNA and/or RNA polymerase).
[0006] Thus, there remains a need in the art for further and improved DNA chemical ligation methodologies that are capable of producing oligonucelotides and oligonucleotide analogues that possess improved binding affinities for complimentary DNA and/or RNA strands.
[0007] The present invention was devised with the foregoing in mind.
SUMMARY OF THE INVENTION
[0008] According to a first aspect of the present invention there is provided a process for preparing a target oligonucleotide or oligonucleotide analogue comprising two or more different phosphodiester mimic inter-nucleoside linkages, as defined herein.
[0009] According to a second aspect of the present invention there is provided an oligonucleotide or oligonucleotide analogue as defined herein.
[0010] According to a third aspect of the present invention, there is provided a use of an oligonucleotide or oligonucleotide, as defined herein, in the synthesis of genes. Suitably, there is provided a use of an oligonucleotide or oligonucleotide, as defined herein, as a template in the synthesis of genes.
[0011] According to a fourth aspect of the present invention, there is provided a use of an oligonucleotide or oligonucleotide, as defined herein, as:
(i) antisense DNA or RNA;
(ii) exon skipping DNA or RNA; or
(iii) interference RNA (e.g. siRNA); or (iv) an RNA component of a CRISPR-Cas system (e.g. crRNA, tracrRNA or gRNA).
[0012] According to a fifth aspect of the present invention there is provided a use of an oligonucleotide or oligonucleotide, as defined herein, in the treatment of a disease or disorder. Suitably, the disease or disorder is cancer, a genetic disorder or an infection.
[0013] According to a sixth aspect of the present invention there is provided a use of an oligonucleotide or oligonucleotide, as defined herein, as:
i) a template for amplification in a polymerase chain reaction (PCR):
ii) as a template in a DNA replication process;
iii) as a template in a transcription process to provide a corresponding RNA
transcript, or as a template in a reverse transcription process to provide a corresponding DNA transcript;
iv) as template in a translation process to produce a corresponding protein or peptide; or
v) to guide one or more proteins of interest to a target DNA or RNA.
[0014] Features, including optional, suitable, and preferred features in relation to one aspect of the invention may also be features, including optional, suitable and preferred features in relation to any other aspects of the invention.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0015] Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of them mean "including but not limited to", and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.
[0016] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.
[0017] The term "alkyl" includes both straight and branched chain alkyl groups. References to individual alkyl groups such as "propyl" are specific for the straight chain version only and references to individual branched chain alkyl groups such as "isopropyl" are specific for the branched chain version only. For example, "(1-6C)alkyl" includes (1-4C)alkyl, (1-3C)alkyl, propyl, isopropyl and f-butyl.
[0018] The term "(m-nC)" or "(m-nC) group" used alone or as a prefix, refers to any group having m to n carbon atoms.
[0019] The term "halo" refers to fluoro, chloro, bromo and iodo.
[0020] Where optional substituents are chosen from "one or more" groups it is to be understood that this definition includes all substituents being chosen from one of the specified groups or the substituents being chosen from two or more of the specified groups.
[0021] The phrase "oligonucleotide or oligonucleotide analogue of the invention" means those oligonucleotides or oligonucleotide analogues which are disclosed herein, both generically and specifically.
[0022] The term "oligonucleotide" refers to a polynucleotide strand. It will therefore be understood that the term oligonucleotide used herein covers both "short" polynucleotide strands comprising between 2 and 500 nucleotide residues and "long" polynucleotide strands comprising greater than 500 nucleotide residues. It will also be appreciated by those skilled in the art that an oligonucleotide has a 5' and a 3' end and comprises a sequence of nucleosides linked together by inter-nucleoside linkages.
[0023] The terms "oligonucleotide analogue" and "nucleotide analogue" refer to any modified synthetic analogues of oligonucleotides or nucleotides respectively that are known in the art. Examples of oligonucleotide analogues include peptide nucleic acids (PNAs), morpholino oligonucleotides, phosphorothioate oligonucleotides, phosphorodithioate oligonucleotides, alkylphosphonate oligonucleotides, acylphosphonate oligonucleotides and phosphoramidate oligonucleotides.
[0024] The term "nucleobase analogue" refers to any analogues of nucleobases known in the art. The skilled person will appreciate there to be numerous natural and synthetic nucleobase analogues available in the art which could be employed in the present invention. As such, the skilled person will readily be able to identify suitable nucleobase analogues for use in the present invention. Commonly available nucleobase analogues are commercially available from a number of sources (for example, see the Glen Research catalogue (http://www.glenresearch.com/Catalog/contents.php). It will also be appreciated that the term "nucleobase analogue" covers: universal/degenerate bases (e.g. 3-nitropyrrole, 5-nitroindole and hypoxanthine); fluorescent bases (e.g. tricyclic cytosine analogues (tCO, tCS) and 2- aminopurine); base analogues bearing reactive groups selected from alkynes, thiols or amines; and base analogues that can crosslink oligonucleotides to DNA, RNA or proteins (e.g. 5-bromouracil or 3-cyanovinyl carbazole).
[0025] The nucleobase or nucleobase analogue is attached to a sugar moiety (typically ribose or deoxyribose) or a ribose or deoxyribose mimic, for example a chemically modified sugar derivative (e.g. a chemically modified ribose or deoxyribose) or a cyclic group that functions as a synthetic mimic of a ribose or deoxyribose sugar moiety (e.g. the morpholino ring present in morpholino oligonucleotides). The term "nucleoside" is used herein to refer to a moiety composed of a sugar / a ribose or deoxyribose mimic bound to a nucleobase/nucleobase analogue. The term nucleoside as used herein excludes the inter-nucleoside linkage that connects adjacent nucleosides together. An "inter-nucleoside linkage" is a linking group that connects the rings of the sugar / ribose or deoxyribose mimic of adjacent nucleosides.
[0026] The terms "locked nucleic acid", "LIMA" or "locked nucleoside" are used herein to refer to nucleic acids or nucleosides comprising a ribose or deoxyribose moiety in which the conformation of the ribose or deoxyribose ring is fixed or locked in a specific conformation, typically by a bridging group. Typically the bridging group connects the 2' and 4' carbon atoms of the ribose or deoxyribose rings and locks the ribose or deoxyribose in the 3'-endo conformation (which is often found in A-form duplexes). Examples of locked nucleic acid/nucleoside structures are well known in the art and are commercially available.
Process of the invention
[0027] According to one aspect of the present invention, there is provided a process for preparing a target oligonucleotide or oligonucleotide analogue comprising two or more different phosphodiester mimic inter-nucleoside linkages, wherein the process comprises the steps of:
A) ligating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula I, shown below, at the point(s) of ligation:
Figure imgf000007_0001
Formula I wherein:
/ a and independently denote the points of attachment to the target oligonucleotide or oligonucleotide analogue;
R1 a, R1 b, R1 c, R1 d, R1 e, R1 f, R19 and R1 h are each independently selected from hydrogen or (1 -4C)alkyl, wherein each (1 -4C)alkyl is optionally substituted with one or more NH2, OH or SH;
V and W are independently selected from O, S or NRX, wherein Rx is selected from hydrogen or (1 -4C)alkyl;
x, xi , z and zi are integers independently selected from 0 to 2; and y and yi are integers independently selected from 0 to 1 ;
and
ligating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula II and/or Formula II I shown below, at the point(s) of ligation:
Figure imgf000007_0002
Formula II I wherein:
tV LV'
/ c d e and ^ f independently denote the points of attachment to the target oligonucleotide or oligonucleotide analogue;
R2a, R2b, R2c, R2d, R3a, R3b, R3c and R3d are independently selected from hydrogen or (1-4C)alkyl, wherein each (1-4C)alkyl is optionally substituted with one or more NH2, OH or SH
R2e and R3e are independently selected from hydrogen or (1-4C)alkyl;
Vi, Wi and W2 are independently selected from O, S or NRZ, wherein Rz is selected from hydrogen or (1-4C)alkyl;
Q is selected from S or O;
n, rii , m and m 1 are integers independently selected from 0 to 2; and p, pi and P2 are integers independently selected from 0 to 1 ;
and wherein steps A) and B) above are conducted in either order;
with the proviso that:
1) the sum of integers x, xi, y, yi , z and zi is either 0, 1 , 2, 3, 4, 5 or 6;
2) the sum of integers n, rii , p and pi are greater than or equal to 2;
3) the sum of integers m, mi and P2 is equal to 0, 1 , 2, 3 or 4; and
Figure imgf000008_0001
[0028] The inventors have discovered a new process for preparing oligonucleotides or oligonucleotide analogues comprising two or more different phosphodiester mimic inter- nucleoside linkages. In exploiting orthogonal chemistries to prepare the two or more different phosphodiester mimic inter-nucleoside linkages, the inventors have provided a cheap and highly efficient process for preparing oligonucleotides and analogues thereof comprising a vast array of modifications. Furthermore, the inventors have also discovered that the two or more different phosphodiester mimic inter-nucleoside linkages used in the present process are fully compatible with and readable by DNA and RNA polymerases, which makes the oligonucleotides and oligonucleotide analogues prepared by the present process of particular use in a number of biological applications.
[0029] It will be appreciated that the process of the present invention also comprises the preparation of long polynucleotides (e.g. genes), wherein the long polynucleotide comprises greater than 500 nucleotide residues. [0030] In certain embodiments, the process of the present invention is conducted in the presence of a template.
[0031] It will be appreciated that any suitable template may be used. A person skilled in the art will be able to select a suitable template having the correct size and nucleoside sequence for hybridisation with the oligonucleotides and oligonucleotide analogues of the present process that are to be ligated together. It will also be understood that the template may comprise both an oligonucleotide and a synthetic oligonucleotide analogue, such as, for example, a peptide nucleic acid (PNA).
[0032] Suitably, the template is a single stranded oligonucleotide or oligonucleotide analogue that the oligonucleotides or oligonucleotide analogues to be ligated bind to, such that, functional groups on the termini of adjacent oligonucleotides or oligonucleotide analogues are available to be ligated together to form an inter-nucleoside linkage of Formula I, II and III as defined herein.
[0033] In other embodiments, the process of the present invention is conducted in the absence of a template. Processes conducted in the absence of a template will be understood to encompass reactions such as, for example, solution phase reactions and/or solid supported reactions.
[0034] In a further embodiment of the present process, at least one of the oligonucleotides or oligonucleotide analogues to be ligated is attached to a solid support.
[0035] It will be appreciated that any solid support that is suitable for use in oligonucleotide synthesis may be used. In an embodiment, the solid support is selected from controlled pore glass (CPG), silica, hydroxylated methacrylic polymer beads (e.g. Toyopearl® beads), grafted copolymers comprising a crosslinked polystyrene matrix onto which polyethylene glycol is grafted (e.g. Tenagel®) or microporous polystyrene (MPPS). Suitably, the solid support is selected from controlled pore glass (CPG) or microporous polystyrene (MPPS). Most suitably, the solid support is a controlled pore glass (CPG) support.
[0036] One of the advantages of the present invention is that it allows multiple (i.e. three or more) oligonucleotides or oligonucleotide analogous to ligated together sequentially. The inventors have advantageously discovered that in exploiting the orthogonal chemistries used to prepare the one or more phosphodiester backbone mimic inter-nucleoside linkages of Formulae I, II or III, oligonucleotides and oligonucleotide analogues can be prepared easily and very efficiently, without the need for any functional group interconversions.
[0037] Thus, it will be understood that the target oligonucleotides or oligonucleotide analogues prepared by the present process may comprise any number of phosphodiester backbone mimic inter-nucleoside linkages of Formula I and any number of phosphodiester backbone mimic inter-nucleoside linkages of Formula II or Formula III.
[0038] In another embodiment of the present invention, the target oligonucleotide or oligonucleotide analogue comprises one or more phosphodiester backbone mimic inter- nucleoside linkages of Formula I and one or more phosphodiester backbone mimic inter- nucleoside linkages of Formula II. Suitably, the target oligonucleotide or oligonucleotide analogue comprises one phosphodiester backbone mimic inter-nucleoside linkage of Formula I and one phosphodiester backbone mimic inter-nucleoside linkage of Formula II.
[0039] In a further embodiment of the present invention, the target oligonucleotide or oligonucleotide analogue comprises one or more phosphodiester backbone mimic inter- nucleoside linkages of Formula I and one or more phosphodiester backbone mimic inter- nucleoside linkages of Formula III. Suitably, the target oligonucleotide or oligonucleotide analogue comprises one phosphodiester backbone mimic inter-nucleoside linkage of Formula I and one phosphodiester backbone mimic inter-nucleoside linkage of Formula III.
[0040] In certain embodiments, the one or more phosphodiester backbone mimic inter- nucleoside linkages of Formula II or Formula III are formed before the one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula I. Thus, in an embodiment, step B) of the present process is conducted before step A).
[0041] It will also be understood that the target oligonucleotide prepared by the present process may also comprises at least one locked nucleoside. Locked nucleosides are well known in the art and include nucleic acids and/or nucleosides comprising a ribose or deoxyribose moiety in which the conformation of the ribose or deoxyribose ring is fixed or "locked" in a specific conformation, typically by a bridging group. A non-limiting list of suitable locked nucleosides which may be used in the present invention are described in K. Singh, S. and J. Wengel (1998). "Universality of LNA-mediated high-affinity nucleic acid recognition." Chemical Communications (12): 1247-1248 and/or Kaur, H., et al. (2007). "Perspectives on Chemistry and Therapeutic Applications of Locked Nucleic Acid (LIMA)." Chemical Reviews 107(1 1): 4672-4697.
[0042] Furthermore, it will also be appreciated that the at least one locked nucleoside may be positioned at either the 3' or 5' end of an inter-nucleoside linkage of Formula I, II or III defined herein, or a locked nucleoside may be positioned at both the 3' and 5' end of an inter- nucleoside linkage of Formula I, II or III defined herein.
[0043] In an embodiment, the at least one locked nucleoside is positioned at the 3' end of an inter-nucleoside linkage of Formula I, II or III defined herein. [0044] In another embodiment, the at least one locked nucleoside is positioned at the 5' end of an inter-nucleoside linkage of Formula I, II or III defined herein.
[0045] In yet another embodiment, the target oligonucleotide or oligonucleotide analogue comprises at least two locked nucleosides, with at least one locked nucleoside positioned at the 3' end of an inter-nucleoside linkage of Formula I, II or III defined herein and at least one locked nucleoside position at 5' end of an inter-nucleoside linkage of Formula I, II or III defined herein.
[0046] It will also be understood that the target oligonucleotides or oligonucleotide analogues prepared by the process of the present invention may be isolated and purified using any suitable techniques known in the art. Suitably, the target oligonucleotides or oligonucleotide analogues formed by the process of the present invention are isolated and purified using column chromatography, such as, for example, using Sephadex® columns (i.e. cross-linked dextran gels).
Step A
[0047] In an embodiment, step A) of the present process involves ligating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula la, shown below, at the point(s) of ligation:
Figure imgf000011_0001
Formula la wherein:
/ a and independently denote the points of attachment to the target oligonucleotide or oligonucleotide analogue;
V and W are independently selected from O, S or NRX, wherein Rx is selected from hydrogen or (1-4C)alkyl;
x, xi , z and zi are integers independently selected from 0 to 2; and
y and yi are integers independently selected from 0 to 1 ; with the proviso that the sum of x, xi , y, yi , z and zi is either 0, 1 , 2, 3, 4, 5 or 6.
[0048] In another embodiment, step A) of the present process involves ligating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula lb, shown below, at the point(s) of ligation:
Figure imgf000012_0001
Formula lb wherein:
/ a and independently denote the points of attachment to the target oligonucleotide or oligonucleotide analogue;
x, xi , z and zi are integers independently selected from 0 to 2; and
y and yi are integers independently selected from 0 to 1 ;
with the proviso that the sum of x, xi , y, yi , z and zi is either 0, 1 , 2, 3, 4 or 5.
[0049] In another embodiment, step A) of the present process involves ligating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages at the point(s) of ligation selected from:
Figure imgf000013_0001
target oligonucleotide or oligonucleotide analogue; and
^ denotes the point of attachment to a 5' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue.
[0050] In another embodiment, step A) of the present process involves ligating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages at the point(s) of ligation selected from:
Figure imgf000014_0001
/ a denotes the point of attachment to a 3' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue;
^ denotes the point of attachment to a 5' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue.
[0051] In another embodiment, step A) of the present process involves ligating two or more oligonucleotides or oligonucleotide analogues together to form a phosphodiester backbone mimic inter-nucleoside linkage at the point of ligation of the formula:
Figure imgf000015_0001
wherein:
/ a denotes the point of attachment to a 3' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue;
^ denotes the point of attachment to a 5' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue.
[0052] It will be understood that the one or more phosphodiester backbone mimic inter- nucleoside linkages of Formulae I, la or lb may be prepared using any suitable technique known in the art. The person skilled in the art will be able to select suitable reaction conditions and reagents to prepare the one or more phosphodiester backbone mimic inter-nucleoside linkages of Formulae I, la or lb.
[0053] In an embodiment, the one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula I are formed at the point(s) of ligation by reacting:
A1) one or more alkyne terminating oligonucleotides or oligonucleotide analogues
comprising a terminal functional group of Formula A shown below:
Figure imgf000015_0002
Formula A
wherein:
^ a' denotes the point of attachment to the alkyne terminating oligonucleotide or oligonucleotide analogue;
V is selected from O, S or NRX, wherein Rx is selected from hydrogen or (1- 4C)alkyl; R1 a, R1 b, R1 c and R1 d are independently selected from hydrogen or (1 - 4C)alkyl, wherein each (1 -4C)alkyl is optionally substituted with one or more NH2, OH or SH;
x and z are integers independently selected from 0 to 2; and
y is an integer selected from 0 to 1 ;
with
A2) one or more azide terminating oligonucleotides or oligonucleotide analogues
comprising a terminal functional group of Formula B, shown below:
Figure imgf000016_0001
Formula B
wherein: ' denotes the point of attachment to the azide terminating oligonucleotide or oligonucleotide analogue;
W is selected from O, S or NRX, wherein Rx is selected from hydrogen or (1 - 4C)alkyl;
R1 e, R1 f, R19 and R1 h are independently selected from hydrogen or (1 -4C)alkyl, wherein each (1 -4C)alkyl is optionally substituted with one or more NH2, OH or SH;
xi and zi are integers independently selected from 0 to 2; and yi is an integer selected from 0 to 1 ;
with the proviso that the sum of integers x, xi , y, yi , z and zi is either 0, 1 , 2, 3, 4, 5 or 6.
[0054] It will be appreciated that in certain embodiments, the alkyne terminal functional group of Formula A may be protected using a suitable protecting group. The person skilled in the art will be able to select suitable protecting groups to use. Non-limiting examples of suitable alkyne protecting groups include, for example, trialkylsilylacetylenes (e.g. trimethylsilylacetylene). In instances where the alkyne terminal functional group of Formula A is protected, it may be necessary to conduct the reaction between the one or more alkyne terminating oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula A and the one or more azide terminating oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula B in the presence of a deprotecting agent. Suitable deprotecting agents will be apparent to those skilled in the art. For instance, when the alkyne terminal functional group of Formula A is protected with a trimethylsilylacetylene, the reaction between the one or more alkyne terminating oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula A and the one or more azide terminating oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula B may optionally be carried out in the presence of tetrabutylammonium fluoride (TBAF) as the deprotecting agent.
[0055] It will also be understood that in other embodiments, the azido terminal functional group of Formula B may be masked in the form of an azide precursor. Again, suitable azide precursors will be known to the person skilled in the art. Non-limiting examples of azide precursors include alkyl halides, tosylates or mesylates. In instances where the azido terminal functional group of Formula B is delivered in the form of an azide precursor, it will be understood that it will be necessary to conduct the reaction between the one or more alkyne terminating oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula A and the one or more azide terminating oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula B in the presence of a suitable azide source (e.g. NaNs), to firstly convert the azide precursor to the terminal functional group of Formula B.
[0056] In an embodiment, the sum of integers x, xi , y, yi , z and zi is either 0, 1 , 2, 3, 4 or 5. Suitably, the sum of integers x, xi , y, yi , z and zi is either 0, 1 , 2, 3 or 4. More suitably, the sum of integers x, xi , y, yi , z and zi is either 1 , 2, 3 or 4. Most suitably, the sum of integers x, xi , y, yi , z and zi is either 1 , 2 or 3.
[0057] In an embodiment, V is selected from O or NRX, wherein Rx is selected from hydrogen or (1-4C)alkyl. Suitably, V is selected from O or NRX, wherein Rx is selected from hydrogen or methyl. Most suitably, V is O.
[0058] In a particular embodiment, y is 1. In another embodiment, y is 0.
[0059] In another embodiment, W is selected from O or NRX, wherein Rx is selected from hydrogen or (1-4C)alkyl. Suitably, W is selected from O or NRX, wherein Rx is selected from hydrogen or methyl. Most suitably, W is O.
[0060] In certain embodiments, yi is 1. In other embodiments, yi is 0.
[0061] In another embodiment, R1a, R1 b, R1c, R1d, R1e, R1f, R19 and R1 h are independently selected from hydrogen or (1-4C)alkyl. Suitably, R1a, R1 b, R1c, R1d, R1 e, R1f, R19 and R1 h are independently selected from hydrogen or methyl. Most suitably, R1a, R1 b, R1c, R1d, R1e, R1f, R19 and R1 h are hydrogen.
[0062] In yet another embodiment, x, xi , z and zi are integers independently selected from 0 to 1.
[0063] In a particular embodiment, ^ a' denotes the point of attachment to a 3' carbon of a
'Τ,-' nucleoside of the alkyne terminating oligonucleotide or oligonucleotide analogue and ^ v denotes the point of attachment to a 5' carbon of a nucleoside of the azide terminating oligonucleotide or oligonucleotide analogue.
[0064] It will be appreciated that step A) of the process may be conducted using any suitable reaction conditions. Furthermore, it will be understood that the reaction conditions used in the step A) of the present process will vary according to the specific oligonucleotide, oligonucleotide analogue and/or functional groups of Formula A and B that are used. A person skilled in the art will be able to select suitable reaction conditions (e.g. temperature, pressures, reaction times, concentration etc.) to use in the present process.
[0065] In an embodiment, the reaction between the one or more alkyne terminating oligonucleotides or oligonucleotide analogues of step A1) and the one or more azide terminating oligonucleotides or oligonucleotide analogues of step A2) is conducted in the presence of a catalyst. Suitably, the catalyst is a copper (I) species. Non-limiting examples of suitable catalysts include copper iodide (Cul), copper bromide (CuBr) or copper iodide-triethyl phosphite (Cul.P(OEt)3).
[0066] It will be appreciated that the catalyst (e.g. the copper (I) species) may be formed in situ upon adding a pre-catalyst complex and reducing agent to the reaction conditions. Accordingly, in an embodiment, the catalyst may be added in the form of a pre-catalyst together with a reducing agent. Non-limiting examples of suitable pre-catalysts include copper sulfate (CuSCU), copper chloride (CuC ), copper bromide (CuBr2), copper formate (Cu(OC(0)H)2), copper hydroxide (CuOhb) and copper nitrate (Cu(NOs)2). A non-limiting example of a suitable reducing agent is sodium ascorbate.
[0067] In an embodiment, the pre-catalyst is copper sulfate and the reducing agent is sodium ascorbate.
[0068] In another embodiment, the step of reacting together the alkyne terminating oligonucleotide or oligonucleotide analogue comprising a terminal functional group of Formula A with the azide terminating oligonucleotide or oligonucleotide analogue comprising a terminal functional group of Formula B may be repeated sequentially more than once, for example, more than twice, more than three times, more than four times or more than five times, to form an oligonucleotide or oligonucleotide analogue comprising the one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula I.
Step B
Phosphodiester backbone mimic inter-nucleoside linkages of Formula II
[0069] In an embodiment, step B) of the present process involves ligating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula lla, shown below, at the point(s) of ligation:
Figure imgf000019_0001
Formula lla
wherein:
/ c and d independently denote the points of attachment to the target oligonucleotide or oligonucleotide analogue;
R2a, R2b, R2c, R2d, R2e and Rz are independently selected from hydrogen or (1- 4C)alkyl;
n and ni are integers independently selected from 0 to 2; and
q is an interger from 0 to 1 ;
with the proviso that n + ni + p = 2, 3 or 4.
[0070] In an embodiment, step B) of the present process involves ligating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula lib, shown below, at the point(s) of ligation:
Figure imgf000019_0002
Formula lib wherein:
/ c and ^ d independently denote the points of attachment to the target oligonucleotide or oligonucleotide analogue;
R2a, R2b, R2c, R2d and R2e are independently selected from hydrogen or (1- 4C)alkyl; and
n and ni are integers independently selected from 0 to 2, with the proviso that
Figure imgf000020_0001
[0071] In yet another embodiment, step B) of the present process involves ligating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages at the point(s) of ligation selected from:
-CH2-C(0)-NH-CH2-;
-C(0)-NH-CH2-CH2-;
-CH2-CH2-C(0)-NH-;
-CH2-NH-C(0)-CH2;
-NH-C(0)-CH2-CH2-;
-CH2-C(0)-NH-0-CH2;
-0-NH-C(0)-CH2- -NH-C(0)-0-CH2- -0-C(0)-NH-CH2- -C(0)-NH-NH-CH2-;
-CH2-C(0)-NH-NH-CH2-;
-CH2-NH-NH-C(0)-;
-NH-NH-C(0)-CH2-;
-NH-C(0)-NH-CH2-;
-S-C(0)-NH-CH2-;
-NH-C(0)-S-CH2-; or
-NH-C(S)-NH-CH2-. [0072] In still another embodiment, step B) of the present process involves ligating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages at the point(s) of ligation selected from:
-CH2-C(0)-NH-CH2-;
-C(0)-NH-CH2-CH2-;
-CH2-CH2-C(0)-NH-;
-CH2-NH-C(0)-CH2;
-NH-C(0)-CH2-CH2-;
-CH2-C(0)-NH-0-CH2;
-0-NH-C(0)-CH2-;
-NH-C(0)-0-CH2-;
-0-C(0)-NH-CH2-;
-C(0)-NH-NH-CH2-;
-CH2-C(0)-NH-NH-CH2-;
-CH2-NH-NH-C(0)-;
-NH-NH-C(0)-CH2-; or
-NH-C(0)-NH-CH2-.
[0073] In a further embodiment, step B) of the present process involves ligating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages at the point(s) of ligation selected from:
Figure imgf000021_0001
wherein: c denotes the point of attachment to a 3' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue; and
^ d denotes the point of attachment to a 5' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue.
[0074] It will be understood that the one or more phosphodiester backbone mimic inter- nucleoside linkages of Formulae II, lla or lib may be prepared using any suitable technique known in the art. The person skilled in the art will be able to select suitable reaction conditions and reagents to prepared the one or more phosphodiester backbone mimic inter-nucleoside linkages of Formulae II, lla or lib.
[0075] It will be appreciated that during the synthesis of the one or more phosphodiester backbone mimic inter-nucleoside linkages of Formulae II, lla or lib, it may be desirable to protect certain substituent groups to prevent their undesired reaction. The skilled chemist will appreciate when such protection is required, and how such protecting groups may be put in place, and later removed.
[0076] By way of example, a suitable protecting group for an amino or alkylamino group is, for example, an acyl group, for example an alkanoyl group such as acetyl, an alkoxycarbonyl group, for example a methoxycarbonyl, ethoxycarbonyl or t-butoxycarbonyl group, an arylmethoxycarbonyl group, for example benzyloxycarbonyl, or an aroyl group, for example benzoyl. The deprotection conditions for the above protecting groups necessarily vary with the choice of protecting group. Thus, for example, an acyl group such as an alkanoyl or alkoxycarbonyl group or an aroyl group may be removed by, for example, hydrolysis with a suitable base such as an alkali metal hydroxide, for example lithium or sodium hydroxide. Alternatively, an acyl group such as a te/f-butoxycarbonyl group may be removed, for example, by treatment with a suitable acid as hydrochloric, sulfuric or phosphoric acid or tnfluoroacetic acid and an arylmethoxycarbonyl group such as a benzyloxycarbonyl group may be removed, for example, by hydrogenation over a catalyst such as palladium-on-carbon, or by treatment with a Lewis acid for example boron tris(trifluoroacetate). A suitable alternative protecting group for a primary amino group is, for example, a phthaloyl group which may be removed by treatment with an alkylamine, for example dimethylaminopropylamine, or with hydrazine.
[0077] A suitable protecting group for a carboxy group is, for example, an esterifying group, for example a methyl or an ethyl group which may be removed, for example, by hydrolysis with a base such as sodium hydroxide, or for example a t-butyl group which may be removed, for example, by treatment with an acid, for example an organic acid such as tnfluoroacetic acid, or for example a benzyl group which may be removed, for example, by hydrogenation over a catalyst such as palladium-on-carbon.
[0078] In an embodiment, the one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula II are formed by reacting:
B1) one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula C shown below:
Figure imgf000023_0001
Formula C
wherein:
/ c denotes the point of attachment to the oligonucleotide or oligonucleotide analogue comprising a terminal functional group of Formula C;
X is a leaving group selected from halo, OS02Rx1, (1-2C)haloalkyl, (1- 2C)haloalkoxy, OR"2, heteroaryl, wherein Rx1 and R*2 are independently selected from H, (1-6C)alkyl, (1-6C)alkanoyl, cycloalkyl, heteroalkyl, aryl, heteroaryl, (1-2C)haloalkyl, and wherein each of (1-6C)alkyl, cycloalkyl, heteroalkyl, aryl, heteroaryl are optionally further substituted with one or more groups selected from (1-4C)alkyl, halo, cyano, nitro or (1-2C)haloalkyl;
Vi is selected from O, S or NRZ, wherein Rz is selected from hydrogen or (1- 4C)alkyl;
Q is selected from O or S;
R2a and R2b are independently selected from hydrogen or (1-4C)alkyl, wherein each (1-4C)alkyl is optionally substituted with one or more NH2, OH or SH; n is an integer selected from 0 to 2; and
p is an integer selected from 0 to 1 ;
with
one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula D, shown below:
Figure imgf000024_0001
Formula D
wherein:
^ d' denotes the point of attachment to the oligonucleotide or oligonucleotide analogue comprising a terminal functional group of Formula D;
R2c and R2d are independently selected from hydrogen or (1-4C)alkyl;
Wi is selected from O or NH;
Xa is selected from NRe or SH, wherein Re is selected from hydrogen or (1- 4C)alkyl;
ni is an integer selected from 0 to 2; and
pi is an integer selected from 0 or 1 ;
and wherein the reaction is optionally conducted in the presence of one or more of the following:
i) one or more peptide coupling reagents;
ii) one or more activating agents; and
iii) a catalyst;
with the proviso that:
1) the sum of integers n, ni , p and pi is equal to or greater than 2; and
Figure imgf000024_0002
[0079] In an embodiment, the sum of integers n, ni , p is equal to 2, 3 or 4. Suitably, the sum of integers n, ni , p is equal to 2 or 3. Most suitably, the sum of integers n, ni , p is equal to 2.
[0080] In an embodiment, X is selected from halo, OS02Rx1 , (1-2C)haloalkyl, (1- 2C)haloalkoxy, OR"2, 5-membered heteroaryl, wherein Rx1 and R*2 are independently selected from H, (1-6C)alkyl, (1-6C)alkonyl, aryl or (1-2C)haloalkyl, and wherein each of (1-6C)alkyl or aryl is optionally further substituted with one or more groups selected from (1-4C)alkyl, halo, cyano, nitro or (1-2C)haloalkyl. Suitably, X is selected from halo, OS02Rx1 , (1-2C)haloalkyl, (1-2C)haloalkoxy, OR"2, triazolyl, wherein Rx1 and R"2 are independently selected from H, (1- 6C)alkyl, (1-6C)alkonyl, phenyl or (1-2C)haloalkyl. More suitably, X is selected from halo, (1- 2C)haloalkyl or OR"2, wherein R*2 is selected from H, (1-6C)alkyl or a (1-6C)alkonyl. Even more suitably, X is selected from OR"2, wherein R"2 is selected from H or (1-6C)alkyl. Most suitably, X is OH.
[0081] In another embodiment, Vi is selected from O or NRZ, wherein Rz is selected from hydrogen or (1-4C)alkyl. Suitably, Vi is selected from O or Nz, wherein Rz is selected from hydrogen or methyl. Most suitably, Vi is O.
[0082] In another embodiment, Q is O.
[0083] In another embodiment, Wi is O.
[0084] In yet another embodiment, n and ni are integers selected from 0 or 1.
[0085] In still a further embodiment, the sum of integers n, ni , p and pi is equal to 2, 3 or 4. Suitably, the sum of integers n, ni , p and pi is equal to 2 or 3. Most suitably, the sum of integers n, ni , p and pi is equal to 2.
[0086] In a further embodiment, R2a, R2b, R2c and R2d are independently selected from hydrogen or (1-4C)alkyl. Suitably, R2a, R2b, R2c and R2d are independently selected from hydrogen or methyl. Most suitably, R2a, R2b, R2c and R2d are hydrogen.
[0087] In a further embodiment, pi is 0.
[0088] In yet a further embodiment, p is 0.
[0089] In still a further embodiment, Xa is NRe, wherein Re is selected from hydrogen or (1- 4C)alkyl. Suitably, Xa is NH2.
[0090] In certain embodiments, the reaction between the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula C and the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula D is conducted at a temperature of between 0 °C and 150 °C. Suitably, the reaction between the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula C and the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula D is conducted at a temperature of between 0 °C and 100 °C. More suitably, the reaction between the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula C and the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula D is conducted at a temperature of between 0 °C and 75 °C.
[0091] In another embodiment, the reaction between the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula C and the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula D is carried out in a polar solvent. The polar solvent may be used to solubilise the oligonucleotides comprising functional groups of Formulae C and D and thereby facilitate reaction therebetween. Accordingly, it will be understood that the polar solvent selected will depend on the specific oligonucleotides selected. Suitable polar solvents may include, but are not limited to, water, an aqueous buffered solution (e.g. a solution of sodium phosphate or sodium carbonate), DMF, DMSO, acetonitrile, tetrahydrofuran (THF) and mixtures thereof with aqueous salt solutions.
[0092] In another embodiment, the reaction between the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula C and the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula D is carried out in an aqueous medium at a pH within the range of 5 to 9. Suitably, the reaction between the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula C and the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula D is carried out at a pH within the range of 6 to 8. Most suitably, the reaction between the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula C and the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula D is carried out at a pH within the range of 6.5 to 7.5.
[0093] In a further embodiment, a suitable buffer is present to maintain the reaction medium within the pH range 5 to 9. In a further embodiment, the buffer maintains the reaction medium within the pH range 6 to 8. In another embodiment, the buffer maintains the reaction medium within the pH range 6.5 to 7.5.
[0094] It will be understood that any suitable buffer may be used. In an embodiment, the buffer is selected from the group comprising: phosphate, acetate, borate, citrate, sulfonic acid, ascorbate, linolenate, carbonate and bicarbonate based buffers. In a further embodiment, the buffer is selected from the group comprising: phosphate, acetate, carbonate and bicarbonate based buffers. In a particular embodiment, the buffer is sodium phosphate or sodium carbonate.
[0095] In a further embodiment, the reaction between the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula C and the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula D is conducted in the presence of a salt (e.g. NaCI). Any suitable concentration of salt may be used. Suitably, the salt is present in a concentration of between 20 mM and 500 mM. More suitably, the salt is present in a concentration between 50 mM and 300 mM. Yet more suitably, the salt is present in a concentration between 100 mM and 250 mM.
[0096] In yet a further embodiment, the reaction between the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula C and the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula D is carried out in the presence of a catalyst. It will be understood that a catalysts may be any suitable reagent that helps to promote the rate of the reaction between the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula C and the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula D. Suitably, the catalyst is an acid and/or a base. Most suitably, the catalyst is a base. Non-limiting examples of suitable bases include NaOH, trimethylamine, diisopropylethylamine and N-methylmorpholine.
[0097] In still a further embodiment, the reaction between the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula C and the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula D is carried out in the presence of one or more peptide coupling agents. Any suitable peptide coupling reagent capable of enhancing the reaction between the functional group of Formula C and the functional group of Formula D may be used. It will be understood that the peptide coupling agent is preferably present when X is OH (i.e. the functional group of Formula C comprises a carboxy group).
[0098] In another embodiment, the peptide coupling reagent is a carbodiimide-based coupling reagent.
[0099] Suitably, the peptide coupling reagent is selected from 1- [Bis(dimethylamino)methylene]-1 H-1 ,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU), 2-(1 H-benzotriazol-1-yl)-1 ,1 ,3,3-tetramethyluronium hexafluorophosphate (HBTU), (Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), 4-(4,6-Dimethoxy-1 ,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM), N-Ethoxycarbonyl-2-ethoxy-1 ,2-dihydroquinoline (EEDQ), Ν,Ν'- dicyclohexylcarbodiimide (DCC), Ν,Ν'-diisopropylcarbodiimide (DIC), 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDCI), N-cyclohexyl-N'-isopropylcarbodiimide (CIC) or Ν,Ν'-dicyclopentylcarbodiimide (CPC). More suitably, the coupling reagent is selected from Ν,Ν'-dicyclohexylcarbodiimide (DCC), Ν,Ν'-diisopropylcarbodiimide (DIC) or 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDCI). Most suitably, the coupling reagent is 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDCI). [00100] Additional activating agents such as, for example, hydroxybenzotriazole (HOBt), N-hydroxy 2-phenyl benzimidazole (HOBI), 1-hydroxy-7-azabenzotriazole (HOAt), N- hydroxysuccinimide (NHS), N-hydroxysulfosuccinimide (Sulfo-NHS), 4-dimethylaminopyridine (DMAP) and ethyl cyano(hydroxyimino)acetate (Oxyma Pure®) may also be used together with the peptide coupling reagent defined hereinabove, to further enhance reactivity between the functional group of Formula C and the functional group of Formula D.
[00101] In an embodiment, the activating agent is N-hydroxysuccinimde (NHS), N- hydroxysulfosuccinimide (Sulfo-NHS) or ethyl cyano(hydroxyimino)acetate (Oxyma Pure®). Suitably, the activating agent is N-hydroxysuccinimde (NHS).
[00102] In a further embodiment, the reaction between the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula C and the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula D is carried out in the presence of both a peptide coupling agent (e.g. EDCI) and an activating agent (e.g. NHS). Suitably, the ratio of peptide coupling agent (e.g. EDCI) to activating agent (e.g. NHS) is from between 10: 1 to 1 : 1. More suitably, the ratio of peptide coupling agent (e.g. EDCI) to activating agent (e.g. NHS) is from between 6: 1 to 1 :1. Most suitably, the ratio of peptide coupling agent (e.g. EDCI) to activating agent (e.g. NHS) is 4: 1.
[00103] In certain embodiments, the following proviso applies:
i) when Q is S, Vi and/or Wi are not S.
[00104] In another embodiment, the step of reacting together the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula C and the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula D may be repeated sequentially more than once, for example, more than twice, more than three times, more than four times or more than five times, to form an oligonucleotide or oligonucleotide analogue comprising the one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula II.
Phosphodiester backbone mimic inter-nucleoside linkages of Formula III
[00105] In another embodiment, step B) of the present process involves ligating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula Ilia, shown below, at the point(s) of ligation:
Figure imgf000029_0001
wherein:
< / "-if
e and / f independently denote the points of attachment to the target oligonucleotide or oligonucleotide analogue;
W2 is selected from O or NH;
m and m 1 are integers independently selected from 0 to 2; and P2 is an integer selected from 0 or 1 ;
with the proviso that the sum of integers m, mi and P2 is equal to 0, 1 , 2 or 3.
[00106] In another embodiment, step B) of the present process involves ligating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula ll lb, shown below, at the point(s) of ligation:
Figure imgf000029_0002
Formula ll lb
wherein:
< / "-if
e and / f independently denote the points of attachment to the target oligonucleotide or oligonucleotide analogue;
R3a, R3b, R3c and R3d are independently selected from hydrogen or (1 -4C)alkyl; and
m and m 1 are integers independently selected from 0 to 2;
with the proviso that the sum of integers m and mi is equal to 0, 1 , 2 or 3.
[00107] In another embodiment, step B) of the present process involves ligating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula lllc, shown below, at the point(s) of ligation:
Figure imgf000030_0001
Formula ll lc
wherein:
< / "-if
e and / f independently denote the points of attachment to the target oligonucleotide or oligonucleotide analogue; and
m and m ^ are integers independently selected from 0 to 2;
with the proviso that the sum of integers m and mi is equal to 0, 1 or 2.
[00108] In another embodiment, step B) of the present process involves ligating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages at the point(s) of ligation of the formula:
Figure imgf000030_0002
wherein: e denotes the point of attachment to a 3' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue; and f denotes the point of attachment to a 5' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue.
[00109] It will be understood that the one or more phosphodiester backbone mimic inter- nucleoside linkages of Formulae I II , Il ia, 1 Mb or ll lc may be prepared using any suitable technique known in the art. The person skilled in the art will be able to select suitable reaction conditions and reagents to prepared the one or more phosphodiester backbone mimic inter- nucleoside linkages of Formulae II I, I lia, 1 Mb or l llc.
[001 10] It will again be appreciated that during the synthesis of the one or more phosphodiester backbone mimic inter-nucleoside linkages of Formulae II I, I l ia, 1Mb or lllc, it may be desirable to protect certain substituent groups to prevent their undesired reaction. The skilled chemist will appreciate when such protection is required, and how such protecting groups may be put in place, and later removed.
[001 11] In an embodiment, the one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula II I are formed by reacting:
B3) one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula E shown below:
Figure imgf000031_0001
Formula E
wherein:
^ e' denotes the point of attachment to the oligonucleotide or oligonucleotide analogue comprising a terminal functional group of Formula E;
R3a and R3b are independently selected from hydrogen or (1 -4C)alkyl, wherein each (1 -4C)alkyl is optionally substituted with one or more NH2, OH or SH; and
m is an integer selected from 0 to 2;
with
one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula F shown below:
Figure imgf000031_0002
Formula F
wherein: f denotes the point of attachment to the oligonucleotide or oligonucleotide analogue comprising a terminal functional group of Formula F;
R3c and R3d are independently selected from hydrogen or (1 -4C)alkyl, wherein each (1 -4C)alkyl is optionally substituted with one or more NH2, OH or SH;
R3e is selected from and hydrogen or (1 -4C)alkyl; W2 is selected from O, S or NRZ, wherein Rz is selected from hydrogen or (1 - 4C)alkyl;
mi is an integer selected from 0 to 2; and
P2 is an integer selected from 0 or 1 ;
and wherein the reaction is optionally conducted in the presence of one or more of the following:
i) one or more peptide coupling reagents;
ii) one or more activating agents; and
iii) a catalyst.
[001 12] In an embodiment, s e denotes the point of attachment to a 3' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue and ^ f denotes the point of attachment to a 5' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue.
[001 13] In an embodiment, R3a, R3b, R3c and R3d are independently selected from hydrogen or (1 -4C)alkyl. Suitably, R3a, R3b, R3c and R3d are independently selected from hydrogen or methyl. Most suitably, R3a, R3b, R3c and R3d are hydrogen.
[001 14] In another embodiment, R3e is selected from hydrogen or methyl. Suitably, R3e is hydrogen.
[001 15] In another embodiment, W2 is selected from O or NH.
[001 16] In an embodiment, P2 is 0.
[001 17] In yet another embodiment, m and mi are integer independently selected from 0 or 1 . Suitably, m and mi are 0.
[001 18] In a further embodiment, the sum of integers m, mi and P2 is equal to 0, 1 , 2, 3 or 4. Suitably, the sum of integers m, mi and p2 is equal to 0, 1 , 2 or 3. More suitably, the sum of integers m, mi and P2 is equal to 0, 1 or 2. Most suitably, the sum of integers m, mi and P2 is equal to 0 or 1 .
[001 19] In a further embodiment, the reaction between the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula E and the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula F is conducted in the presence of one or more peptide coupling reagents. Suitable peptide coupling reagents are analogous to those described in paragraphs [0098] and [0099] hereinabove. [00120] Additional activating may also be used together with the peptide coupling reagent defined hereinabove, to further enhance reactivity between the functional group of Formula E and the functional group of Formula F. Suitable activating agents are analogous to those described in paragraphs [00100] and [00101] hereinabove.
[00121] In another particular embodiment, the step of reacting together the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula E and the one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula F may be repeated sequentially more than once, for example, more than twice, more than three times, more than four times or more than five times, to form an oligonucleotide or oligonucleotide analogue comprising the one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula III.
Particular embodiments of the process of the present invention
[00122] In a particular embodiment of the present process, the one or more alkyne terminating oligonucleotides comprising a terminal functional group of Formula A, or the one or more azide terminating oligonucleotides comprising a terminal functional group of Formula B, further comprise a terminal functional group selected from Formula C, Formula D, Formula E or Formula F, as defined hereinabove.
[00123] In another particular embodiment of the present process, the one or more alkyne terminating oligonucleotides comprising a terminal functional group of Formula A, further comprise a terminal functional group selected from Formula D or Formula F, as defined hereinabove.
[00124] In another particular embodiment of the present process, the one or more azide terminating oligonucleotides comprising a terminal functional group of Formula B, further comprise a terminal functional group selected from Formula C or Formula E, as defined hereinabove.
[00125] In another particular embodiment of the present process, the one or more alkyne terminating oligonucleotides comprising a terminal functional group of Formula A, further comprise a terminal functional group selected from Formula C or Formula E, as defined hereinabove.
[00126] In another particular embodiment of the present process, the one or more azide terminating oligonucleotides comprising a terminal functional group of Formula B, further comprise a terminal functional group selected from Formula D or Formula F, as defined hereinabove. [00127] In another particular embodiment of the present invention, there is provided a process for preparing a target oligonucleotide or oligonucleotide analogue comprising two or more different phosphodiester mimic inter-nucleoside linkages, wherein the process comprises the steps of:
A) ligating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula la shown below, at the point(s) of ligation:
Figure imgf000034_0001
Formula la wherein:
/ a and independently denote the points of attachment to the target oligonucleotide or oligonucleotide analogue;
V and W are independently selected from O, S or NRX, wherein Rx is selected from hydrogen or (1 -4C)alkyl;
x, xi , z and zi are integers independently selected from 0 to 2; and y and yi are integers independently selected from 0 to 1 ;
and
B) ligating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula I la and/or Formula Il ia shown below at the point(s) of ligation:
Figure imgf000034_0002
Formula l la
Figure imgf000035_0001
wherein:
tV LV'
/ c d e and ^ f independently denote the points of attachment to the target oligonucleotide or oligonucleotide analogue;
R2a, R2b, R2c, R2d, R2e, R3a, R3b, R3c and R3d are independently selected from hydrogen or (1-4C)alkyl,
W2 is selected from O or NH;
n, rii, m and m 1 are integers independently selected from 0 to 2; and p and P2 are integers independently selected from 0 to 1 ;
and wherein steps A) and B) above are conducted in either order;
with the proviso that:
1) the sum of integers x, xi, y, yi , z and zi is either 0, 1 , 2, 3, 4 or 5;
2) the sum of integers n, rii, p is equal to 2, 3 or 4; and
3) the sum of m, mi and P2 is equal to 0, 1 , 2, 3 or 4.
[00128] In another particular embodiment of the present invention, there is provided a process for preparing a target oligonucleotide or oligonucleotide analogue comprising two or more different phosphodiester mimic inter-nucleoside linkages, wherein the process comprises the steps of:
A) ligating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula lb, shown below, at the point(s) of ligation:
Figure imgf000036_0001
Formula lb wherein:
/ a and independently denote the points of attachment to the target oligonucleotide or oligonucleotide analogue;
x, xi , z and zi are integers independently selected from 0 to 2; and y and yi are integers independently selected from 0 to 1 ;
and
gating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula lib and/or Formula ll lc shown below, at the point(s) of ligation:
Figure imgf000036_0002
Formula lib
Figure imgf000036_0003
Formula l llc
/ c d e and / f independently denote the points of attachment to the target oligonucleotide or oligonucleotide analogue;
R2a, R2b, R2c, R2d and R2e are independently selected from hydrogen or (1 - 4C)alkyl, n, Πι , m and m ^ are integers independently selected from 0 to 2; and and wherein steps A) and B) above are conducted in either order;
with the proviso that:
1) the sum of integers x, xi , y, yi , z and zi is either 0, 1 , 2, 3, 4 or 5;
2) the sum of integers n and ni is equal to 2, 3 or 4; and
3) the sum of m and mi is equal to 0, 1 , 2 or 3.
[00129] In another particular embodiment of the present invention, there is provided a process for preparing a target oligonucleotide or oligonucleotide analogue comprising two or more different phosphodiester mimic inter-nucleoside linkages, wherein the process comprises the steps of:
A) ligating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages at the point(s) of ligation selected from:
Figure imgf000037_0001
Figure imgf000038_0001
wherein:
/ a denotes the point of attachment to a 3' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue;
^ denotes the point of attachment to a 5' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue;
and
ligating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages at the point(s) of ligation selected from:
Figure imgf000038_0002
wherein:
/ c denotes the point of attachment to a 3' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue; ^ d denotes the point of attachment to a 5' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue; e denotes the point of attachment to a 3' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue; f denotes the point of attachment to a 5' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue; and
wherein steps A) and B) above are conducted in either order.
[00130] In another particular embodiment of the present invention, there is provided a process for preparing a target oligonucleotide or oligonucleotide analogue comprising two or more different phosphodiester mimic inter-nucleoside linkages, wherein the process comprises the steps of:
A) ligating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula la shown below, at the point(s) of ligation:
Figure imgf000039_0001
Formula wherein the one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula la are formed at the point(s) of ligation by reacting:
A1 ) one or more alkyne terminating oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula A1 shown below:
Figure imgf000039_0002
Formula A1
with one or more azide terminating oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula B1 , shown below:
Figure imgf000040_0001
Formula B1
in the presence of a catalyst (e.g. a copper (I) species);
wherein:
/ a and independently denote the points of attachment to the target oligonucleotide or oligonucleotide analogue;
^ a' denotes the point of attachment to the alkyne terminating oligonucleotide or oligonucleotide analogue
'Τ,-'
' denotes the point of attachment to the azide terminating oligonucleotide or oligonucleotide analogue;
V and W are independently selected from O, S or NRX, wherein Rx is selected from hydrogen or (1 -4C)alkyl;
x, xi , z and zi are integers independently selected from 0 to 2; and y and yi are integers independently selected from 0 to 1 ; and
ligating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula I la and/or Formula 1Mb shown below, at the point(s) of ligation:
Figure imgf000040_0002
Formula l la
Figure imgf000041_0001
Formula lllb
wherein the one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula lla are formed at the point(s) of ligation by reacting:
B1) one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula C1 shown below:
Figure imgf000041_0002
with
B2) one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula D1 , shown below:
Figure imgf000041_0003
Formula D1
optionally, in the presence of one or more peptide coupling reagents; and the one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula lllb are formed at the point(s) of ligation by reacting:
B3) one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula E1 shown below:
Figure imgf000041_0004
Formula E1 with
B4) one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula F1 shown below:
Figure imgf000042_0001
Formula F1
wherein:
tV LV'
/ c d e and ^ f independently denote the points of attachment to the target oligonucleotide or oligonucleotide analogue;
/ c denotes the point of attachment to the oligonucleotide or oligonucleotide analogue comprising a terminal functional group of Formula C1 ;
^ d' denotes the point of attachment to the oligonucleotide or oligonucleotide analogue comprising a terminal functional group of Formula D1 ;
^ e' denotes the point of attachment to the oligonucleotide or oligonucleotide analogue comprising a terminal functional group of Formula E1 ; f denotes the point of attachment to the oligonucleotide or oligonucleotide analogue comprising a terminal functional group of Formula F1 ;
X is selected from OR"2, wherein R"2 is selected from hydrogen or (1 - 6C)alkyl;
R2a, R2b, R2c, R2d, R2e, R3a, R3b, R3c and R3d are independently selected from hydrogen or (1 -4C)alkyl,
n, ni , m and m ^ are integers independently selected from 0 to 2; and p is an integer selected from 0 to 1 ;
and wherein steps A) and B) above are conducted in either order; with the proviso that:
1) the sum of integers x, xi , y, yi , z and zi is either 0, 1 , 2, 3, 4 or 5;
2) the sum of integers n, ni , p is equal to 2, 3 or 4; and
3) the sum of m and mi is equal to 0, 1 , 2 or 3.
[00131] In another particular embodiment of the present invention, there is provided a process for preparing a target oligonucleotide or oligonucleotide analogue comprising two or more different phosphodiester mimic inter-nucleoside linkages, wherein the process comprises the steps of:
A) ligating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula lb shown below, at the point(s) of ligation:
Figure imgf000043_0001
Formula lb wherein the one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula lb are formed at the point(s) of ligation by reacting:
A1) one or more alkyne terminating oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula A2 shown below:
Figure imgf000043_0002
Formula A2
with
A2) one or more azide terminating oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula B2, shown below:
Figure imgf000044_0001
Formula B2
in the presence of a copper (I) catalyst species;
wherein:
/ a and independently denote the points of attachment to the target oligonucleotide or oligonucleotide analogue;
^ a' denotes the point of attachment to the alkyne terminating oligonucleotide or oligonucleotide analogue
'Τ,-'
' denotes the point of attachment to the azide terminating oligonucleotide or oligonucleotide analogue;
x, xi , z and zi are integers independently selected from 0 to 2; and y and yi are integers independently selected from 0 to 1 ; and
ligating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula lib and/or Formula ll lc shown below, at the point(s) of ligation:
Figure imgf000044_0002
Formula lib
Figure imgf000044_0003
Formula ll lc wherein the one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula lib are formed at the point(s) of ligation by reacting:
B1) one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula C2 shown below:
Figure imgf000045_0001
Formula C2
with
one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula D2, shown below:
Figure imgf000045_0002
Formula D2
in the presence of one or more peptide coupling reagents; and the one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula Ilia are formed at the point(s) of ligation by reacting:
B3) one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula E2 shown below:
Figure imgf000045_0003
Formula E2
with
one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula F2 shown below:
Figure imgf000045_0004
Formula F2 optionally, in the presence of one or more peptide coupling reagents (e.g. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide);
wherein:
tV LV'
/ c d e and ^ f independently denote the points of attachment to the target oligonucleotide or oligonucleotide analogue;
/ c denotes the point of attachment to the oligonucleotide or oligonucleotide analogue comprising a terminal functional group of Formula C2;
^ d' denotes the point of attachment to the oligonucleotide or oligonucleotide analogue comprising a terminal functional group of Formula D2;
^ e' denotes the point of attachment to the oligonucleotide or oligonucleotide analogue comprising a terminal functional group of Formula E2;
^ f denotes the point of attachment to the oligonucleotide or oligonucleotide analogue comprising a terminal functional group of Formula F2;
X is OH;
R2a, R2b, R2c and R2d are independently selected from hydrogen or methyl,
n, rii , m and m ^ are integers independently selected from 0 to 2; and
and wherein steps A) and B) above are conducted in either order; with the proviso that:
1) the sum of integers x, xi , y, yi , z and zi is either 0, 1 , 2, 3, 4 or 5;
2) the sum of integers n and rii is equal to 2, 3 or 4; and
3) the sum of m and mi is equal to 0, 1 or 2. Oligonucleotide of the present invention
[00132] According to another aspect of the present invention, there is provided an oligonucleotide or oligonucleotide analogue comprising:
A) one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula I shown below:
Figure imgf000047_0001
Formula I wherein:
/ a and independently denote the points of attachment to the target oligonucleotide or oligonucleotide analogue;
R1 a, R1 b, R1 c, R1 d, R1 e, R1 f, R19 and R1 h are each independently selected from hydrogen or (1 -4C)alkyl, wherein each (1 -4C)alkyl is optionally substituted with one or more NH2, OH or SH;
V and W are independently selected from O, S or NRX, wherein Rx is selected from hydrogen or (1 -4C)alkyl;
x, xi, z and zi are integers independently selected from 0 to 2; and y and yi are integers independently selected from 0 to 1 ;
and
one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula II and/or Formula I I I, shown below:
Figure imgf000047_0002
Formula I I
Figure imgf000048_0001
Formula III
wherein:
tV LV' "T-^ "V
/ c d e and ^ f independently denote the points of attachment to the target oligonucleotide or oligonucleotide analogue;
R2a, R2b, R2c, R2d, R3a, R3b, R3c and R3d are independently selected from hydrogen or (1-4C)alkyl, wherein each (1-4C)alkyl is optionally substituted with one or more NH2, OH or SH
R2e and R3e are independently selected from hydrogen or (1-4C)alkyl;
Vi, Wi and W2 are independently selected from O, S or NRZ, wherein Rz is selected from hydrogen or (1-4C)alkyl;
Q is selected from S or O;
n, ni, m and mi are integers independently selected from 0 to 2; and p, pi and P2 are integers independently selected from 0 to 1 ;
with the proviso that:
1) the sum of integers x, xi, y, yi , z and zi is either 0, 1 , 2, 3, 4, 5 or 6;
2) the sum of integers n, ni, p and pi are greater than or equal to 2;
3) the sum of integers m, mi and P2 is equal to 0, 1 , 2, 3 or 4; and
Figure imgf000048_0002
[00133] In an embodiment, the oligonucleotide or oligonucleotide analogue comprises one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula I and one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula II. Suitably, the oligonucleotide or oligonucleotide analogue comprises one phosphodiester backbone mimic inter-nucleoside linkage of Formula I and one phosphodiester backbone mimic inter- nucleoside linkage of Formula II.
[00134] In another embodiment, the oligonucleotide or oligonucleotide analogue comprises one or more phosphodiester backbone mimic inter-nucleoside linkage of Formula I and one or more phosphodiester backbone mimic inter-nucleoside linkage of Formula III. Suitably, the oligonucleotide or oligonucleotide analogue comprises one phosphodiester backbone mimic inter-nucleoside linkages of Formula I and one phosphodiester backbone mimic inter- nucleoside linkages of Formula III.
[00135] It will be understood that features, including optional, suitable, and preferred features in relation to any one of the aspects of the present invention detailed above (i.e. the process of the present invention) may also be features, including optional, suitable and preferred features in relation to any other aspects of the invention (i.e. the oligonucleotides of the present invention).
[00136] In an embodiment, R1a, R1 b, R1c, R1d, R2a, R2b, R2c, R2d, R3a, R3b, R3c and R3d are independently selected from hydrogen or (1-4C)alkyl. Suitably, R1a, R1 b, R1c, R1d, R2a, R2b, R2c, R2d, R3a, R3b, R3c and R3d are independently selected from hydrogen or methyl. Most suitably, R1a, R1 b, R1c, R1d, R2a, R2b, R2c, R2d, R3a, R3b, R3c and R3d are hydrogen.
[00137] In another embodiment, V and W are independently selected from O or NRX, wherein Rx is selected from hydrogen or (1-4C)alkyl. Suitably, V and W are independently selected from O or NRX, wherein Rx is selected from hydrogen or methyl. More suitably, V and W are independently selected from O or NH2. Most suitably, V and W are both O.
[00138] In another embodiment, the sum of integers x, xi , y, yi , z and zi is either 0, 1 , 2, 3, 4 or 5. Suitably, the sum of integers x, xi , y, yi , z and zi is either 0, 1 , 2, 3 or 4. More suitably, the sum of integers x, xi , y, yi , z and zi is either 1 , 2, 3 or 4. Most suitably, the sum of integers x, xi , y, yi , z and zi is either 1 , 2 or 3.
[00139] In another embodiment, Q is oxygen.
[00140] In another embodiment, pi is 0.
[00141] In another embodiment, p is 0.
[00142] In another embodiment, Vi and Wi are independently selected from O or NRZ, wherein Rz is selected from hydrogen or (1-4C)alkyl. Suitably, Vi and Wi are independently selected from O or NRZ, wherein Rz is selected from hydrogen or methyl. More suitably, Vi and Wi are independently selected from O or NH. Most suitably, Vi and Wi are both oxygen.
[00143] In another embodiment, the sum of integers n, ni , p is equal to 2, 3 or 4. Suitably, the sum of integers n, ni , p is equal to 2 or 3. Most suitably, the sum of integers n, ni , p is equal to 2.
[00144] In another embodiment, the sum of integers m and mi is equal to 0, 1 , 2, 3 or 4. Suitably, the sum of integers m and mi is equal to 0, 1 , 2 or 3. More suitably, the sum of integers m and mi is equal to 0, 1 or 2. Most suitably, the sum of integers m and mi is equal to 0 or 1.
[00145] In another embodiment, s e denotes the point of attachment to a 3' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue and ^ f denotes the point of attachment to a 5' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue.
[00146] In a further embodiment, the one or more phosphodiester backbone mimic inter- nucleoside linkages of Formula I are selected from one of the following:
Figure imgf000050_0001
wherein:
Zi and∑2 are independently selected from O or NH; a denotes the point of attachment to a 3' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue;
^ denotes the point of attachment to a 5' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue.
[00147] In a further embodiment, the one or more phosphodiester backbone mimic inter- nucleoside linkages of Formula I I are selected from one of the following:
Figure imgf000051_0001
wherein:
/ c denotes the point of attachment to a 3' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue;
^ d denotes the point of attachment to a 5' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue.
[00148] In a further embodiment, the one or more phosphodiester backbone mimic inter- nucleoside linkages of Formula II I is of the following formula:
Figure imgf000051_0002
wherein: e denotes the point of attachment to a 3' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue; and f denotes the point of attachment to a 5' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue. Particular embodiments of the oligonucleotides and oligonucleotide analogues
[00149] In a particular embodiment, there is provided an oligonucleotide or oligonucleotide analogue comprising:
A) one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula lb shown below:
Figure imgf000052_0001
Formula lb wherein:
/ a and independently denote the points of attachment to the target oligonucleotide or oligonucleotide analogue;
x, xi , z and zi are integers independently selected from 0 to 2; and y and yi are integers independently selected from 0 to 1 ;
and
one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula lib and/or Formula ll lc, shown below:
Figure imgf000052_0002
Formula lib
Figure imgf000052_0003
Formula ll lc
wherein: tV LV'
/ c d e and ^ f independently denote the points of attachment to the target oligonucleotide or oligonucleotide analogue;
R2a, R2b, R2c and R2d are independently selected from hydrogen or (1- 4C)alkyl;
n, ni , m and m ^ are integers independently selected from 0 to 2; and with the proviso that:
1) the sum of integers x, xi , y, yi , z and zi is either 0, 1 , 2, 3, 4 or 5;
2) the sum of integers n and ni are equal to 2, 3 or 4; and
3) the sum of integers m and mi are equal to 0 , 1 , 2 or 3.
[00150] In another particular embodiment, there is provided an oligonucleotide or oligonucleotide analogue comprising:
A) one or more phosphodiester backbone mimic inter-nucleoside linkages selected from:
Figure imgf000053_0001
Figure imgf000054_0001
wherein:
Zi and ∑2 are independently selected from O or NH;
/ a denotes the point of attachment to a 3' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue;
^ denotes the point of attachment to a 5' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue;
and
B) one or more phosphodiester backbone mimic inter-nucleoside linkages selected from:
or
Figure imgf000054_0002
wherein:
"c denotes the point of attachment to a 3' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue;
^ d denotes the point of attachment to a 5' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue; e denotes the point of attachment to a 3' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue; and f denotes the point of attachment to a 5' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue.
Uses and applications
[00151] It is well known that oligonucleotides may be used therapeutically for the treatment of various diseases and disorders, such as, for example, cancer, genetic disorders and infection. Thus, in one aspect, the present invention provides a use of an oligonucleotide or oligonucleotide analogue, as defined herein, in the treatment of a disease or disorder. In an embodiment, the disease or disorder is cancer. In another embodiment, the disease or disorder is a genetic disorder. In yet another embodiment, the disease or disorder is an infection.
[00152] In another aspect there is provided a method for the treatment of a disease or disorder, said method involving administering a therapeutically effective amount of an oligonucleotide or oligonucleotide analogue, as defined herein, or a pharmaceutically acceptable salt or solvate thereof. In an embodiment, the disease or disorder is cancer. In a further embodiment, the disease or disorder is a genetic disorder. In another embodiment, the disease or disorder is an infection.
[00153] According to another aspect of the present invention, there is provided a use of an oligonucleotide or oligonucleotide, as defined herein, in the synthesis of genes. Suitably, there is provided a use of an oligonucleotide or oligonucleotide, as defined herein, as a template in the synthesis of genes.
[00154] According to another aspect of the present invention, there is provided a use of an oligonucleotide or oligonucleotide, as defined herein, as:
(i) antisense DNA or RNA;
(ii) exon skipping DNA or RNA;
(iii) interference RNA (e.g. siRNA); or
(iv) an RNA component of a CRISPR-Cas system (e.g. crRNA, tracrRNA or
gRNA).
[00155] According to another aspect of the present invention, there is provided a use of an oligonucleotide or oligonucleotide analogue, as defined herein, as:
i) a template for amplification in a polymerase chain reaction (PCR): ii) as a template in a DNA replication process;
iii) as a template in a transcription process to provide a corresponding RNA
transcript, or as a template in a reverse transcription process to provide a corresponding DNA transcript;
iv) as template in a translation process to produce a corresponding protein or peptide; or
v) to guide one or more proteins of interest to a target DNA or RNA.
[00156] According to another aspect of the present invention, there is provided a method for amplifying an oligonucleotide or oligonucleotide analogue sequence, the method comprising the steps of:
1) providing an oligonucleotide or oligonucleotide analogue as defined herein; and
2) carrying out a polymerase chain reaction (PCR) using the oligonucleotide or oligonucleotide analogue of step 1 as a template.
[00157] According to another aspect of the present invention, there is provided a method for replicating an oligonucleotide or oligonucleotide analogue sequence, the method comprising the steps of:
1) providing an oligonucleotide or oligonucleotide analogue as defined iherein; and
2) carrying out a replication reaction using the oligonucleotide or oligonucleotide analogue of step 1 as a template.
[00158] According to another aspect of the present invention, there is provided a method for producing a ribonucleic acid (RNA) sequence comprising the steps of:
1) providing an oligonucleotide or oligonucleotide analogue as herein; and
2) transcribing the oligonucleotide or oligonucleotide analogue of step 1 to form a
ribonucleic acid (RNA) transcript.
[00159] According to another aspect of the present invention, there is provided a method for producing a deoxyribonucleic acid (DNA) sequence comprising the steps of:
1) providing an oligonucleotide or oligonucleotide analogue as herein; and
2) reverse-transcribing the oligonucleotide or oligonucleotide analogue of step 1 to form a complementary deoxyribonucleic acid (cDNA) sequence.
[00160] According to another aspect of the present invention, there is provided a method for preparing a protein or peptide comprising the steps of: 1) providing an oligonucleotide or oligonucleotide ananlogue as defined herein; and
2) translating the oligonucleotide or oligonucleotide analogue of step 1 to form the protein or peptide.
Illustrative Examples of oligonucleotides in CRISPR-Cas systems
[00161] In general terms, there are two main classes of CRISPR-Cas systems (Makarova et al. Nat Rev Microbiol. 13:722-736 (2015)), which encompass five major types and 16 different subtypes based on cas gene content, cas operon architecture, Cas protein sequences, and process steps (Makarova et al. Biol Direct. 6:38 (201 1); Makarova and Koonin Methods Mol Biol. 131 1 :47-75 (2015); Barrangou, R. Genome Biology 16:247 (2015)). This classification in either Class 1 or Class 2 is based upon the Cas genes involved in the interference stage.
[00162] Class 1 systems have a multi-subunit crRNA-effector complex such as Cascade- Cas3, whereas Class 2 systems have a crRNA-effector complex having a single Cas protein, such as Cas9, Cas12 (previously referred to as Cpfl) and Cas 13a (previously referred to as C2c2). For Type II systems there is a second RNA component tracrRNA which hybridises to crRNA to form a crRNA:tracr RNA duplex, these two RNA components may be linked to form single guide RNA.
[00163] RNA components in such CRISPR-Cas systems may be adapted to be an oligonucleotide in accordance with the invention or a dinucleotide of the invention may be comprised within an RNA components of a CRISPR-Cas system. It would be a matter of routine for a person of ordinary skill in the art to synthesise a crRNA, pre-crRNA, tracrRNA or guideRNA comprising a dinucleotide of the invention or having at least one inter-nucleoside linkage which is a triazole linker moiety between two nucleosides with a locked nucleoside positioned at the 3' end of the triazole linker moiety, and which retains the desired function of the RNA component (e.g., to guide the crRNA:effector complex to a target site). Standard methods are known in the art for testing whether oligonucleotides of the invention when used as such CRISPR RNA components retain the desired function (e.g. by comparing the desired function to that of a control CRISPR RNA component which has the same nucleosides without any-triazole linker moieties between nucleosides or locked nucleosides).
[00164] The term "CRISPR RNA components" or "RNA component of a CRISPR-Cas system" is used herein, as in most CRISPR-Cas systems, the nucleic acid sequences which guide the effector protein(s) to a desired target sequence are RNA components. However, CRISPR hybrid DNA/RNA polynucleotides which can also function to guide effector protein(s) to a desired target site in a DNA or RNA sequence are also known in the art - see for example Rueda et al. (Mapping the sugar dependency for rational generation of a DNA-RNA hybrid- guided Cas9 endonuclease, Nature Communications 8, Article Number: 1610 (2017)). Accordingly, reference to CRISPR RNA components herein may also encompass hybrid RNA/DNA components such as crDNA/RNA, tracrDNA/RNA or gDNA/RNA.
[00165] Advantageously the oligonucleotides of the invention may have particular utility in in vivo gene therapy applications. For example, one way of carrying out in vivo therapy using a Type II CRISPR-Cas system involves delivering the Cas9 and tracrRNA via a virus, which can assemble inactive complexes inside of cells. The crRNA can then be administered later to assemble and selectively activate CRISPR/Cas9 complexes, which would then go on to target and edit specific sites in the human genome, such as disease relevant genes (Gagnon and Corey, Proc. Natl. Acad. Sci. USA 1 12: 15536-15537, 2015; Rahdar, et al, Proc. Natl. Acad. Sci. USA 112Έ71 10-71 17, 2015). For this gene therapy approach to work the crRNA should be extremely resistant to nucleases and cellular degradation, as well as confer high activity and specificity to the assembled CRISPR/Cas9 complex. Hence, the increased stability of the oligonucleotides of the invention to degradation is highly desirable. Alternatively, crRNA:effector complexes (i.e. CRISPR-Cas complexes, such as CRISPR/Cas9) can be assembled in vitro and directly transfected into cells for genome editing (Liang, et al, J. Biotechnol. 208:44-53, 2015; Zuris, et al, Nat. Biotechnol. 33:73-80, 2015). Special transfection reagents, such as CRISPRMAX (Yu, et al, Biotechnol. Lett. 38:919-929, 2016), have been developed for this purpose. Oligonucleotides of the invention when used as crRNAs may improve this approach by offering stability against degradation.
[00166] Accordingly, the oligonucleotides of the invention when used as CRISPR RNA components can advantageously be used for the various applications of CRISPR-Cas systems known in the art including: gene-editing, gene activation (CRISPRa) or gene interference (CRISPRi), base-editing, multiplex engineering (CRISPRm), DNA amplification, diagnostics (e.g. SKERLOCK or DETECTR), cell recording (e.g. CAMERA), typing bacteria, antimicrobial applications, synthesising new chemicals etc..
[00167] Suitably, in diagnostic applications such as SHERLOCK and DETECTR the oligonucleotides of the invention can be used as RNA components such as the "sacrificial RNA molecules" used to create a signal.
EXAMPLES
[00168] Embodiments of the invention will be described, by way of example only, with reference to the accompanying drawings, in which:
Figure 1 shows: A) a schematic representation of the templated simultaneous orthogonal phosphoramidate and CuAAC ligation reactions; and B) a schematic representation of the concept of single tube gene assembly by phosphoramidate ligation followed by transcription of modified DNA.
Figure 2 shows the 12% denaturing PAGE analysis of 3'-phosphate/5'-amine oligonucleotides ligation to give the phosphoramidate-containing product. Lane 1 ; phosphoramidate reaction mixture (ODN 1 , 81-mer), lane 2; reference starting material ODN 3. An excess of the amine oligonucleotide (ODN 3) was used, resulting in a residual lower band and complete consumption of the phosphate oligonucleotide.
Figure 3 shows the 12% denaturing PAGE analysis for optimisation of 3'-phosphate/5'-amine oligonucleotides ligation to give the phosphoramidate template (ODN 1 , 81-mer) top bands. Lanes 1-8; reaction mixture after 5, 10, 15, 30, 45, 60 120 and 360 min. An excess of the amine oligonucleotide was used resulting in a residual lower band and consumption of the phosphate oligonucleotide
Figure 4 shows the PCR amplification of the 81-mer phosphoramidate DNA template (ODN 1). Lane 1 ; 50 bp DNA ladder, lane 2; PCR using the phosphoramidate-containing template ODN 1 , lane 3; control PCR without a phosphoramidate linkage.
Figure 5 shows the 6% denaturing PAGE analysis of 3'-phosphate/5'-amine oligonucleotide ligation to give the product containing two phosphoramidate linkages (ODN 5, 303-mer). Lane 1 ; 0.2 M NaCI, 25 mM MgCI2, lane 2; 50 mM Tris (pH=8.5), 25 mM MgCI2, 0.2 M NaCI, lane 3; 10 mM phosphate (pH=7.0), 25 mM MgCI2, 0.2 M NaCI, lane 4; 0.2 M HEPES (pH=7.2), 0.4 M NaCI, 25 mM MgCI2, lane 5; 0.2 M HEPES (pH=7.2), 0.4 M NaCI, lane 6; 0.4 M NaCI.
Figure 6 shows the PCR amplification of the 303-mer DNA template ODN 5. Lane 1 ; 50 bp DNA ladder, lane 2; control PCR without phosphoramidate linkage, lane 3 and 4; PCR using the phosphoramidate-containing template ODN 5 (303-mer).
Figure 7 shows the sequence alignment of 20 Clones from PCR of ODN 5 (2x phosphoramidate linkages in 303-mer section of EGFP gene, in red with the ligation points in blue). All the sequences are identical indicating the biocompatibility of the phosphoramidate linkage. Only a few mutations were observed and these are far from the ligation points (see Table 1). The mutations could have occurred during sequencing or during oligonucleotide synthesis and purification.
Figure 8 shows: A) PCR amplification of the double stranded phosphoramidate EGFP gene (762-mer). Lane 1 ; 100 bp DNA ladder, lane 2; PCR using the double strand phosphoramidate EGFP gene (762-mer), lane 3; control PCR for individual oligos without ligation. B) Representative sequencing data from cloning of the PCR product of the phosphoramidate EGFP gene. The data show that the polymerase faithfully copied the bases around the phosphoramidate ligation sites (shown in red in the sequence text).
Figure 9 shows the data from cloning and sequencing of the PCR product from the phosphoramidate EGFP gene (762-mer) showing the faithful copying at the ligation points (shown in red in the sequence text) (A) and the water mark GTACA (B). All clones show the water mark which was inserted into the sequence of the synthesised EGFP gene as a unique signature to differentiate it from potential contaminant DNA.
Figure 10 shows the data from cloning and sequencing of the PCR product of the phosphoramidate EGFP gene (762-mer). The data show that the polymerase copied the gene faithfully including the bases around the phosphoramidate ligation points (shown in red in the inserted sequence text). Only one deletion mutation was found in this clone.
Figure 11 shows the transcription of 79-mer unmodified and phosphoramidate-containing DNA templates. Lane 1 and 2, reaction using phosphoramidate template (ODN 27) and short coding strand (ODN 33) for 2 and 4 h respectively; lane 3; template ODN 31 lane 4 and 5, reaction using control template (ODN 31) and short coding strand (ODN 33) for 2 and 4 h respectively; Lane 6 and 7, reaction using phosphoramidate template (ODN 27) and long coding strand (ODN 32) for 2 and 4 h respectively; lane 8 and 9, reaction using control template (ODN 31) and long coding strand (ODN 32) for 2 and 4 h respectively. 15% polyacrylamide gel.
Figure 12 shows the ES- Mass spectra of A), the RNA transcripts from the phosphoramidate- containing template (ODN 27) and B), the normal template (ODN 31). The transcripts have the expected 5'-triphosphate and an additional 3'-cytidine. Required mass = 17.236 KD. Found mass, 17.239 (transcript with 5'-triphosphate), 17.261 (transcript with 5'-triphosphate + Na+) and 17.566 (transcript with 5'-triphosphate and 3'-cytidine).
Figure 13 shows the orthogonal phosphoramidate and CuAAC reactions for ligation of three oligonucleotides to make a 303-mer product. Lane 1 ; ODN 6, Iane2; 2 x CuAAC reactions, lane 3; 2 x phosphoramidate reactions, lane 4; orthogonal phosphoramidate and CuAAC reactions. Denaturing 8% polyacrylamide gel-electrophoresis.
Figure 14 shows the orthogonal phosphoramidate and CuAAC reactions for ligation of three oligonucleotides to make a fluorescent 331 -mer product. Lane 1 ; starting material ODN 39, Iane2; orthogonal phosphoramidate and CuAAC reactions using ODN 39 (3'-phosphate, 5'- Cy3), ODN 38 (3'-propargyl and 5'-amine) and ODN 40 (5'-azide). Denaturing 8% polyacrylamide gel-electrophoresis.
Figure 15 shows a schematic representation of the orthogonal phosphoramidate and CuAAC reactions for ligation of three oligonucleotides to make a 303-mer product. Also shown is the gel electrophoresis trace. Lane 1 ; orthogonal phosphoramidate and CuAAC reactions using ODN 6 (3'-phosphate), ODN 38 (3'-propargyl and 5'-amine) and ODN 37 (5'-azide), Iane2; starting material ODN 6. Denaturing 8% polyacrylamide gel-electrophoresis.
Figure 16 shows a schematic representation of how the process of the present invention may be applied using solid supported chemistry.
Experimental
General method for oligonucleotide synthesis and purification
[00169] Standard DNA phosphoramidites, solid supports, and additional reagents were purchased from Link Technologies Ltd and Applied Biosystems Ltd. 5'- Monomethoxytritylamino-2'-deoxythymidine,3'-[(2-cyanoethyl)-(N,N-diisopropyl)]- phosphoramidite was purchased from Glen Research (Catalog Number: 10-1932-90).
[00170] All oligonucleotides were synthesized on an Applied Biosystems 394 automated DNA/ RNA synthesizer using a standard 0.2 or 1.0 /ymole phosphoramidite cycle of acid- catalyzed detritylation, coupling, capping, and iodine oxidation. Stepwise coupling efficiencies and overall yields were determined by the automated trityl cation conductivity monitoring facility and in all cases were >98.0%.
[00171] All β-cyanoethyl phosphoramidite monomers were dissolved in anhydrous acetonitrile to a concentration of 0.1 M immediately prior to use. The coupling time for normal A, G, C, and T monomers was 60 s, and the coupling time for the 5'-amino dT phosphoramidite monomer was extended to 600 s. Cleavage of the oligonucleotides from the solid support and deprotection was achieved by exposure to concentrated aqueous ammonia solution for 60 min at room temperature followed by heating in a sealed tube for 5 h at 55 °C.
[00172] Purification of oligonucleotides was carried out by reversed-phase HPLC on a Gilson system using a Brownlee Aquapore column (C8, 8 mm x 250 mm, 300A pore) with a gradient of acetonitrile in triethylammonium bicarbonate (TEAB) increasing from 0% to 50% buffer B over 30 min with a flow rate of 4 mL/min (buffer A: 0.1 M triethylammonium bicarbonate, pH 7.0, buffer B: 0.1 M triethylammonium bicarbonate, pH 7.0 with 50% acetonitrile). Elution of oligonucleotides was monitored by ultraviolet absorption at 295 or 300 nm. After HPLC purification, oligonucleotides were freeze dried then dissolved in water without the need for desalting.
[00173] For long oligonucleotides, polyacrylamide gel electrophoresis was used for purification. Oligonucleotide bands were then visualized using a UV lamp and the desired bands excised, crushed and soaked in water overnight at 37 °C. After evaporation, samples were desalted using NAP-25 followed by NAP-10 columns (G.E. Healthcare Life Sciences). All oligonucleotides were characterised by electrospray mass spectrometry using a Bruker micrOTOF II focus ESI-TOF MS instrument in ESI" mode. Data were processed using MaxEnt.
Demonstration of the compatibility of the phosphoramidate linkage with DNA and RNA polymerase
Synthesis of 81-mer (ODN 1) template with one phosphoramidate ligation point
[00174] Oligonucleotides ODN 2 (1.0 nmol), ODN 3 (1.1 nmol) and splint ODN 4 (1.0 nmol) in 0.2 M HEPES (pH=7.2) with 0.4 M NaCI (80 μΙ_) were annealed by heating at 90 °C for 5 min then cooling slowly to room temperature. A solution of 1-(2-hydroxyethyl) imidazole (1.0 M, 10 μΙ_) (0.1 M final concentration) and EDC.HCI (6.0 M, 10 μΙ_) (0.6 M final concentration) was added to the annealed oligonucleotides and the reaction mixture was kept at room temperature for 2 h. Reagents were removed using NAP-25 gel-filtration column and the ligated DNA was analysed and purified by denaturing 12% polyacrylamide gel electrophoresis (Figure 2). The reaction was scaled up 10-fold and aliquots were taken at different time points (Figure 3).
[00175] The gel showed that the reaction was complete within 45 min and there was no difference in intensity of the product after 2 h.
PCR amplification of the 81 -mer phosphoramidate template (ODN 1)
[00176] GoTaq DNA polymerase was used to generate a PCR product from the 81 -mer template (ODN 1) which includes one phosphoramidate linkage. Reagents and conditions: 4 μΙ_ of 5x buffer (Promega green PCR buffer) was used in a total reaction volume of 20 μΙ_ with 5 ng of the DNA template, 0.5 mM of each primer, 0.2 mM dNTP and 1.0 unit of GoTaq polymerase. The reaction mixture was loaded onto a 2% agarose gel in 1xTBE buffer. PCR cycling conditions: 95 °C (initial denaturation) for 2 min then 25 cycles of 95 °C (denaturation) for 15 s, 54 °C (annealing) for 20 s and 72 °C (extension) for 30 s. The reaction was then left at 72 °C for 5 min then loaded onto a 2% agarose gel in 1 X Tris/Borate/EDTA buffer (TBE) (Figure 4).
[00177] 5 X Promega green PCR buffer was provided with the enzyme (Promega GoTaq DNA polymerase), pH 8.5 containing 7.5 mM MgC to give a final Mg2+ concentration of 1.5 mM. The buffer contains Tris.HCI, KCI and two dyes (blue and yellow) that separate during electrophoresis to monitor the migration process.
Synthesis of 303-mer (ODN 5) template with double phosphoramidate ligation points
[00178] Oligonucleotides ODN 6, ODN 7, ODN 8 with splints ODN 9 and ODN 10 (0.5 nmol of each) in 0.2 M HEPES (pH=7.2) with 0.4 M NaCI (80 μΙ_) were annealed by heating at 90 °C for 5 min then cooling slowly to room temperature. A solution of 1-(2-hydroxyethyl) imidazole (1.0 M, 10 μΙ_) (0.1 M final concentration) and EDC.HCI (6.0 M, 10 μΙ_) (0.6 M final concentration) was added to the annealed oligonucleotides and the reaction mixture was kept at room temperature for 2 h. Reagents were removed using NAP-25 gel-filtration column and the ligated DNA was analysed by denaturing 6% polyacrylamide gel electrophoresis.
[00179] A mixture of 3 nmoles of each oligonucleotide and splints were dissolved in water and then divided into 6 samples and each mixed with 2x buffer. A solution of 1-(2-hydroxyethyl) imidazole (1.0 M, 10 μΙ_) (0.1 M final concentration) and EDC.HCI (6.0 M, 10 μΙ_) (0.6 M final concentration) was added to the annealed oligonucleotides and the reaction mixture was kept at room temperature for 1 h then analysed by denaturing 6% polyacrylamide gel electrophoresis (Figure 5).
[00180] The following buffer systems were used:
50 mM Tris (pH=8.5), 25 mM MgCI2, 0.2 M NaCI; 10 mM phosphate (pH=7.0), 25 mM MgCI2, 0.2 M NaCI; 0.2 M HEPES (pH=7.2), 0.4 M NaCI, 25 mM MgCI2 and 0.2 M HEPES (pH=7.2), 0.4 M NaCI.
PCR amplification of the 303-mer double phosphoramidate template ODN 5 using GoTaq DNA polymerase, cloning and sequencing
[00181] Following the above method for PCR amplification of 81-mer ODN 1 , the PCR product was purified by extraction from a 2% agarose gel (Figure 6) using a QIAquick Gel Extraction kit. It was then inserted into vector pCR2.1. TOPO for subcloning. Cloning was carried out using a standard TOPO cloning protocol. Standard automated Sanger DNA sequencing was performed and the data shown in Figure 7.
Table 1 - Mutations found in sequence data in Figure 7
TC NMC MC IM DM SM LPM TB TM
20 13 7 1 10 1 0 6060 12 TC: total clones, NMC: non-mutant clones, MC: mutant clones, IM: insertion mutation, DM: deletion mutation, SM: substituted mutation, LPM: ligation point mutation, TB: total number of bases, TM: total number of mutation.
Single tube synthesis of the entire EGFP gene
[00182] The synthesis of the entire EGFP gene in one tube was achieved by mixing 0.1 nmole of each of the 10 oligonucleotides (ODN 15-ODN 24 for both forward and reverse strands), freeze drying them together then re-dissolving them in 100 μΙ_ HEPES Buffer (0.2 M, pH=7.2) with 0.4 M NaCI. The oligonucleotide mixture was annealed by heating at 90 °C for 5 min then cooled slowly to room temperature. EDC.HCI (30 mg) and a solution of 1-(2-hydroxyethyl) imidazole (1.0 M, 30 μΙ_) were added to the annealed oligonucleotides and the reaction mixture was kept at room temperature for 2 h. Reagents were removed using NAP-25 gel-filtration column and the ligated DNA was analysed by denaturing 4% polyacrylamide gel electrophoresis. The band was cut and DNA was extracted then used in PCR.
PCR amplification of the double stranded phosphoramidate EGFP gene by phosphoramidate ligation using GoTaq DNA polymerase
[00183] A PCR product from the whole EGFP gene duplex was generated using GoTaq DNA polymerase under the same conditions explained above for PCR of 81-mer ODN 1. The PCR product was purified by extraction from a 2% agarose gel (Figure 8A) using a QIAquick Gel Extraction kit. It was then inserted into the vector pCR2.1. Cloning into the TOPO vector was done with a standard TOPO cloning protocol. Automated Sanger DNA sequencing was performed; and the data is shown in Figure 9 and Figure 10. This procedure was carried out by Eurofins GmbH.
[00184] Expected EGFP sequence (SeqID 1) with the ligation points in bold and a watermark in underlined text:
TCGACGGTACCGCGGGCCCGGGATCCACCGGTCGCCACCATGGTGAGCAAGGGCGA
GGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGG
CCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGAC
CCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACC
ACCCTGACCTACGGTGTACAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACG
ACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAA
GGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGT
GAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCA CAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAG
AACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGC
TCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCG
ACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCG
ATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGA
GCTGTACAAGTAAAGC.
[00185] Found EGFP sequence (SeqID 2) watermark underlined and only one deletion mutation in bold (Figure 10):
CGGTACCGCGGGCCCGGGATCCACCGGTCGCCACCATGGTGAGCAAGGGCGAGGAG
CTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCAC
AAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTG
AAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCC
TGACCTACGGTGT JAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTT
CTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGAC
GACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAAC
CGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAG
CTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACG
GCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGC
CGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAA
CCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCA
CATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTcGGCATGGACGAGCT
GTACAAGTAAAGC.
[00186] Two more clones were sequenced and similar results were obtained with a small number of mutations which were far from the ligation points. Two deletion mutations were found in one clone whereas in the other clone, 4 deletion mutation and 2 substitution mutation were found. The mutation could have occurred during sequencing or oligonucleotide synthesis and purification. The mutation rate is consistent with that expected from oligonucleotide syntheses. All clones show the water mark which was inserted in the sequence of the synthesised EGFP as a unique signature to differentiate it from potential contaminant DNA. (Figure 9).
Transcription of phosphoramidate template ODN 27 and control ODN 31 [00187] MegaScript T7 Transcription Kit (ThermoFislher Scientific, cat. no. AM 1333) was used according to the manufacturer's recommended protocol. Reaction mixtures were prepared in the following order at room temperature: phosphoramidate template ODN 27 (5 μΜ, 2.5 μΙ), long coding strand ODN 32 (5 μΜ, 2.75 μΙ), water (14.75 μΙ), reaction buffer (10x, 5 μΙ), ATP (5 μΙ), CTP (5 μΙ), GTP (5 μΙ), UTP (5 μΙ) and enzyme mix (5 μΙ). The reaction mixture was then incubated at 37 °C and 10 μΙ aliquots removed at the specified times and mixed with an equal volume of formamide before storing at -80 °C. Samples were then loaded on 12% denaturing polyacrylamide gel (1x TBE, 7 M urea, W x D x H = 18 x 0.2 x 24.4 cm) at 20 W for 2 h.
[00188] The same reaction was repeated using short coding strand ODN 33 and gave similar results.
[00189] For comparison of efficiency, the experiments were also performed using the control unmodified template ODN 31 using long coding strand ODN 32 and short coding strand ODN 33 (Figure 11).
[00190] Oligonucleotide bands were then visualized using a UV lamp and the desired bands excised, crushed and soaked in buffer (50 mM Tris-HCI, pH 7.5, 25 mM NaCI) overnight at 37 °C. After evaporation of the solvent, samples were desalted using two NAP-25 columns (G.E. Healthcare Life Sciences, cat. no. 17-0852-01). The expected product was confirmed by mass spectrometry of transcripts formed from phosphoramidate-containing and control strands using the long coding strand.
[00191] Transcription reaction (70 μΙ total volume) was performed as above using phosphoramidate (ODN 27) or control (ODN 31) template and long coding strand (ODN 32). The reaction mixture was left for 16 h before mixing with a mixture of phenol:chloroform:isoamyl alcohol (25:24: 1 ,v/v) (from Invitrogen) to remove excess reagents. The mixture was mixed vigorously and the top layer (transcripts) was removed. The RNA transcripts were precipitated by adding sodium acetate (3 M, 50 μΙ) followed by isopropanol (150 μΙ). The mixture was left at -80 °C for 3 h then centrifuged at 4 °C and 13 RPM for 10 min. The RNA was dried then dissolved in 20 μΙ water where 0.5 μΙ was analysed by mass spectrometry. The crude transcripts gave the same (expected) mass for phosphoramidate and control templates.
Demonstration of the compatibility of the triazole-based and amide-based linkages with DNA and RNA polymerase [00192] The compatibility of the one or more phosphodiester backbone mimic inter- nucleoside linkages of Formula I (i.e. the triazole based linkages) and the one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula II (i.e. the amido based linkages) with DNA and RNA polymerase is suitably described in Proc. Natl. Acad. Sci. USA., 2011 , 108, 11338-11343 and J. Am. Chem. Soc, 2017, 139 (4), pp 1575-1583 respectively, the entire contents of which are incorporated herein by reference.
Orthogonality of phosphoramidate and CuAAC reactions to form 303-mer and 331-mer DNA strands
[00193] For CuAAC reactions, 0.5 nmole of ODN 35 and ODN 36 and ODN 37 with 3'-alkyne and 5'-azide and splints ODN 9 and ODN 10 in 0.2 NaCI (40 μΙ_) were annealed by heating at 90 °C for 5 min then cooling slowly to room temperature. A solution of Cu' click catalyst was prepared from fr/s-hydroxypropyltriazole (0.7 μηιοΙ in 0.2 M NaCI, 17.0 μΙ_), and sodium ascorbate (1.0 μηιοΙ in 0.2 M NaCI, 2.0 μΙ_) and CuS04.5H20 (0.1 μηιοΙ in 0.2 M NaCI, 1.0 μΙ_) was added to the above annealed oligonucleotides. The mixture was kept at room temperature for 2 h before analysis by denaturing 4% polyacrylamide gel electrophoresis.
[00194] For phosphoramidate reactions, 0.5 nmole of ODN 6 and ODN 7 and ODN 8 with 3'- phosphate and 5'-amine and splints ODN 9 and ODN 10 in 40 uL buffer (0.2 M HEPES, 0.4 M NaCI) were annealed by heating at 90 °C for 5 min then cooling slowly to room temperature. EDC.HCI (10 mg) and a solution of 1-(2-hydroxyethyl) imidazole (1.0 M, 10 μΙ_) were added to the annealed oligonucleotides and the reaction mixture was kept at room temperature for 2 h before it was analysed by denaturing 4% polyacrylamide gel electrophoresis.
[00195] For orthogonal CuAAC and phosphoramidate reactions, 0.5 nmole of ODN 6 (3'- phosphate), ODN 38 (3'-propargyl and 5'-amine), ODN 37 (5'-azide) and splints ODN 9 and ODN 10 in 40 uL buffer (0.2 M HEPES, 0.4 M NaCI) were annealed by heating at 90 °C for 5 min then cooling slowly to room temperature. A solution of Cu' click catalyst was prepared from fr/s-hydroxypropyltriazole (0.35 μηιοΙ in 0.2 M NaCI, 17.0 μΙ_), sodium ascorbate (1.0 μηιοΙ in 0.2 M NaCI, 1.0 μΙ_) and CuS04.5H20 (0.1 μηιοΙ in 0.2 M NaCI, 1.0 μΙ_) followed by EDC.HCI (10 mg) and a solution of 1-(2-hydroxyethyl) imidazole (1.0 M, 10 μΙ_) were added to the annealed oligonucleotides and the reaction mixture was kept at room temperature for 2 h before being analysed by denaturing 8% polyacrylamide gel electrophoresis. Figure 13 shows similar results for all three reactions indicating the orthogonality of CuAAC click and phosphoramidate ligations. [00196] The orthogonal CuAAC and phosphoramidate reactions were repeated under the same conditions using fluorescently labelled oligonucleotide ODN 39, and gave a similar result as indicated by denaturing 8% polyacrylamide gel-electrophoresis (Figure 14).
Table 2 - Oligonucleotides used in this study
NT= 5'-amino dT, p=3'-phosphate, X=3'-propargyl-5-Me-dC\
: SeqID No. Code Oligonucleotide sequences (5'-3'j
I SeqI D 3 I GCATTCGAGCAACGTAAGATCGCTAGCACACAATCTCACACTCTGG I
ODN 1
AATTCACACTGACAATACTGCCGACACACATAACC
j SeqI D 4 j ODN 2 I GCATTCGAGCAACGTAAGATCGCj)
I SeqI D 5 'iJAGCACACAATCTCACACTCTGGAATTCACACTGACAATACTGCCG |
ODN 3
ACACACATAACC
I SeqI D 6 j ODN 4
TGTGTGCTAGCGATCTTA splint j SeqI D 7 j AAGCTTTATTAAAATGTCTAAAGGTGAAGAATTATTCACTGGTGTTG ;
j TCCCAATTTTGGTTGAATTAGATGGTGATGTTAATGGTCACAAATTT ;
TCTGTCTCCGGTGAAGGTGAAGGTGATGCTACTTACGGTAAATTGA !
ODN 5,
j CCTTAAAATTTATTTGTACTACTGGTAAATTGCCAGTTCCATGGCCA j
303-mer
! ACCTTAGTCACTACTTTCGGTTATGGTGTTCAATG I I I I GCTAGATA j j CCCAGATCATATGAAACAACATGACTTTTTCAAGTCTGCCATGCCAG j AAGGTTATGTTCAAGAAAGAAC
I SeqI D 8 j AAGCTTTATTAAAATGTCTAAAGGTGAAGAATTATTCACTGGTGTTG |
ODN 6 j TCCCAATTTTGGTTGAATTAGATGGTGATGTTAATGGTCACAAATTT |
TCTGTCfi
I SeqI D 9 j ^TCCGGTGAAGGTGAAGGTGATGCTACTTACGGTAAATTGACCTTA
ODN 7 ; AAATTTATTTGTACTACTGGTAAATTGCCAGTTCCATGGCCAACCTT |
AGTCACTACg
j SeqI D 10 j H'TTCGGTTATGGTGTTCAATGTTTTGCTAGATACCCAGATCATATG |
ODN 8 I AAACAACATGACTTTTTCAAGTCTGCCATGCCAGAAGGTTATGTTCA |
AGAAAGAAC
j SeqI D 1 1 ODN 9 CCATAACCGAAAGTAGTGACTAAG Splint for 303-mer template ligation
I SeqI D 12 ODN 10 j ACCTTCACCGGAGACAGAAAATTT Splint for 303-mer template ligation ;
I SeqI D 13 \ ODN 11 GTTCTTTCTTGAACATAA PCR Primer 1 for 303-mer template j SeqI D 14 j ODN 12 ; AAGCTTT ATTAAAAT GTCTA PCR Primer 2 for 303-mer template SeqID 15 pTCGACGGTACCGCGGGCCCGGGATCCACCGGTCGCCACCATGGT
GAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGG
TCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCG
GCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAG
TTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTC
GTGACCACCCTGACCTACGGTGTACAGTGCTTCAGCCGCTACCCC
GACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAA
ODN 13,
GGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAAC EGFP
TACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTG
forward
AACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAAC
strand
ATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCT
ATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAA
GATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCA
CTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCC
CGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCC
CAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGC
CGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAAGC
SeqID 16 pGGCCGCTTTACTTGTACAGCTCGTCCATGCCGAGAGTGATCCCGG
CGGCGGTCACGAACTCCAGCAGGACCATGTGATCGCGCTTCTCGT
TGGGGTCTTTGCTCAGGGCGGACTGGGTGCTCAGGTAGTGGTTGT
CGGGCAGCAGCACGGGGCCGTCGCCGATGGGGGTGTTCTGCTGG
TAGTGGTCGGCGAGCTGCACGCTGCCGTCCTCGATGTTGTGGCGG
ATCTTGAAGTTCACCTTGATGCCGTTCTTCTGCTTGTCGGCCATGAT
ATAGACGTTGTGGCTGTTGTAGTTGTACTCCAGCTTGTGCCCCAGG
ODN
ATGTTGCCGTCCTCCTTGAAGTCGATGCCCTTCAGCTCGATGCGGT EGFP
TCACCAGGGTGTCGCCCTCGAACTTCACCTCGGCGCGGGTCTTGT
reverse
AGTTGCCGTCGTCCTTGAAGAAGATGGTGCGCTCCTGGACGTAGC
strand
CTTCGGGCATGGCGGACTTGAAGAAGTCGTGCTGCTTCATGTGGT
CGGGGTAGCGGCTGAAGCACTGTACACCGTAGGTCAGGGTGGTCA
CGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGATG
AACTTCAGGGTCAGCTTGCCGTAGGTGGCATCGCCCTCGCCCTCG
CCGGACACGCTGAACTTGTGGCCGTTTACGTCGCCGTCCAGCTCG
ACCAGGATGGGCACCACCCCGGTGAACAGCTCCTCGCCCTTGCTC
ACCATGGTGGCGACCGGTGGATCCCGGGCCCGCGGTACCG
SeqID 17 gTCGACGGTACCGCGGGCCCGGGATCCACCGGTCGCCACCATGGT
ODN 15 GAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGG
TCGAGCTGGACGGCGACGTAAACGGCCACAAGp 120-mer
SeqID 18 !iTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAA
ODN 16
GCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCC CTGGCCCACCCTCGTGACCACCCTGACCTACGGTGTACAGTGCTT
CAGCCGCTACCCCGACCACAfi 154-mer
I SeqI D 19 I ^TGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACG
TCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGAC
ODN 17
CCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCAT CGAGCTGAAGGGCATCGACg 155-mer
j SeqI D 20 j ;ilTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAA
CTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAAC
ODN 18
GGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGC AGCGTGCAGCTCGCCGACCAC > 156-mer
I SeqI D 21 ! :'TACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCC I
CGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCC
ODN 19
CAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGC CGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAAGC
I 177-mer
I SeqI D 22 GGCCGCTTTACTTGTACAGCTCGTCCATGCCGAGAGTGATCCCGG j
CGGCGGTCACGAACTCCAGCAGGACCATGTGATCGCGCTTCTCGT
ODN 20
j TGGGGTC I I I GCTCAGGGCGGACTGGGTGCTCAGGTAGTGGTTGT | CGGGCAGCAGCACGGGGCC^3 155-mer
I SeqI D 23 ! :'r!'CGCCGATGGGGGTGTTCTGCTGGTAGTGGTCGGCGAGCTGCAC I
GCTGCCGTCCTCGATGTTGTGGCGGATCTTGAAGTTCACCTTGATG
ODN 21
CCGTTCTTCTGCTTGTCGGCCATGATATAGACGTTGTGGCTGTTGT AGTTGTACTCCAGCTTOjS 153-mer
I SeqI D 24 j ^TGCCCCAGGATGTTGCCGTCCTCCTTGAAGTCGATGCCCTTCAGC
TCGATGCGGTTCACCAGGGTGTCGCCCTCGAACTTCACCTCGGCG
ODN 22
CGGGTCTTGTAGTTGCCGTCGTCCTTGAAGAAGATGGTGCGCTCCT GGACGTAGCCTjS 147-mer
j SeqI D 25 I :'TCGGGCATGGCGGACTTGAAGAAGTCGTGCTGCTTCATGTGGTCG I
GGGTAGCGGCTGAAGCACTGTACACCGTAGGTCAGGGTGGTCACG !
ODN 23
AGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGATGAA CTTCAGGGTCAGCTTGCCG^ 153-mer
j SeqI D 26 'il.AGGTGGCATCGCCCTCGCCCTCGCCGGACACGCTGAACTTGTG |
j GCCG I I I ACGTCGCCGTCCAGCTCGACCAGGATGGGCACCACCCC |
ODN 24
GGTGAACAGCTCCTCGCCCTTGCTCACCATGGTGGCGACCGGTGG ATCCCGGGCCCGCGGTACCG 154-mer
I SeqI D 27 \ ODN 25 TCGACGGTACCGCGGGCC PCR primer for EGFP forward strand j j SeqI D 28 j ODN 26 ; GCTTTACTTGTACAGCTCGTCC PCR primer for EGFP reverse strand ; 1 SeqlD 29 I CACCCCGGTGAACAGCTCCTCGCCCTTGCTCACCATGGTGGCGAC j
ODN 27 TTCTCCCTATAGTGAGTCGTATTAGGACCAGCGT transcription template
SeqID 30 j ^TCGCCCTTGCTCACCATGGTGGCGACTTCTCCCTATAGTGAGTCG
ODN 28
TATTAGGACCAGCGT
1 SeqID 31 j ODN 29 CACCCCGGTGAACAGCTCCo
j SeqID 32 ODN 30 GCAAGGGCGAGGAGCTGTTC splint
I SeqID 33 CACCCCGGTGAACAGCTCCTCGCCCTTGCTCACCATGGTGGCGAC j
ODN 31 TTCTCCCTATAGTGAGTCGTATTAGGACCAGCGT control for transcription
I SeqID 34 j ACGCTGGTCCTAATACGACTCACTATAGGGAGAAGTCGCCACCATG
ODN 32
GTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTG long coding strand
SeqID 35 j ACGCTGGTCCTAATACGACTCACTATAGGGAGAAGTCGCC short
ODN 33
coding strand
SeqID 36 j pppGGGAGAAGUCGCCACCAUGGUGAGCAAGGGCGAGGAGCUGU j
ODN 34
UCACCGGGGUGc transcript
j SeqID 37 j AAGCTTTATTAAAATGTCTAAAGGTGAAGAATTATTCACTGGTGTTG j
ODN 35
j TCCCAATTTTGGTTGAATTAGATGGTGATGTTAATGGTCACAAATTT j TCTGT
I SeqID 38 I ; TCCGGTGAAGGTGAAGGTGATGCTACTTACGGTAAATTGACCTTAA j
ODN 36
j AATTTA I I I GTACTACTGGTAAATTGCCAGTTCCATGGCCAACCTTA | GTCACTAX
I SeqID 39 j JTTTCGGTTATGGTGTTCAATGTTTTGCTAGATACCCAGATCATATGA |
ODN 37
I AACAACATGACTTTTTCAAGTCTGCCATGCCAGAAGGTTATGTTCAA | GAAAGAAC
j SeqID 40 j ;irCCGGTGAAGGTGAAGGTGATGCTACTTACGGTAAATTGACCTTA
ODN 38
I AAATTTATTTGTACTACTGGTAAATTGCCAGTTCCATGGCCAACCTT j AGTCACTAX
j SeqID 41 YGGCCGCTTTACTTGTACAGCTCGTCCATGCCGAGAGTGATCCCG I
GCGGCGGTCACGAACTCCAGCAGGACCATGTGATCGCGCTTCTCG
ODN 39
j TTGGGGTC I I I GCTCAGGGCGGACTGGGTGCTCAGGTAGTGGTTG j TCGGGCAGCAGCACGGGGCCGg
I SeqID 42 j iTTCAGCCGGTGCGTGATCAGAAAGGCGAGCTGCAATACTTTATTG |
ODN 40
GGGTTCAGTTAGATGGATCCGATCATGTGG
I SeqID 43 I TCACCTTCACCTTCACCGGACGGCCCCGTGCTGCTGCCCG splint 1
ODN 41
for 331 click amidate ligation Seql D 44 CTGATCACGCACCGGCTGAAGTAGTGACTAAGGTTGGCCA splint 1
ODN 41
for 331 click amidate ligation
Table 3 - Oligonucleotide mass spectra
Figure imgf000072_0001
[00197] While specific embodiments of the invention have been described for the purpose of reference and illustration, various modifications will be apparent to a person skilled in the art without departing from the scope of the invention as defined by the appended claims. REFERENCES
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2. M. M. Suzuki and A. Bird, Nature Rev. Genet., 2008, 9, 465-476.
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Shapland, Z. Serber, J. Dean, J. D. Newman and S. S. Chandran, ACS Syn. Biol., 2014, 3, 97-106.
4. A. S. Xiong, R. H. Peng, J. Zhuang, J. G. Liu, F. Gao, J. M. Chen, Z. M. Cheng and Q. H.
Yao, Biotechnol. Adv., 2008, 26, 121-134.
5. A. H. El-Sagheer, A. P. Sanzone, R. Gao, A. Tavassoli and T. Brown, Proc. Natl. Acad.
Sci. USA., 201 1 , 108, 1 1338-1 1343.
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Sci. USA., 2011 , 108, 1 1338-1 1343.

Claims

1 . A process for preparing a target oligonucleotide or oligonucleotide analogue comprising two or more different phosphodiester mimic inter-nucleoside linkages, wherein the process comprises the steps of:
A) ligating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula I shown below, at the point(s) of ligation:
Figure imgf000074_0001
Formula I wherein:
/ a and independently denote the points of attachment to the target oligonucleotide or oligonucleotide analogue;
R1 a, R1 b, R1 c, R1 d, R1 e, R1 f, R19 and R1 h are each independently selected from hydrogen or (1 -4C)alkyl, wherein each (1 -4C)alkyl is optionally substituted with one or more NH2, OH or SH;
V and W are independently selected from O, S or NRX, wherein Rx is selected from hydrogen or (1 -4C)alkyl;
x, xi , z and zi are integers independently selected from 0 to 2; and y and yi are integers independently selected from 0 to 1 ;
and
B) ligating two or more oligonucleotides or oligonucleotide analogues together to form one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula II and/or Formula II I shown below, at the point(s) of ligation: R2e R2c R2d
Formula II
Figure imgf000075_0001
Formula III
wherein:
tV LV' "T-^ "V
/ c d e and ^ f independently denote the points of attachment to the target oligonucleotide or oligonucleotide analogue;
R2a, R2b, R2c, R2d, R3a, R3b, R3c and R3d are independently selected from hydrogen or (1-4C)alkyl, wherein each (1-4C)alkyl is optionally substituted with one or more N H2, OH or SH
R2e and R3e are independently selected from hydrogen or (1-4C)alkyl;
Vi , Wi and W2 are independently selected from O, S or NRZ, wherein Rz is selected from hydrogen or (1-4C)alkyl;
Q is selected from S or O;
n, rii , m and mi are integers independently selected from 0 to 2; and p, pi and P2 are integers independently selected from 0 to 1 ;
wherein steps A) and B) above are conducted in either order;
and with the proviso that:
1) the sum of integers x, xi , y, yi , z and zi is either 0, 1 , 2, 3, 4, 5 or 6;
2) the sum of integers n, rii , p and pi is greater than or equal to 2;
3) the sum of integers m, mi and P2 is equal to 0, 1 , 2, 3 or 4; and
4) when p is 1 , pi is 0.
2. A process according to claim 1 , wherein the one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula I are formed at the point(s) of ligation by reacting:
A1 ) one or more alkyne terminating oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula A shown below:
Figure imgf000076_0001
Formula A
wherein:
^ a' denotes the point of attachment to the alkyne terminating oligonucleotide or oligonucleotide analogue;
V is selected from O, S or NRX, wherein Rx is selected from hydrogen or (1 - 4C)alkyl;
R1 a, R1 b, R1 c and R1 d are independently selected from hydrogen or (1 - 4C)alkyl, wherein each (1 -4C)alkyl is optionally substituted with one or more NH2, OH or SH;
x and z are integers independently selected from 0 to 2; and
y is an integer selected from 0 to 1 ;
with
one or more azide terminating oligonucleotides or oligonucleotide analog comprising a terminal functional group of Formula B, shown below:
Figure imgf000076_0002
Formula B
wherein:
'Τ,-'
' denotes the point of attachment to the azide terminating oligonucleotide or oligonucleotide analogue; W is selected from O, S or NRX, wherein Rx is selected from hydrogen or (1- 4C)alkyl;
R1e, R1f, R19 and R1 h are independently selected from hydrogen or (1-4C)alkyl, wherein each (1-4C)alkyl is optionally substituted with one or more NH2, OH or SH;
xi and zi are integers independently selected from 0 to 2; and yi is an integer selected from 0 to 1 ;
with the proviso that the sum of integers x, xi , y, yi, z and zi is either 0, 1 , 2, 3, 4, 5 or 6.
3. A process according to claim 2, wherein the reaction between the one or more alkyne terminating oligonucleotides or oligonucleotide analogues and the one or more azide terminating oligonucleotides or oligonucleotide analogues is conducted in the presence of a catalyst.
4. A process according to claim 3, wherein the catalyst is a copper (I) species.
5. A process according to any one of claims 1 to 4, wherein R1a, R1 b, R1c, R1d, R1e, R1f, R19 or R1 h are independently selected from hydrogen or (1-4C)alkyl.
6. A process according to any one of claims 1 to 5, wherein V and W are independently selected from O or NRX, wherein Rx is selected from hydrogen or (1-4C)alkyl.
7. A process according to any one of claims 1 to 6, wherein x, xi , z and zi are integers independently selected from 0 to 1 ; and y and yi are integers independently selected from 0 to 1.
8. A process according to any one of claims 1 to 7, wherein the one or more
phosphodiester backbone mimic inter-nucleoside linkages of Formula I formed at the point(s) of ligation are selected from one of the following:
Figure imgf000078_0001
target oligonucleotide or oligonucleotide analogue;
^ denotes the point of attachment to a 5' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue.
9. A process according to any one of claims 1 to 8, wherein the one or more
phosphodiester backbone mimic inter-nucleoside linkages of Formula II are formed by reacting: one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula C shown below:
Figure imgf000079_0001
Formula C
wherein:
/ c denotes the point of attachment to the oligonucleotide or oligonucleotide analogue comprising a terminal functional group of Formula C;
X is a leaving group selected from halo, OS02Rx1 , (1-2C)haloalkyl, (1- 2C)haloalkoxy, OR"2, heteroaryl, wherein Rx1 and R*2 are independently selected from H, (1-6C)alkyl, (1-6C)alkanoyl, cycloalkyl, heteroalkyi, aryl, heteroaryl, (1-2C)haloalkyl, and wherein each of (1-6C)alkyl, cycloalkyl, heteroalkyi, aryl, heteroaryl are optionally further substituted with one or more groups selected from (1-4C)alkyl, halo, cyano, nitro or (1-2C)haloalkyl;
Vi is selected from O, S or NRZ, wherein Rz is selected from hydrogen or (1- 4C)alkyl;
Q is selected from O or S;
R2a and R2b are independently selected from hydrogen or (1-4C)alkyl, wherein each (1-4C)alkyl is optionally substituted with one or more NH2, OH or SH; n is an integer selected from 0 to 2; and
p is an integer selected from 0 to 1 ;
with
one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula D, shown below:
Figure imgf000079_0002
Formula D wherein:
^ d' denotes the point of attachment to the oligonucleotide or oligonucleotide analogue comprising a terminal functional group of Formula D;
R2c and R2d are independently selected from hydrogen or (1-4C)alkyl;
Wi is selected from O or NH;
Xa is selected from NRe or SH, wherein Re is selected from hydrogen or (1- 4C)alkyl;
ni is an integer selected from 0 to 2; and
pi is an integer selected from 0 or 1 ;
and wherein the reaction is optionally conducted in the presence of one or more of the following:
i) one or more peptide coupling reagents;
ii) one or more activating agents; and
iii) a catalyst;
with the proviso that:
1) the sum of integers n, ni , p and pi is equal to or greater than 2; and
Figure imgf000080_0001
10. A process according to any one of claims 1 to 9, wherein X is selected from halo,
OS02Rx1 , (1-2C)haloalkyl, (1-2C)haloalkoxy or OR*2, wherein Rx1 and R*2 are independently selected from H or (1-6C)alkyl.
1 1. A process according to any one of claims 1 to 10, wherein p is 0.
12. A process according to any one of claims 1 to 11 , wherein n and ni are integers
selected from 0 or 1.
13. A process according to any one of claims 1 to 12, wherein R2a, R2b, R2c and R2d are independently selected from hydrogen or (1-4C)alkyl. A process according to any one of claims 1 to 13, wherein the one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula II are selected from one of the following:
Figure imgf000081_0001
wherein:
/ c denotes the point of attachment to a 3' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue;
^ d denotes the point of attachment to a 5' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue.
15. A process according to any one of claims 1 to 14, wherein the one or more
phosphodiester backbone mimic inter-nucleoside linkages of Formula III are formed by reacting:
B3) one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula E shown below:
Figure imgf000081_0002
Formula E
wherein:
^ e' denotes the point of attachment to the oligonucleotide or oligonucleotide analogue comprising a terminal functional group of Formula E;
R3a and R3b are independently selected from hydrogen or (1-4C)alkyl, wherein each (1-4C)alkyl is optionally substituted with one or more NH2, OH or SH; and m is an integer selected from 0 to 2;
with
B4) one or more oligonucleotides or oligonucleotide analogues comprising a terminal functional group of Formula F shown below:
Figure imgf000082_0001
Formula F
wherein: f denotes the point of attachment to the oligonucleotide or oligonucleotide analogue comprising a terminal functional group of Formula F;
R3c and R3d are independently selected from hydrogen or (1 -4C)alkyl, wherein each (1 -4C)alkyl is optionally substituted with one or more NH2, OH or SH;
R3e is selected from and hydrogen or (1 -4C)alkyl;
W2 is selected from O, S or NRZ, wherein Rz is selected from hydrogen or (1 - 4C)alkyl;
mi is an integer selected from 0 to 2; and
P2 is an integer selected from 0 or 1 ;
and wherein the reaction is optionally conducted in the presence of one or more of the following:
i) one or more peptide coupling reagents;
ii) one or more activating agents; and
iii) a catalyst.
A process according to any one of claims 1 to 15, wherein R3a, R3b, R3c and R3d are independently selected from hydrogen or (1 -4C)alkyl.
A process according to any one of claims 1 to 16, wherein m and mi are integers independently selected from 0 or 1.
18. A process according to any one of claims 1 to 17, wherein p2 is 0.
A process according to any one of claims 1 to 18, wherein s e denotes the point of attachment to a 3' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue and ^ f denotes the point of attachment to a 5' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue.
A process according to any one of claims 1 to 19, wherein the one or more
phosphodiester backbone mimic inter-nucleoside linkages of Formula I I I is of the following formula:
Figure imgf000083_0001
wherein: e denotes the point of attachment to a 3' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue; and f denotes the point of attachment to a 5' carbon of a nucleoside of the target oligonucleotide or oligonucleotide analogue.
A process according to any one of claims 9 or 15, wherein the reaction is carried out in the presence of one or more peptide coupling reagents.
22. A process according to claim 21 , wherein the one or more peptide coupling reagent is selected from Ν, Ν'-dicyclohexylcarbodiimide (DCC), N. N'-diisopropylcarbodiimide (DIC), 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), N-cyclohexyl-N'- isopropylcarbodiimide (CIC) or Ν, Ν'-dicyclopentylcarbodiimide (CPC).
A process according to any one of claims 1 to 22, wherein the process is conducted in the presence of a template.
A process according to claim 23, wherein the template is a single stranded
oligonucleotide or oligonucleotide analogue that the oligonucleotides or oligonucleotide analogues to be ligated bind to such that the functional groups on the termini of adjacent oligonucleotides or oligonucleotide analogues are available to be ligated together to form an inter-nucleoside linkage of Formula I, II and III as defined in claim 1.
25. A process according to any one of claims 1 to 22, wherein the process is conducted in the absence of a template.
26. A process according to any one of claims 15 to 25, wherein: i) the one or more alkyne terminating oligonucleotides comprising a terminal
functional group of Formula A; or
ii) the one or more azide terminating oligonucleotides comprising a terminal
functional group of Formula B;
further comprise a terminal functional group selected from one of Formula C, Formula D, Formula E or Formula F, as defined in claims 15 to 25 above.
27. A process according to any one of claims 1 to 26, wherein the target oligonucleotide or oligonucleotide analogue comprises one phosphodiester backbone mimic inter- nucleoside linkages of Formula I and one phosphodiester backbone mimic inter- nucleoside linkages of Formula II.
28. A process according to any one of claims 1 to 26, wherein the target oligonucleotide or oligonucleotide analogue comprises one phosphodiester backbone mimic inter- nucleoside linkages of Formula I and one phosphodiester backbone mimic inter- nucleoside linkages of Formula III.
29. A process according to any one of claims 1 to 28, wherein the target oligonucleotide or oligonucleotide analogue comprises one or more locked nucleosides.
30. A process according to claim 29, wherein the one or more locked nucleosides are
positioned at the 3' end of an inter-nucleoside linkage.
31. A process according to claim 29, wherein the one or more locked nucleosides are
positioned at the 5' end of an inter-nucleoside linkage
32. A process according to claim 29, wherein the target oligonucleotide or oligonucleotide analogue comprises at least two locked nucleosides, and wherein at least one locked nucleoside is positioned at the 3' end of an inter-nucleoside linkage and at least one locked nucleoside is positioned at the 5' end of an inter-nucleoside linkage.
33. A process according to any one of claims 1 to 32, wherein the one or more
phosphodiester backbone mimic inter-nucleoside linkages of Formula I I or Formula I II are formed before the one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula I .
34. A process according to any one of claims 1 to 22 or 25 to 33, wherein at least one of the oligonucleotides to be ligated together is attached to a solid support.
35. A process according to claim 34, wherein the solid support is selected from controlled pore glass (CPG), silica, hydroxylated methacrylic polymer beads (e.g. Toyopearl® beads) or microporous polystyrene (MPPS).
36. An oligonucleotide or oligonucleotide analogue comprising:
A) one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula I shown below:
Figure imgf000085_0001
Formula I wherein:
/ a and independently denote the points of attachment to the target oligonucleotide or oligonucleotide analogue;
R1 a, R1 b, R1 c, R1 d, R1 e, R1 f, R19 and R1 h are each independently selected from hydrogen or (1 -4C)alkyl, wherein each (1 -4C)alkyl is optionally substituted with one or more NH2, OH or SH; V and W are independently selected from O, S or NRX, wherein Rx is selected from hydrogen or (1-4C)alkyl;
x, xi, z and zi are integers independently selected from 0 to 2; and y and yi are integers independently selected from 0 to 1 ;
and
one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula II and/or Formula III, shown below:
Figure imgf000086_0001
Formula II
Figure imgf000086_0002
Formula III
wherein: y c _ y d _ / e and f independently denote the points of attachment to the target oligonucleotide or oligonucleotide analogue;
R2a, R2b, R2c, R2d, R3a, R3b, R3c and R3d are independently selected from hydrogen or (1-4C)alkyl, wherein each (1-4C)alkyl is optionally substituted with one or more NH2, OH or SH
R2e and R3e are independently selected from hydrogen or (1-4C)alkyl;
Vi, Wi and W2 are independently selected from O, S or NRZ, wherein Rz is selected from hydrogen or (1-4C)alkyl;
Q is selected from S or O;
n, rii , m and m 1 are integers independently selected from 0 to 2; and p, pi and P2 are integers independently selected from 0 to 1 ; with the proviso that:
1 ) the sum of integers x, xi , y, yi , z and zi is either 0, 1 , 2, 3, 4, 5 or 6;
2) the sum of integers n, ni , p and pi are greater than or equal to 2;
3) the sum of integers m, mi and p2 is equal to 0, 1 , 2, 3 or 4; and
Figure imgf000087_0001
37. An oligonucleotide or oligonucleotide analogue according to claim 36, wherein the
oligonucleotide or oligonucleotide analogue comprises one phosphodiester backbone mimic inter-nucleoside linkage of Formula I and one phosphodiester backbone mimic inter-nucleoside linkage of Formula II.
38. An oligonucleotide or oligonucleotide analogue according to claim 36, wherein the
oligonucleotide or oligonucleotide analogue comprises one phosphodiester backbone mimic inter-nucleoside linkage of Formula I and one phosphodiester backbone mimic inter-nucleoside linkage of Formula III.
39. An oligonucleotide or oligonucleotide analogue according to any one of claims 36 to 38, wherein R1a, R1 b, R1c, R1d, R2a, R2b, R2c, R2d, R3a, R3b, R3c and R3d are independently selected from hydrogen or (1-4C)alkyl.
40. An oligonucleotide or oligonucleotide analogue according to any one of claims 36 to 39, wherein V and W are independently selected from O or NRX, wherein Rx is selected from hydrogen or (1-4C)alkyl.
41. An oligonucleotide or oligonucleotide analogue according to any one of claims 36 to 40, wherein x, xi , z and zi are integers independently selected from 0 to 1 ; and y and yi are an integers independently selected from 0 to 1.
42. An oligonucleotide or oligonucleotide analogue according to any one of claims 36 or 41 , wherein the one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula I are selected from one of the following:
Figure imgf000088_0001
oligonucleotide or oligonucleotide analogue;
^ denotes the point of attachment to a 5' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue.
An oligonucleotide or oligonucleotide analogue according to any one of claims 36 or 42, wherein p is 0.
44. An oligonucleotide or oligonucleotide analogue according to any one of claims 36 or 43, wherein n and ni are integers selected from 0 or 1.
An oligonucleotide or oligonucleotide analogue according to any one of claims 36 or 44, wherein the one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula I I are selected from one of the following:
Figure imgf000089_0001
wherein:
/ c denotes the point of attachment to a 3' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue;
^ d denotes the point of attachment to a 5' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue.
46. An oligonucleotide or oligonucleotide analogue according to any one of claims 36 or 45, wherein m and mi are integers independently selected from 0 or 1.
47. An oligonucleotide or oligonucleotide analogue according to any one of claims 36 or 46, wherein s e denotes the point of attachment to a 3' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue and ^ f denotes the point of attachment to a 5' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue.
48. An oligonucleotide or oligonucleotide analogue according to any one of claims 36 or 47, wherein the one or more phosphodiester backbone mimic inter-nucleoside linkages of Formula I I I is of the following formula:
Figure imgf000090_0001
wherein: e denotes the point of attachment to a 3' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue; and f denotes the point of attachment to a 5' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue.
An oligonucleotide or oligonucleotide analogue according to any one of claims 36 to 48, wherein the oligonucleotide or oligonucleotide analogue comprises:
i) one phosphodiester backbone mimic inter-nucleoside linkages of the formula:
Figure imgf000090_0002
wherein: denotes the point of attachment to a 3' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue; and
^ denotes the point of attachment to a 5' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue;
and
one phosphodiester backbone mimic inter-nucleoside linkages of the formula:
Figure imgf000090_0003
wherein: e denotes the point of attachment to a 3' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue; and f denotes the point of attachment to a 5' carbon of a nucleoside of the oligonucleotide or oligonucleotide analogue.
50. Use of an oligonucleotide or oligonucleotide analogue according to any one of claims 36 to 49, as:
(i) antisense DNA or RNA;
(ii) exon skipping DNA or RNA; or
(iii) interference RNA (e.g. siRNA).
Use of an oligonucleotide or oligonucleotide analogue according to any one of claims 36 to 49 in the synthesis of a gene.
Use of an oligonucleotide or oligonucleotide analogue according to any one of claims 36 to 49 in the treatment of a disease or disorder.
53. Use of an oligonucleotide or oligonucleotide analogue according to claim 52, wherein the disease or disorder is cancer.
54. Use of an oligonucleotide or oligonucleotide analogue according to claim 52, wherein the disease or disorder is a genetic disorder.
55. Use of an oligonucleotide or oligonucleotide analogue according to claim 52, wherein the disease or disorder is an infection.
56. Use of an oligonucleotide or oligonucleotide analogue according to any one of claims 36 to 49 as:
i) a template for amplification in a polymerase chain reaction (PCR):
ii) as a template in a DNA replication process; iii) as a template in a transcription process to provide a corresponding RNA transcript, or as a template in a reverse transcription process to provide a corresponding DNA transcript; or
iv) as template in a translation process to produce a corresponding protein or peptide.
Use of an oligonucleotide or oligonucleotide analogue according to any one of claims 36 to 49, as an RNA component of a CRISPR-Cas system (e.g. crRNA, tracrRNA or gRNA).
Use of an oligonucleotide or oligonucleotide analogue according to any one of claims 36 to 49, to guide one or more proteins of interest to a target DNA or RNA.
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