WO1998033808A2 - Procede stoechiometrique reversible servant a la conjugaison de biomolecules - Google Patents

Procede stoechiometrique reversible servant a la conjugaison de biomolecules Download PDF

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WO1998033808A2
WO1998033808A2 PCT/US1998/002007 US9802007W WO9833808A2 WO 1998033808 A2 WO1998033808 A2 WO 1998033808A2 US 9802007 W US9802007 W US 9802007W WO 9833808 A2 WO9833808 A2 WO 9833808A2
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functionality
nucleic acid
composition according
chelate
comprised
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PCT/US1998/002007
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WO1998033808A3 (fr
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Hubert Koster
Andreas Ruppert
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Hubert Koster
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Priority to AU61416/98A priority Critical patent/AU748806B2/en
Priority to EP98906096A priority patent/EP1000081A2/fr
Priority to CA002284463A priority patent/CA2284463A1/fr
Priority to DE19882066T priority patent/DE19882066T1/de
Priority to JP53319598A priority patent/JP2001526776A/ja
Publication of WO1998033808A2 publication Critical patent/WO1998033808A2/fr
Publication of WO1998033808A3 publication Critical patent/WO1998033808A3/fr
Priority to NO993746A priority patent/NO993746L/no

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes

Definitions

  • Another reversible linkage which is particularly amenable for linkage of nucleic acids, can be accomplished via heterobifunctional trityl groups, which can be cleaved under acidic conditions (E. Leinnie, F. Barnekow and H. K ⁇ ster, Heterobifunctional Trityl Derivatives as Linking Agents for the Recovery of Nucleic Acids after Labeling and Immobilization (1995) Tetrahedron 51, 3793-3802; H. K ⁇ ster, J.M. Coull and B. Gildea, Succinimidyl Trityl Compounds and a Process for Preparing Same, Protecting Groups for Natural Products, US Patent 5,410,068).
  • compositions comprised of at least two biopolymers (e.g. nucleic acids or polypeptides), which are conjugated to an insoluble support by two different reversible linkages, which are cleavable under selective conditions.
  • biopolymers e.g. nucleic acids or polypeptides
  • the invention features novel methods and components for specifically conjugating biomolecules under completely controlled stoichiometry based on the specific and strong interaction between chelators in the presence of metal ions.
  • imidazolyl moieties are introduced via the introduction of t histidine residues (e.g. oligo-His) into a polypeptide (e.g. by recombinant DNA techniques).
  • the oligo-His polypeptide can then interact in the presence of a metal with a nucleic acid carrying a chelator functionality at a position which is exposed and does not interfere with Watson-Crick base pairing of the nucleic acid.
  • the nucleic acid in another embodiment, which is particularly well-suited for the attachment of biomolecules other than polypeptides or for the reversible immobilization of nucleic acid molecules, can carry a series of imidazolyl functionalities in a format which makes them available for chelation and which does not interfere with Watson-Crick base pairing; in which case, the other conjugating partner molecule can carry the chelator functionality.
  • Figure 1 (a) and (c) pictorially depict two general approaches of the invention in which a spacer molecule, A, linked to a polymer support, P, forms a reversible linkage, I, to a nucleic acid or protein/peptide molecule, B, which itself is linked by another reversible linkage, II, to either a nucleic acid, protein/peptide or small molecule (e.g. reporter molecule).
  • Linkage I can be a heterobifunctional trityl group or a hydrophobic interaction stable under aqueous conditions or a photocleavable bond and II can be a bond, which is generated through a chelate complex.
  • the two parts which form the linkage can be reversed (I 1 , 11') as shown in (b) and (d).
  • Figure 2 schematically depicts a nucleic acid molecule, B, which is linked through a spacer, A, via a reversible linkage, I, to a polymer support, P.
  • B interacts via Watson-Crick complementarity with a nucleic acid molecule, C, which in turn through another reversible linkage II allows interaction with a reporter functionality D which can be a protein (enzyme), a nucleic acid or a small detector molecule.
  • Figure 3 schematically depicts the same approach as in Figure 2 with the exception that B is linked to the polymer support through a spacer A with a non- reversible linkage.
  • Figure 4 shows an example of the chelate complex formed between a six residue histidine (his 6 ) tail and nitrilotriacetic acid (NTA) in the presence of Ni 2+ .
  • Figure 5 schematically depicts a reaction, wherein a synthesized, protected N,N-dicarboxymethyl-serine phosphoamidite is synthesized as a chemical building block to introduce the NTA functionality into synthetic oligonucleotides.
  • Figure 6 shows the synthesis of a chelate-linked oligonucleotide to a his 6 - BAP (bacterially generated alkaline phosphatase) conjugate by use of the phosphoamidite chelate precursor.
  • Figure 7 shows the synthesis of a chelate-linked oligonucleotide to his 6 - BAP conjugate via retritylation and subsequent substitution with a chelate building block.
  • Figure 8 shows the structure of imidazolyl phosphoamidite building blocks for the single or multiple addition of an imidazolyl moiety during chemical oligonucleotide synthesis.
  • Figure 9 depicts the introduction of an imidazolyl moiety through an imidazolylnucleoside phosphoamidite.
  • Figure 10 shows the introduction of multiple imidazolyl moieties through chemical peptide synthesis of oligohistidine onto an oligonucleotide during solid phase chemical synthesis of oligonucleotides.
  • Figure 11 shows the chelate modified uracil and adenine nucleoside triphosphates for the enzymatic introduction of chelate functionalities into nucleic acids.
  • Corresponding derivatives can be envisioned for cytidine, guanine or modified nucleosides.
  • Figure 12 shows imidazolyl modified uracil and adenine nucleoside triphosphates for the enzymatic introduction of imidazolyl moieties into nucleic acids.
  • Corresponding derivatives can be envisioned for cytidine, guanine and modified nucleosides.
  • Figure 13 schematically depicts solid phase separation/detection using NHS-DMT oligonucleotides linked to a solid phase and subsequently linked to a BAP- his 6 detector molecule via the LCR (Ligase Chain Reaction).
  • FIG. 14 schematically depicts the detection of Polymerase Chain Reaction (PCR) products via the process of the invention.
  • Insoluble supports or “soluble supports” as used herein can be flat such as membranes, glass plates, metals, plastic films and composites thereof with a homogeneously functionalized surface or functionalized to result in an array format including flat supports with pits, wells, combs, microtiter plates, microtiter filter plates; flat supports can also be magnetic or with an array shaped (checkered) magnetic field; solid supports can also be used as beads from different plastic materials, inorganic supports such as silica, GPG (Controlled Pore Glass), metal, different polymeric material, cellulose, Sephadex, Sepharose; the beads can be porous or non-porous, of different diameter and magnetic or non-magnetic.
  • GPG Controlled Pore Glass
  • Compound A can be a spacer, a nucleic acid sequence (or nucleic acid analog mimetic) or a protein or peptide sequence
  • B can be a nucleic acid (or a nucleic acid analog/mimetic) or a peptide or protein
  • C can be nucleic acid (or a nucleic acid analog/mimetic), protein/peptide or a small reporter molecule.
  • A is a spacer and I is a heterobifunctional trityl group which is coupled to a nucleic acid B;
  • B carries a chelate functionality which interacts with the poly-his tail of a recombinant alkaline phosphatase (his 6 -AP), which carries e.g. a sequence of six histidine residues at the C-terminal end of the polypeptide chain.
  • a chromogenic or fluorogenic substrate is added, for example, dephosphorylation generates color or light thereby providing a nucleic acid detection system.
  • the advantage of this system is that the detection can be done either on the insoluble support or after releasing B from the support by cleavage of bond I (or I'). It is therefore possible to remove all side-products from a reaction by filtration due to the attachment to a solid phase before performing the analytical step in solution. This leads to a robust, reproducible performance.
  • Figure 2 shows schematically how amplification (e.g. polymerase chain reaction (PCR) or ligase chain reaction (LCR) products B-C can be captured specifically, purified and subsequently detected on the support or in solution.
  • the first reversible linkage I (or V) e.g. a heterobifunctional trityl group anchors one strand of the LCR or PCR product via a spacer A to the support through an acid labile tritylether bond the precursor of which has been introduced by an appropriately functionalized primer during the LCR or PCR reaction.
  • the strand C carries e.g. the chelate functionality also introduced by using an appropriately functionalized primer during PCR or LCR.
  • the chelated moiety can then interact with a reporter functionality e.g. his 6 -AP for subsequent detection and quantification of amplification product.
  • B can also be a cDNA molecule which can be linked through its 5'-end to the polymer support. With appropriate primers, solid phase DNA sequencing can be performed. Considering an array format, this could be used for high throughput genetic and expression profiling experiments.
  • B could also be a specific (or oligo-dT) capture sequence to fish mRNA.
  • the cDNA can be directly synthesized since the capture sequence simultaneously can act as a primer for the RNA dependent DNA polymerase.
  • the RNA can be removed, the cDNA purified by washing and filtration steps and either released or directly used for subsequent DNA sequencing.
  • the capture sequence while serving as a primer for the RNA dependent DNA polymerase can be used directly to generate sequencing ladders employing ddNTP's as terminators. After purification of the sequencing ladders by washing and filtration, the bond to the polymer support is cleaved and the purified sequencing ladders subjected to either gel electrophoretic or mass spectrometric separation (H.
  • Figure 3 shows a simplified version of Figure 2 in that nucleic acid fragment B is immobilized through a non-reversible bond via a spacer A to the solid support whereas nucleic acid C carries the reporter functionality via a reversible linkage so that detection can be performed either on the support or in solution.
  • biopolymer C or D could be synthetic peptides linked to an immobilized nucleic acid B or B-C respectively via a reversible linkage as described (heterobifunctional trityl, photocleavable, chelate, hydrophobic interaction) which is then detected by mass spectrometry.
  • Various defined peptide sequences can form a specific mass tag which can be used as a specific nucleic acid identifier.
  • specific nucleic acid sequences can be used as mass tags (specific identifiers) for proteins immobilized through a spacer A.
  • nucleic acids can be single stranded or double stranded polynucleotides (including oligonucleotides), whether natural or synthetic, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) or DNA RNA hybrids, DNA containing ribonucleotides and/or dideoxyribonucleotides and
  • nucleic acid RNA containing deoxyribonucleotides.
  • nucleic acid also encompassed by the term “nucleic acid” are modified nucleotides (e.g. phosphorothioate modified) as well as nucleic acid mimetics or analogs, such as peptide nucleic acids (PNAs).
  • PNAs peptide nucleic acids
  • Proteins can be antibodies, enzymes, receptor molecules; peptides could be of natural or synthetic origin with oligo-his tail, a functionality for hydrophobic interaction, a photocleavable functionality or chelator functionality and displaying different properties such as being adhesive or representing specific ligand-receptor or specific protease cleavage sites.
  • oligo his tail or poly his tail refers to a chain of conjugated histidine residues.
  • Preferred oligo his tails contain 2-10 histidine residues.
  • Particularly preferred oligo his tails are in the range of about 4 to about 8 his residues.
  • Reversible linkages can be formed by hydrophobic interaction between e.g. a trityl group (i.e. with long aliphatic alkyl chains) and a long aliphatic chain e.g. attached to a polymer support or a hydrophobic polymer surface such as that of polystyrene. Since most biochemical and molecular biological reactions are performed in aqueous solution such hydrophobic interaction might be of sufficient stability. Addition of organic solvents such as alcohols, acetonitrile, N.N-dimethylformamide and the like will destabilize (if necessary in conjunction with heat) the hydrophobic interaction and release the attached molecules.
  • a reversible linkage which can independently be addressed could also be a functionality which is cleavable under photolytic conditions (see e.g. J. Olejnik, E. Krzymanska-Olejnik and K. J. Rothschil, Photocleavable Biotin Phosphoramidites for 5'- End-labeling, Affinity purification and Phosphorylation of Synthetic Oligonucleotides (1996) Nucleic Acids Res., 24, 361-366). If the wavelength needed for photocleavage is in the range of the laser wavelength used in MALDI mass spectrometry, this bond can be cleaved during mass spectrometric signal acquisition.
  • a reversible linkage can also be formed from a chelator functionality which interacts with another chelator (e.g oligo-imidazolyl or other oligopeptide moieties) in the presence of a metal ion.
  • chelator refers to a single molecule, which comprises at least two Lewis basic atoms that are capable of associating simultaneously with a Lewis acidic atom, moelcule or ion— either simple or complex.
  • Lewis base is an art recognized term that refers to chemical moieties, which are capable of donating to another atom or moiety at least one pair of unshared electrons.
  • Examples include uncharged functional groups such as alcohols, ethers, carbonyls, thiols, sulfides, amines, imines, and pyridine and imidazole nitrogens; and charged functional groups, such as alkoxides, thiolates, carboxylates and a variety of other anions.
  • Lewis acid is an art recognized term that refers to chemical moieties, which are capable of accepting from another atom or moiety (e.g. a Lewis basic atom or moiety) at least one pair of unshared electrons.
  • Lewis acid moieties include transition metal halides, with at least one vacant d orbital, alkali metal cations, alkaline-earth metal cations, and trivalent boron or aluminum compounds.
  • a "bidentate chelator”, “tridentate chelator” and “tetradentate chelator” refers to chelators comprising two, three and four Lewis basic moieties, respectively, capable of simultaneous donation of at least an equal number of unshared electron pairs to another atom, ion or moiety.
  • Figure 4 depicts a specific example in which the chelator functionality is a nitrilotriacetic acid (NTA) which coordinates with divalent metal cations such as Ni 2+ and forms a strong complex with six imidazolyl groups from a his 6 tail linked to one of the conjugating partner molecules.
  • NTA nitrilotriacetic acid
  • divalent metal cations such as Ni 2+
  • imidazolyl residue or “imidazoyl group” refers to any substituted or unsubstituted form of imidazole (i.e. l,3-diaza-2,4- cyclopentadiene).
  • the side chain of the amino acid histidine comprises an imidazolyl residue.
  • nucleic acid molecule or the protein determines which of the two necessary functions is attached to the nucleic acid molecule or the protein depends on the ease and convenience of introduction of either functionality (e.g. NTA or his 6 tail). In case of proteins the site- specific introduction of a chelator molecule seems to be difficult whereas the his 6 tail can be introduced through recombinant DNA technologies. In contrast to currently available procedures, for linking nucleic acids to proteins (e.g. chemical linkage using either maleimide-thiol coupling (S.S. Gosh et al. (1990), Bioco ⁇ jugate Chem. I, 71-76), disulfide bonds (B.C.F. Chu L.E. Orgel (1988) Nucleic Acids Res.
  • alkaline phosphatase (EC 3.1.3.1) is a versatile enzyme for many molecular biological applications. It catalyzes the hydrolysis of ester bonds in phosphomonoesters and is used in recombinant DNA technology to remove 5-phosphate groups from DNA fragments to prevent self-litigation of vector DNA molecules. Coupled to antibodies or oligonucleotides, it replaces radioactively labeled compounds by serving as a reporter and signal amplifying enzyme which cleaves chromogenic or fluorogenic substrates in diagnostic applications for the specific detection of DNA (Southern blot: E.M. Southern (1975) J. MolBiol. 98, 503- 517) or proteins (Western blots: W.N. Burnett (1981) Anal. Biochem. 112, 195-203).
  • AP is isolated from calf intestine (OP) or the bacterium E coli. (BAP).
  • BAP bacterium E coli.
  • AP consists of a homodimer.
  • the stability of the enzyme can lead to severe problems in cloning experiments. Residual AP activity from the dephosphorylation of vector DNA can result in dephosphorylation of the DNA to be inserted so that no or only low yields of ligation products are obtained. Heat inactivation very often is not sufficient so that time-consuming removal is necessary using treatment with proteinase K and subsequent extraction from phenol/chloroform. This lengthy procedure will also drastically reduce the yield of the product.
  • AP isolated from species living at low temperatures are employed; here heat inactivation is possible, however, reduced stability is disadvantageous for diagnostic applications.
  • a modified BAP derived from E. coli was genetically designed with a his 6 tail at its carboxy terminus.
  • the his 6 tail was introduced using inverse PCR by which six histidine codons followed by a stop codon were placed at the 3' end of the gene (E. Blum et al. (1994) Biochem Biophys J. 22, 113-121).
  • the region coding for the signal peptide of AP together with the untranslated 5' and 3' regions were exchanged with homologous sequences from the E. coli ompA gene.
  • the expression of the resulting protein construct was under the control of the IPTG ( ⁇ -D-isopropyl-thio-galactoside) inducible ptac-promoter.
  • the BAP-his 6 synthesized in the E.coli cell can easily be isolated from an unpurified cell extract through affinity chromatography using commercially available Ni- NTA resins (Qiagen) to which it forms a strong and specific chelate complex via its his 6 tail.
  • the modified enzyme is therefore now available in high yields, high purity and reproducible batch-to-batch quality.
  • BAP-his 6 is able to form with chelate-modified nucleic acids, a stable complex which for the first time makes available specific conjugates between proteins (here BAP) and nucleic acids in a reproducible 1:1 stoichiometry.
  • peptides When peptides are generated by chemical synthesis, the his 6 tail can be directly incorporated during peptide synthesis. Chemical synthesis of peptides also allows the alternative approach in which a chelator functionality is attached to the synthetic peptide either at the N- or C- terminus or one of the side chains depending on which part of the peptide sequence is needed for the biochemical function.
  • the nucleic acid molecule can be functionalized either with the imidazoyl moieties or with the chelator functionalities.
  • the chelator functionality can be introduced in different ways.
  • An amino acid such as serine, cysteine or lysine can be transformed into a ⁇ -cyanoethylphosphoamidite (N.D. Sinha, J. Biernat, J. McManus and H. K ⁇ ster (1984) Nucleic Acids Res. 12, 4539-4477) carrying a precursor of the chelator functionality (e.g. NTA as described in Figure 5 and 6 with serine as starting material).
  • Figure 7 shows the introduction through a heterobifunctional trityl group.
  • the oligonucleotide is, after regular final detritylation, retritylated with a heterobifunctional trityl group bearing an active ester moiety derived from either e.g. N- hydroxysuccinimide or employing active esters such as p-nitrophenyl esters.
  • the active ester functionality is then reacted with a chelator molecule derived from e.g. lysine.
  • the imidazolyl functionality can be introduced during oligonucleotide synthesis employing an appropriate ⁇ -cyanoethylphosphoamidite as shown in Figure 8; single or multiple imidazolyl residues can be incorporated.
  • a imidazolylnucleoside as shown in Figure 9 or a histidine peptide sequence covalently attached to the oligonucleotide chain ( Figure 10) can also be used to introduce the necessary imidazolyl moieties for interaction with the chelator functionality.
  • the chelator and oligoimidazolyl functionalities can also be introduced in high molecular weight nucleic acids using either DNA dependent DNA or RNA polymerases or RNA dependent DNA polymerases using appropriately modified nucleoside triphosphates (either NTPs, 2'-dNTP, 3'-dNTPs, ddNTPs) as depicted in Figure 11.
  • the base will carry either the chelator or the oligoimidazoyl functionality ( Figure 12) in case of pyrimidine bases at C5 and in case of purine bases at C8 so that Watson-Crick base pairing is possible.
  • nucleoside triphosphates those functionalities can either be introduced internally (NTP for RNA synthesis or 2'- dNTP for DNa synthesis) or at the 3'-end (3'-dNTP for RNA synthesis, ddNTP for DNA synthesis).
  • the incorporation can be performed during amplification procedures such as PCR, SDA or during DNA sequencing.
  • Those skilled in the art will realize other approaches to introduce either chelator or oligo-imidazolyl moieties into nucleic acids.
  • Detection of the immobilized nucleic acid-protein/peptide conjugates can be achieved either directly on the polymer support or after selective cleavage of either reversible bond I (I 1 ) or II (11').
  • the signal can be detected by any of a number of means
  • XL including radioactivity, fluorescence, chemiluminescence (using e.g. 1,2-dioxetan derivatives) or colorimetric (using e.g. BCIP/NBT) methods depending on the substrates used as C or D Fig. 1, 2 and 3).
  • D can be an enzyme such as AP which triggers upon contact with a substrate through its enzymatic activity the signal generation.
  • C and D can also be detected through their molecular weight by employing mass spectrometric methods.
  • Preferred mass spectrometer formats for use in analyzing the translation products include ionization (I) techniques, including but not limited to matrix assisted laser desorption (MALDI), continuous or pulsed electrospray (ESI) and related methods (e.g.
  • TOF linear or non-linear reflectron time-of-flight
  • FTICR Fourier Transform ion cyclotron resonance
  • ion trap e.g., ion-trap/time-of-flight
  • MALDI matrix/wavelength combinations
  • ESI solvent combinations
  • Subattomole leels of protein have been detected, for example, using ESI (Valaskovic, G. A. et al., (1996) Science 273: 1199-1202) or MALDI (Li, L. et al., (1996
  • the phoA gene coding for the BAP of E. coli was derived from E. coli strain HB 101.
  • the his 6 fusion at the carboxyterminus was generated via inverse PCR with six his codons followed by a stop codon derived from plasmid pHis 1 (E. Blum et al. (1994) Biochem. Biophys. J. 29_, 113-121).
  • a solution containing DNA fragments is incubated with beads carrying immobilized metal ions complexed with BAP-his 6 protein.
  • filtration or centrifugation removes beads with adsorbed enzyme.
  • a solution containing DNA fragment can be filtered through a derivatized membrane, carrying immobilized metal ions complexed with B AP- his 6 protein.
  • BAP-his 6 as a reporter enzyme for LCR is carried out in the wells (96 or more) of a microtiter filter plate (MTFP) with 96 samples with oligos A-D ( Figure 13).
  • One of the oligos (oligo A being the marker oligo, Fig. 13) carries at its 5'- end a chelating group.
  • the marker oligo is incorporated into one strand, the marker strand, consisting of oligos A and B, with B ligated to the 3'-end of oligo A.
  • ligation products, oligos and other smaller by-products are transferred by suction into a second MTFP with a derivatized filter membrane.
  • oligo D or part of it with sequence complementary to oligo B is coupled via NHS-DMT (heterobifunctional trityl derivative) linkage.
  • Hybridization occurs between membrane bound oligo D and oligo B or the marker strand AB. After removal of supernatant and washing, only oligo A incorporated in the marker strand AB by ligation remains in the wells of the MTFP.
  • B AP-his 6 and a divalent cation such as Ni 2+ are incubated in the wells under adequate conditions to allow coupling of BAP-his 6 to the marker strand.
  • chromogenic or fluorescent AP substrates are added. Only wells containing the LCR product show AP activity as a positive result, bound D alone or the single strand CD cannot give rise to any signal.
  • the experimental setup allows multiplex LCR by employing a mixture of oligos in the LCR and subsequent transfer of the LCR products by suction through a stack of different MTFP with specific bound oligo sequences. This experiment setup is amenable to automation, since the reaction can be carried out e.g. in filter tubes or filter plates, which allow removal of contaminating agents, buffer changes and even detection in situ by dispensing and filtration of different liquids.
  • PCR is carried out in crude cell lysates with a derivatized oligonucleotide primer ( Figure 14). After denaturing, the PCR reaction is filtrated through a membrane derivatized with a capture oligo. It can contain any sequence, which is complementary to the expected PCR fragment and hybridizes with strand elongated from derivatized oligo. Although any nucleic acid containing the sequence complementary to the capture oligo will be retained on the membrane, only PCR products containing the derivatized oligonucleotide primer can bind the modified B AP-his 6 enzyme. The PCR product is detected by BAP activity retained on the membrane after adequate washing procedure.
  • This setup allows PCR with crude lysates, since contaminating agents can be removed by filtration and only the PCR products retained by hybridization to the membrane bound oligonucleotide give rise to a detectable signal.
  • This setup is also amenable to multiplexing (see above).

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Abstract

L'invention concerne des compositions renfermant au moins deux biopolymères (p. ex. des acides nucléiques ou des polypeptides), qui sont conjugués à un support insoluble par deux liaisons réversibles différentes, ces liaisons pouvant être coupées dans des conditions sélectives. L'invention concerne également des procédés et des composants destinés à la production de ces compositions.
PCT/US1998/002007 1997-02-04 1998-02-04 Procede stoechiometrique reversible servant a la conjugaison de biomolecules WO1998033808A2 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
AU61416/98A AU748806B2 (en) 1997-02-04 1998-02-04 A reversible stoichiometric process for conjugating biomolecules
EP98906096A EP1000081A2 (fr) 1997-02-04 1998-02-04 Procede stoechiometrique reversible servant a la conjugaison de biomolecules
CA002284463A CA2284463A1 (fr) 1997-02-04 1998-02-04 Procede stoechiometrique reversible servant a la conjugaison de biomolecules
DE19882066T DE19882066T1 (de) 1997-02-04 1998-02-04 Reversibles stöchiometrisches Verfahren zum Konjugieren von Biomolekülen
JP53319598A JP2001526776A (ja) 1997-02-04 1998-02-04 生体分子を結合するための可逆性化学量論的工程
NO993746A NO993746L (no) 1997-02-04 1999-08-03 En reversibel, stökiometrisk prosess for konjugering av biomolekyler

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US3716597P 1997-02-04 1997-02-04
US60/037,165 1997-02-04

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WO1998033808A2 true WO1998033808A2 (fr) 1998-08-06
WO1998033808A3 WO1998033808A3 (fr) 1999-02-25

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6225061B1 (en) 1999-03-10 2001-05-01 Sequenom, Inc. Systems and methods for performing reactions in an unsealed environment
US6730517B1 (en) 1999-04-02 2004-05-04 Sequenom, Inc. Automated process line
EP1485707A2 (fr) * 2001-07-16 2004-12-15 HK Pharmaceuticals, Inc. Composes de capture, recueils de ceux-ci et procedes d'analyse du proteome et compositions complexes
SG153699A1 (en) * 2003-01-16 2009-07-29 Caprotec Bioanalytics Gmbh Capture compounds, collections thereof and methods for analyzing the proteome and complex compositions
US7917301B1 (en) 2000-09-19 2011-03-29 Sequenom, Inc. Method and device for identifying a biological sample
US8315805B2 (en) 2001-04-20 2012-11-20 Sequenom, Inc. Systems and methods for testing a biological sample
US8975082B2 (en) 2008-05-13 2015-03-10 University Of Kansas Metal abstraction peptide (MAP) tag and associated methods
US9187735B2 (en) 2012-06-01 2015-11-17 University Of Kansas Metal abstraction peptide with superoxide dismutase activity
WO2019081680A1 (fr) * 2017-10-25 2019-05-02 Institut Pasteur Immobilisation d'acides nucléiques à l'aide d'un mimétique d'étiquette histidine enzymatique pour des applications de diagnostic

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9394565B2 (en) 2003-09-05 2016-07-19 Agena Bioscience, Inc. Allele-specific sequence variation analysis
CN101233240A (zh) 2004-03-26 2008-07-30 斯昆诺有限公司 与质量分析结合的甲基化特异性扩增产物的碱基特异性切割

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1985004674A1 (fr) * 1984-04-05 1985-10-24 Life Technologies, Inc. Immobilisation d'acides nucleiques
DE3644346A1 (de) * 1986-12-19 1987-05-21 Saeulentechnik Dr Ing Herbert Matrixgebundene kronenetherliganden als trennmaterial in der affinitaetschromatographie von nukleinsaeuren
WO1989005616A1 (fr) * 1987-12-24 1989-06-29 Bio-Metric Systems, Inc. Revetements biocompatibles
WO1990001564A1 (fr) * 1988-08-09 1990-02-22 Microprobe Corporation Procedes d'analyse a cibles multiples par hybridation d'acides nucleiques
US5410068A (en) * 1989-10-23 1995-04-25 Perseptive Biosystems, Inc. Succinimidyl trityl compounds and a process for preparing same
US5547835A (en) * 1993-01-07 1996-08-20 Sequenom, Inc. DNA sequencing by mass spectrometry
US5582981A (en) * 1991-08-14 1996-12-10 Gilead Sciences, Inc. Method for identifying an oligonucleotide aptamer specific for a target
WO1998020020A2 (fr) * 1996-11-06 1998-05-14 Sequenom, Inc. Immobilisation haute densite d'acides nucleiques

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1985004674A1 (fr) * 1984-04-05 1985-10-24 Life Technologies, Inc. Immobilisation d'acides nucleiques
DE3644346A1 (de) * 1986-12-19 1987-05-21 Saeulentechnik Dr Ing Herbert Matrixgebundene kronenetherliganden als trennmaterial in der affinitaetschromatographie von nukleinsaeuren
WO1989005616A1 (fr) * 1987-12-24 1989-06-29 Bio-Metric Systems, Inc. Revetements biocompatibles
WO1990001564A1 (fr) * 1988-08-09 1990-02-22 Microprobe Corporation Procedes d'analyse a cibles multiples par hybridation d'acides nucleiques
US5410068A (en) * 1989-10-23 1995-04-25 Perseptive Biosystems, Inc. Succinimidyl trityl compounds and a process for preparing same
US5582981A (en) * 1991-08-14 1996-12-10 Gilead Sciences, Inc. Method for identifying an oligonucleotide aptamer specific for a target
US5547835A (en) * 1993-01-07 1996-08-20 Sequenom, Inc. DNA sequencing by mass spectrometry
WO1998020020A2 (fr) * 1996-11-06 1998-05-14 Sequenom, Inc. Immobilisation haute densite d'acides nucleiques

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ARSHADY E: "BEADED POLYMER SUPPORTS AND GELS II. PHYSICO-CHEMICAL CRITERIA AND FUNCTIONALIZATION" JOURNAL OF CHROMATOGRAPHY, vol. 586, no. 2, 22 November 1991, pages 199-219, XP000247969 *
P.D.GERSHON ET AL.: "Stable Chelating Linkage for Reversible Immobilization of Oligohistidine Tagged Proteins in the BIAcore Surface Plasmon Resonance Detector." JOURNAL OF IMMUNOLOGICAL METHODS, vol. 183, 1995, pages 65-76, XP002081778 *
PON R T ET AL: "DERIVATIZATION OF CONTROLLED PORE GLASS BEADS FOR SOLID PHASE OLIGONUCLEOTIDE SYNTHESIS" BIOTECHNIQUES, vol. 6, no. 8, 1 January 1988, pages 768-770, 773 - 775, XP000562920 *
SCOUTEN W H ET AL: "REVERSIBLE IMMOBILIZATION OF ANTIBODIES ON MAGNETIC BEADS" ANALYTICAL BIOCHEMISTRY, vol. 205, no. 2, 1 September 1992, pages 313-318, XP000296795 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6225061B1 (en) 1999-03-10 2001-05-01 Sequenom, Inc. Systems and methods for performing reactions in an unsealed environment
US6730517B1 (en) 1999-04-02 2004-05-04 Sequenom, Inc. Automated process line
US7917301B1 (en) 2000-09-19 2011-03-29 Sequenom, Inc. Method and device for identifying a biological sample
US8315805B2 (en) 2001-04-20 2012-11-20 Sequenom, Inc. Systems and methods for testing a biological sample
EP1485707A2 (fr) * 2001-07-16 2004-12-15 HK Pharmaceuticals, Inc. Composes de capture, recueils de ceux-ci et procedes d'analyse du proteome et compositions complexes
EP1485707A4 (fr) * 2001-07-16 2005-11-16 Hk Pharmaceuticals Inc Composes de capture, recueils de ceux-ci et procedes d'analyse du proteome et compositions complexes
EP2053406A3 (fr) * 2001-07-16 2009-06-24 caprotec bioanalytics GmbH Composés de capture, collections associées et procédés d'analyse protéomique et compositions complexes
SG153699A1 (en) * 2003-01-16 2009-07-29 Caprotec Bioanalytics Gmbh Capture compounds, collections thereof and methods for analyzing the proteome and complex compositions
US8975082B2 (en) 2008-05-13 2015-03-10 University Of Kansas Metal abstraction peptide (MAP) tag and associated methods
US9096652B2 (en) 2008-05-13 2015-08-04 University Of Kansas Metal abstraction peptide (MAP) tag and associated methods
US9187735B2 (en) 2012-06-01 2015-11-17 University Of Kansas Metal abstraction peptide with superoxide dismutase activity
WO2019081680A1 (fr) * 2017-10-25 2019-05-02 Institut Pasteur Immobilisation d'acides nucléiques à l'aide d'un mimétique d'étiquette histidine enzymatique pour des applications de diagnostic

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NO993746L (no) 1999-10-01
JP2001526776A (ja) 2001-12-18
AU748806B2 (en) 2002-06-13
CA2284463A1 (fr) 1998-08-06
WO1998033808A3 (fr) 1999-02-25
NO993746D0 (no) 1999-08-03
DE19882066T1 (de) 2000-01-05
EP1000081A2 (fr) 2000-05-17

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