WO2017097973A1

WO2017097973A1 - Modified nucleosides

Info

Publication number: WO2017097973A1
Application number: PCT/EP2016/080420
Authority: WO
Inventors: Andreas Marx; Moritz WELTER; Jana BALINTOVÁ
Original assignee: Universitaet Konstanz
Priority date: 2015-12-09
Filing date: 2016-12-09
Publication date: 2017-06-15

Abstract

The present invention relates to modified nucleosides which are linked to cargo molecules and which can be incorporated by DNA or RNA polymerases in strand synthesis. The present invention also relates to use of such modified nucleosides as substrates for DNA or RNA polymerases. The present invention also relates to the use of such modified nucleosides in diagnosis and prognosis of diseases or disorders associated with a target sequence. The present invention also relates to methods for producing such modified nucleosides, kits comprising such nucleosides, and in vitro methods for diagnosing or predicting diseases or disorders associated with a target sequence. The present invention also relates to modified nucleosides for use in diagnosing, predicting or treating diseases or disorders associated with a target sequence.

Description

Modified Nucleosides

The present invention relates to modified nucleosides which are linked to cargo molecules and which can be incorporated by DNA or RNA polymerases in strand synthesis. The present invention also relates to use of such modified nucleosides as substrates for DNA or RNA polymerases. The present invention also relates to the use of such modified nucleosides in diagnosis and prognosis of diseases or disorders associated with a target sequence. The present invention also relates to methods for producing such modified nucleosides, kits comprising such nucleosides, and in vitro methods for diagnosing or predicting diseases or disorders associated with a target sequence. The present invention also relates to modified nucleosides for use in diagnosing, predicting or treating diseases or disorders associated with a target sequence.

DNA as an information storage system is simple and at the same time complex owing to the various different arrangements of the four natural nucleotides. Sequencing and identifying target sequences within nucleic acids (DNA or RNA) is essential for research on the one hand and diagnostic methods for detecting diseases or disorders associated with certain target sequences on the other hand. For this purpose, several nucleotide analogues equipped with various marker groups (e.g., dyes, tags or spin labels) have been employed (see, e.g., Asseline, Curr Org Chem (2006), 10: 491 ) in DNA polymerase catalysed reactions to increase the application scope of DNA (e.g., sequencing, structural characterization, and immobilization); Obeid et al., Angew Chem (2008), 120: 6886; Liu et al., Angew Chem (2010), 122: 3385. However, these marker groups share the inherent disadvantage of requiring PCR-based technologies which involve sophisticated instrumentation. Hence, such markers do not allow appropriate device portability, e.g., for point-of-care testing (POCT) or the detection of pathogens in the field (Andras et al., Mol Biotechnol (2001 ), 19: 29; Jeong et al., Cell Mol Life Sci (2009), 66: 3325).

The template dependent replication of a DNA strand by a DNA polymerase is exploited for research and diagnostics as the intrinsic fidelity of the enzyme strongly favors the formation of the correct Watson-Crick base pair over mismatch pairs. This characteristics are key for employing modified nucleotides in order to gain sequence information, label the complementary DNA strand or to detect the presence of a particular, i.e. pathogen-derived nucleic acid (DNA or RNA) sequence (Obeid et al., Angewandte Chemie Int Edition (2008), 47(36): 6782-6785).

Corresponding modified nucleosides were examined with regard to their capability of being substrates for DNA polymerases. Such modifications comprised the addition of small molecules such as nitroxides or amines as well as larger molecules with sizes up to the diameter of DNA polymerase (dendrons, structured and non-structured oligonucleotides). However, incorporation of modified nucleosides carrying such molecules by DNA polymerase was either impaired, did not allow naked-eye detection or exhibited strong inhibitory effects when double-stranded oligonucleotides were attached (Obeid et al., PNAS USA (2010), 107(50), 21327-21331 ; Bergen et al., J Am Chem Soc (2012), 134: 1 1840-1 1843; Baccaro et al., Angew Chem Int Ed (2012), 51 : 254-257; Chem Commun (2015), 51 : 7379-7381 ).

Accordingly, the technical problem underlying the present invention was to comply with the disadvantages set out above.

The present invention addresses the technical problem by providing modified nucleosides linked to cargo molecules, uses thereof and methods applying such modified nucleosides as set forth herein below and as defined by the claims. Accordingly, the present invention relates to a modified nucleoside which comprises a structure represented by formula (I) below

Y-L-X (I)

wherein

Y is a pyrimidine or purine nucleoside, wherein

L is a linker, and

X is a cargo having a volume of 15000 A³ or more,

wherein said modified nucleoside is incorporated by DNA or RNA polymerase in strand synthesis.

Alternatively or additionally, the present invention relates to a modified nucleoside which comprises a structure represented by formula (I) below

Y-L-X (I)

wherein

Y is a pyrimidine or purine nucleoside, wherein

L is a linker, and X is a cargo that has at least 90 amino acids for cargo X being a protein,

wherein said modified nucleoside is incorporated by DNA or RNA polymerase in strand synthesis. That is, the modified nucleoside of the present invention is suitable to be incorporated by DNA or RNA polymerases in strand synthesis. Accordingly, the modified nucleoside of the present invention is preferably recognized by DNA or RNA polymerases as defined and exemplified herein without abolishing the catalytic activity of the polymerases. In other words, the modified nucleoside of the present invention is preferably a substrate for DNA or RNA polymerases as defined and exemplified herein. That is, DNA or RNA polymerases may attach the modified nucleoside (or nucleotide comprising it as described and defined herein) to the 3'- or 5' end of a nucleic acid molecule. As such, the modified nucleoside of the present invention can be incorporated by DNA or RNA polymerases into strand synthesis, e.g., during strand elongation in 5' to 3'-direction or - particularly for proof-reading polymerases, e.g., exonucleases, in 3' to 5'-direction. In one embodiment of the present invention, nucleotides comprising the modified nucleosides of the present invention are incorporated into strand synthesis. For example, nucleotides comprising said modified nucleosides are connected by said DNA or RNA polymerase to a nucleic acid molecule during strand synthesis via ester bonds between the sugar (e.g., pentose such as (desoxy)ribose) moiety of the growing strand and the phosphate moiety of the nucleotide to be incorporated or between the sugar (e.g., pentose such as (desoxy)ribose) moiety of the nucleotide to be incorporated and the phosphate moiety of the growing strand.

As has surprisingly be found in context with the present invention, nucleosides that are conjugated to a bulky globular cargo protein such as an enzyme are substrates to nucleic acid polymerases (DNA or RNA polymerases) without compromising the enzymatic activity, i.e. neither of the polymerase nor of the cargo enzyme. For example, as shown and further described herein, when conjugating horseradish peroxidase (HRP, a -40 kDa glycoenzyme derived from Amoracia rusticana having catalytic properties to produce a colorimetric signal by oxidation of dye substrates) to a nucleoside, such modified nucleosides can readily be used for naked-eye detection of DNA and RNA at single-nucleotide resolution. This is in sharp contrast to what has been found for oligonucleotide-modified nucleotides which were up to 40-times larger than the natural substrate for DNA polymerase where such nucleotides exhibited strong inhibitory effects on the DNA polymerase when double-stranded oligonucleotides were attached (Baccaro et al., Angew Chem Int Ed (2012), 51 (1 ): 254-257). That is, the gist of the present invention lies in the surprising finding that bulky cargo molecules of 15000 A³ or more (or alternatively at least 90 amino acids for cargo X being a protein) can be attached to nucleosides via a linker molecule without compromising the capability of RNA or DNA polymerase to introduce such modified nucleosides in strand synthesis, while also keeping the activity of the cargo molecule. Herein below, the present invention is further described and exemplified for proteins (particularly enzymes) to be linked to nucleosides. Yet, the finding of the present invention also allows linkage of different bulky cargo molecules to the nucleosides to obtain modified nucleosides according to the present invention, e.g., antibodies or other molecules allowing employment of the modified nucleosides for methods and applications as described and provided herein. As used herein- the term "naked-eye" detection particularly means that a molecule comprising the modified nucleoside of the present invention may be employed in a system (e.g., a colorimetric system) where the presence or binding of said molecule can be detected with the naked eye, i.e. without the necessity of technical aid. Generally, the advantage of the modified nucleosides of the present invention and their uses and methods employing said modified nucleosides comprise the possibility to conduct a naked-eye readout and, this, allow point-of-care testing (POCT) of samples, e.g., samples comprising target sequences to be detected as described herein.

As it is shown and described in accordance with the present invention, the conjugation of a cargo X (e.g., an enzyme E) to a purine or pyrimidine nucleoside Y as described and defined herein generates a versatile tool that allows easy application to any nucleic acid sequence context without change of the general setup as described and exemplified herein. Furthermore, the utilization of DNA or RNA polymerases as defined herein for genotyping (i.e. detecting and binding target sequences) according to the present invention allows a high discrimination between match and mismatch template. Also, as has been found in context with the present invention, the use of inter alia mesophilic DNA polymerases allows the assay to be carried out without any laboratory equipment like thermocyclers making it suitable for in field analysis and point-of-care testing (POCT). Exemplary for the present invention, without being bound by theory, well-tolerated modifications (i.e. addition of bulky cargo X) which are attached via a flexible linker L in, e.g., C5-position of pyrimidines and, e.g., C7-position of 7-deaza-purines may be able to accommodate the cargo through defined channels of the DNA polymerase. Thereby the suited linker may place the cargo of the nucleotide outside of the DNA polymerase active site and thus, may prevent significant misalignment of the active site due to steric clash of the modification with the protein. Generally, unless specifically defined otherwise, the term "modified nucleoside" as used herein encompasses structures comprising pyrimidine or purine nucleosides (naturally occurring or artificially modified nucleosides as well as derivatives or analogues thereof, including phosphates (e.g., mono-, di-, or triphosphates) thereof as also further defined and described herein), a linker L, and a cargo molecule X of 15000 A³ or more (or alternatively at least 90 amino acids for cargo X being a protein), wherein said modified nucleoside is incorporated by DNA or RNA polymerase in strand synthesis. That is, as the person of skill in the art can readily recognize, "modified nucleosides" also encompass structures further comprising additional molecules and/or groups. For example, "modified nucleosides" as used herein may further encompass structures comprising one or more phosphate groups, i.e. nucleoside phosphates (e.g., mono-, di-, or triphosphates) or nucleotides. As such, a "modified nucleoside" as used herein is not limited to structures comprising nucleosides consisting of a base (purine or pyrimidine and derivatives or analogues thereof) and a sugar moiety (e.g., a pentose, such as, e.g., (desoxy)ribose), but may inter alia also refer to a structure comprising nucleotides (i.e. nucleoside + one or more phosphate groups). For example, if there are 1 , 2 or 3 phosphate groups attached to a nucleoside, the "modified nucleoside" of the present invention encompasses structures comprising NMPs (nucleosidemonophosphate, a nucleotide with 1 phosphate group), NDPs (nucleosidediphosphate, a nucleotide with 2 phosphate groups), or NTPs (nucleosidetriphosphate, a nucleotide with 3 phosphate groups), as well as a linker L and a cargo molecule X of 15000 A³ or more (or alternatively at least 90 amino acids for cargo X being a protein), wherein said modified nucleoside is incorporated by DNA or RNA polymerase in strand synthesis. Also, "modified nucleosides" according to the present invention may encompass structures comprising multiple nucleotides, e.g., nucleic acid molecules (nucleic acid strands) such as DNA or RNA strands.

Accordingly, in one aspect of the present invention, the modified nucleoside described and provided herein may comprise a structure represented by formula (II) below

R¹-Y-L-X (II)

wherein R¹ is H, or a (poly)phosphate represented by , with n being an integer from 1 to 20, and Z being selected from the group consisting of H, free electron, and a pentose selected from the group consisting of ribose, desoxyribose, arabinose, and methylribose (2-O-methyribose), for example a ribose or a desoxyribose. In one embodiment of the present invention, n is 1 , 2, or 3 (preferably 3), and/or Z is H.

The modified nucleosides as part as described and provided herein may be generally encompass structures comprising any purine or pyrimidine nucleoside and derivatives or analogues thereof. That is, "purine nucleoside" or "pyrimidine nucleoside" (herein also referred to as "Y") as used in context with the modified nucleoside of the present invention generally comprises any kind of purine or pyrimidine as well as derivatives or analogues thereof as described herein (all these purines, pyrimidines, and derivatives or analogues thereof herein also referred to as "B"), respectively, as well as a pentose.

In one aspect of the present invention, the purine nucleoside of the modified nucleoside may be selected from the group consisting of (deoxy)adenosine, inosine, and (deoxy)guanosine and derivatives or analogues thereof. A derivative may be, e.g., a nucleoside with a purine selected from the group consisting of a deazapurine, an azidopurine, an alkylpurine, a thiopurine, a bromopurine, an O-alkylpurine, and an isopurine, for example a deazapurine such as, e.g., 7-deazapurine. That is, in one aspect of the present invention, the purine nucleoside of the modified nucleoside may be a nucleoside with a purine selected from the group consisting of a deazapurine, an azidopurine, an alkylpurine, a thiopurine, a bromopurine, an O-alkylpurine, and an isopurine, for example a deazapurine such as, e.g., 7- deazapurine. In another aspect of the present invention, the purine nucleoside of the modified nucleoside may be selected from the group consisting of 1 - methyl(deoxy)adenosine, 2-methyl-(deoxy)adenosine, N⁶-methyl(deoxy)adenosine, N⁶,N⁶- dimethyl(deoxy)adenosine, 7-deaza(deoxy)adenosine, 7-deaza-8-aza(deoxy)adenosine, 7- deaza-7-bromo(deoxy)adenosine, 7-deaza-7-iodo(deoxy)adenosine, 8- azido(deoxy)adenosine, 8-bromo(deoxy)adenosine, 8-iodo(deoxy)adenosine, 8-bromo-2'- deoxy(deoxy)adenosine, 2'-0-methyladenosin, inosin, 1 -methylinosin, 2'-0-methylinosin, 1 - methyl(deoxy)guanosine, 7-methyl(deoxy)guanosine, N²-methyl(deoxy)guanosine, N²,N² dimethyl-guanosine, isoguanosine, 7-deaza(deoxy)guanosine, 7-deaza-8- aza(deoxy)guanosine, 7-deaza-7-bromo(deoxy)guanosine, 7-deaza-7- iodo(deoxy)guanosine, 6-thio(deoxy)guanosine, 06-methyl(deoxy)guanosine, 8- azido(deoxy)guanosine, 8-bromo(deoxy)guanosine, 8-iodo(deoxy)guanosine, 2'-0- methylguanosine, 8-azidoinosine, 7-azainosine,8-bromoinosine, 8- iodoinosine, 1 - methylinosine, and 4-methylinosine. In a further aspect of the present invention, the purine nucleosides may be selected from the group consisting of a queuosine, an archaeosine, a wyosine and a N⁶-threonylcarbamoyladenosine.

In one aspect of the present invention, the pyrimidine nucleoside of the modified nucleoside may be selected from the group consisting of (deoxy)cytidine, (deoxy)thymidine, (deoxy)ribothymidine, (deoxy)uridine, and derivatives thereof, preferably deoxythymidine. A derivative may be, e.g., a nucleoside with a pyrimidine selected from the group consisting of an alkylpyrimidine, a thiopyrimidine, a bromopyrimidine, an O-alkylpyrimidine, an isopyrimidine, an acetylpyrimidine hydropyrimidine, and a pseudopyrimidine. That is, in one aspect of the present invention, the pyrimidine nucleoside of the modified nucleoside may be a nucleoside with a pyrimidine selected from the group consisting of an alkylpyrimidine, a thiopyrimidine, a bromopyrimidine, an O-alkylpyrimidine, an isopyrimidine, an acetylpyrimidine hydropyrimidine, and a pseudopyrimidine. In another aspect of the present invention, the pyrimidine nucleoside of the modified nucleoside may be selected from the group consisting of 3-methyl-(deoxy)cytidine, N⁴-methyl(deoxy)cytidine, N⁴,N⁴- dimethyl(deoxy)cytidine, iso(deoxy)cytidine, pseudo(deoxy)cytidine, pseudoiso(deoxy)cytidine, 2-thio(deoxy)cytidine, N⁴-acetyl(deoxy)cytidine, 3- methyl(deoxy)uridine, pseudo(deoxy)uridine, 1 -methyl-pseudo(deoxy)uridine, 5,6- dihydro(deoxy)uridine, 2-thio(deoxy)uridine, 4-thio(deoxy)uridine, 5- bromodeoxy(deoxy)uridine, 2'-deoxyuridine, 4-thio(deoxy)thymidine, 5,6- dihydro(deoxy)thymidine, 04-methylthymidine, difluortolune, and other nucleobase surrogates. In a particular aspect of the present invention, the pyrimidine nucleoside is 2'- deoxyuridine.

All purines, pyrimidines, and derivatives and analogies thereof as described and defined herein are collectively also referred to herein as "B".

As mentioned, the nucleosides as part of the modified nucleosides as described and provided herein generally comprise a purine or pyrimidine or derivative or analogue thereof as described herein above (also referred to herein as "B") and below as well as sugar moiety such as, e.g., a pentose. Generally, the pentose as part of the purine or pyrimidine nucleoside or derivative or analogue thereof as described herein above (Y) may be, inter alia, ribose, deoxyribose, arabinose, or methylribose (2-O-methyribose), for example a ribose or a deoxyribose. That is, the nucleoside as part of the modified nucleoside of the present invention may be, e.g., a (ribosyl)nucleoside, a desoxy(ribosyl)nucleoside, an arabinosylnucleoside or an (methylribosyl)nucleoside, for example a (ribosyl)nucleoside or a deoxy(ribosyl)nucleoside. As used herein, the terms "desoxy" and "deoxy" as prefixes of molecule terms are used synonymously and indicate the absence of an oxygen atom or a hydroxyl-group, e.g., in a given pentose such as ribose or others as exemplified and defined herein. In context with the present invention, the linker (herein also referred to as "L") between the purine or pyrimidine nucleoside (herein also referred to as "Y") and the cargo molecule having a volume of 15000 A³ (herein also referred to as "X") or more (or alternatively at least 90 amino acids for cargo X being a protein) serves as a spacer between Y and X. That is, L is generally not limited to a specific structure as long as it is able to serve as a spacer and to avoid steric affections between the DNA or RNA polymerase and X which would affect or diminish the respective abilities of the polymerase and X (e.g., enzymatic activity for X as an enzyme, or binding activity for X as an antibody). In one aspect of the present invention, Y, L and X are linked to each other via covalent bonds.

In one aspect of the present invention, the linker L does not comprise an amino acid. As used herein, the term "amino acid" comprises naturally occurring (peptidogen and non- peptidogen) amino acids as well as artificial (or non-naturally occurring) amino acids. An amino acid as used herein generally comprises an acid group and an amine group and is capable to form amide bonds (peptide bonds) with other amino acids. Examples for natural occurring, peptidogen amino acids comprise Histidine, Isoleucine, Leucine, Methionine, Phenylalanine, Threonine, Tryptophan, Valine, Alanine, Arginine, Asparagine, Aspartic Acid, Cysteine, Glutamic Acid, Glutamine, Glycine, Proline, Selenocysteine, Serine, Tyrosine, Pyrrolysine, and /V-formylmethionine. Examples for non-peptidogen amino acids comprise Carnitine, GABA, Hydroxyproline, Selenomethionine, Ornithine, Citrulline, Pyroglutamic Acid, and others. Artificial amino acids are amino acids that exhibit an acid group and an amine group and are capable of forming peptide bonds (amide bonds), but are not synthesized by living organisms. For example, the linker L as part of the modified nucleoside according to the present invention may comprise 6-aminohexan acid.

The linker L of the modified nucleoside of the present invention is preferably covalently bound to the purine or pyrimidine or derivatives or analogues thereof as defined herein (i.e. linked to B) as part of the modified nucleoside of the present invention. Methods for binding linkers L as described herein to the modified nucleoside of the present invention are known in the art (cf., e.g., Hocek, Org Chem (2014), 79(21 ): 9914-9921 ) and also described and exemplified herein.

A general structure for purines and derivatives or analogues thereof as comprised by the collective term "B" as used herein is shown in formula (1 ) below:

formula (1 )

Formula (1 ) herein serves as indication for specific positions where one or moieties, substitutions or additions may take place for the purines, derivatives or analogues thereof as described herein under the collective term "B". Formula (1 ) must not be construed as limiting the purines, derivatives or analogues thereof as described herein under the collective term "B" to compounds according to formula (1 ). Unsaturated bonds and potential substituents or additional moieties are not shown in formula (1 ). The collective term "B" as used herein also comprises further purine analogues or derivatives according to formula (1 ). For example, for 7-deazapurine, there is no N at position 7 of formula (1 ) but a C (-CH₂-). Also, for example, for guanine, there is an amine group (-NH₂) at position 2 and a keto group (=0) at position 6, while the bond between C2 and Λ/3 as well as the bond between N7 and C8 are unsaturated (H substituents not shown). Preferred positions where the linker L is covalently bound to B are positions 6, 7, and 8, more preferably positions 7 and 8, and is most preferably position 7 of formula (1 ) for modified nucleosides of the present invention where Y is a purine nucleoside or derivative or analogue thereof. In this context, in a certain aspect of the present invention, the linker L may be covalently bound to CI of 7-deazapurine. The sugar moiety (e.g., a pentose such as (desoxy)ribose)) of a purine or pyrimidine nucleoside Y is preferably also covalently bound to B, e.g., via a glycosidic bond (e.g., C- or - preferably - /V-glycosdic bond). For example, the sugar moiety (e.g., a pentose such as (desoxy)ribose)) may be covalently bound to positions 8 or 9, preferably to position 9 of formula (1 ) for modified nucleosides of the present invention where Y is a purine nucleoside or derivative or analogue thereof. A general structure for pyrimidines and derivatives or analogues thereof as comprised by the collective term "B" as used herein is shown in formula (2) below:

formula (2) Formula (2) herein serves as indication for specific positions where one or moieties, substitutions or additions may take place for the pyrimidines, derivatives or analogues thereof as described herein under the collective term "B". Formula (2) must not be construed as limiting the pyrimidines, derivatives or analogues thereof as described herein under the collective term "B" to compounds according to formula (2). Unsaturated bonds and potential substituents or additional moieties are not shown in formula (2). The collective term "B" as used herein also comprises further pyrimidine analogues or derivatives according to formula (2). For example, for thymine, there is a keto group (=0) at positions 2 and 4 and a methyl group (-CH₃) at position 5, while the bond between C5 and C6 is unsaturated (H substituents not shown). A preferred position where linker L is covalently bound to B is position (C)5 of formula (2) for modified nucleosides of the present invention where Y is a pyrimidine nucleoside or derivative or analogue thereof. The sugar moiety (e.g., a pentose such as (desoxy)ribose)) of a purine or pyrimidine nucleoside Y is preferably also covalently bound to B. Preferably, the sugar moiety (e.g., a pentose such as (desoxy)ribose)) may be covalently bound to position 1 of formula (2) for modified nucleosides of the present invention where Y is a pyrimidine nucleoside or derivative or analogue thereof.

Accordingly, in one aspect, the present invention relates to a modified nucleoside as described and provided herein, which comprises a structure represented by formula (III) below

wherein R² is -OH, -H or -0(CH₂)_n-CH₃, with n being an integer from 0 to 20;

wherein R³ is H, or , with n being an integer from 1 to 20 (preferably 1 , 2 or 3; more preferably 3), and Z being selected from the group consisting of H, free electron, and ribose or desoxyribose (preferably H);

wherein B is a purine, a purine derivative, a pyrimidine or a pyrimidine derivative as described herein;

wherein L is a linker as described herein; and wherein X is a cargo having a volume of 15000 A³ or more (or alternatively at least 90 amino acids for cargo X being a protein), and wherein X is preferably an enzyme E.

In a further aspect, the present invention relates to a modified nucleoside as described and provided herein, wherein the purine or the purine nucleoside Y is comprised by a structure represented by formula (IV) below

wherein R⁴ is -H, a free electron pair, or -CH₃;

wherein R⁵ is H, -CH₃, -NH₂, -N(CH₃)₂ or =0;

wherein R⁶ is a free electron pair or H;

wherein R⁷ is -H, -NH₂, -CH₃, -N(CH₃)₂, =0 or L;

wherein R⁸ is a free electron pair or -CH₃ or L;

wherein R⁹ is a linker L as described herein or -H;

wherein R¹⁰ is selected from the group consisting of ribose, ribose-5-phosphate, ribose-5- diphosphate, ribose-5-triphosphate, 2'-0-Methyl-ribose, 2'-0-Methyl-ribose-5-phosphate, 2'- O-Methyl-ribose-5-diphosphate, 2'-0-Methyl-ribose-5-triphosphate, deoxyribose, deoxyribose-5-phosphate, deoxyribose-5-diphosphate, and deoxyribose-5-triphosphate; and wherein at least one L is present in the purine or purine nucleoside.

In a further aspect, the present invention relates to a modified nucleoside as described and provided herein, wherein the purine or purine nucleoside is comprised by a structure represented by formulas (V) or (VI) below

or

wherein R³ is H, or ,with n being an integer from 1 to 20 (e.g., 1 , 2 or 3; preferably 3), and Z being selected from the group consisting of H, free electron, and ribose or a desoxyribose, preferably H;

wherein R⁵ is H, -CH₃, -NH₂, -N(CH₃)₂ or =0; e.g., H or -NH₂;

wherein R⁸ is L or a free electron pair;

wherein R⁹ is a linker L as described herein or -H; and

wherein L is a linker and wherein at least one of R⁸ or R⁹ is L, preferably wherein R⁸ is L.

In a further aspect, the present invention relates to a modified nucleoside as described and provided herein, wherein the purine nucleoside is represented by formula (VII) below

wherein R is H, or , with n being an integer from 1 to 20 (e.g., 1 , 2, or 3; preferably 3), and Z being selected from the group consisting of H, free electron, and ribose or a desoxyribose, preferably H;

wherein R¹¹ is -H, or a heteroatom containing group;

wherein R¹² is -NH₂, or -H; and

wherein L is a linker as described herein.

In this context, the heteroatom containing group may be selected from the group consisting of a nitrogen containing moiety, an oxygen containing moiety, or a halogen containing moiety, wherein the halogen may be selected from the group consisting of a fluorine (F), a chlorine (CI), a bromine (Br), and an iodine (I). In a further aspect, the present invention relates to a modified nucleoside as described and provided herein, wherein the purine nucleoside is represented by formula (VIII) below

wherein R is H or , with n being an integer from 1 to 20 (e.g., 1 , 2, or 3; preferably 3), and Z being selected from the group consisting of H, free electron, and ribose or a desoxyribose (preferably H); and

wherein L is a linker as described herein.

In a further aspect, the present invention relates to a modified nucleoside as described and provided herein, wherein the pyrimidine or pyrimidine nucleoside is represented by formula (IX) below

wherein R¹³ is selected from the group consisting of ribose, ribose-5-phosphate, ribose-5- diphosphate, ribose-5-triphosphate, 2'-0-Methyl-ribose, 2'-0-Methyl-ribose-5-phosphate, 2'- O-Methyl-ribose-5-diphosphate, 2'-0-Methyl-ribose-5-triphosphate, deoxyribose, deoxyribose-5-phosphate, deoxyribose-5-diphosphate, and deoxyribose-5-triphosphate; wherein R¹⁴ is a free electron pair, =0, -NH₂, or =S;

wherein R¹⁵ is a free electron pair, -H or -CH₃;

wherein R¹⁶ is -NH₂, -CH₃, -N(CH₃)₂, =0 or -NH-CO-CH₃; and

wherein R¹⁷ is a linker L as described herein.

In a further aspect, the present invention relates to a modified nucleoside as described and provided herein, wherein the pyrimidine or pyrimidine nucleoside is represented by formula (X) below

wherein R¹³ is selected from the group consisting of ribose, ribose-5-phosphate, ribose-5- diphosphate, ribose-5-triphosphate, 2'-0-Methyl-ribose, 2'-0-Methyl-ribose-5-phosphate, 2'- O-Methyl-ribose-5-diphosphate, 2'-0-Methyl-ribose-5-triphosphate, deoxyribose, deoxyribose-5-phosphate, deoxyribose-5-diphosphate, and deoxyribose-5-triphosphate; and wherein L is a linker as described herein.

In a further aspect, the present invention relates to a modified nucleoside as described and provided herein, wherein the pyrimidine or pyrimidine nucleoside is represented by formula (XI) below

In a further aspect, the present invention relates to a modified nucleoside as described and provided herein, wherein the pyrimidine or pyrimidine nucleoside is represented by formula (XII) below

wherein R¹³ is selected from the group consisting of ribose, 2'-0-Methyl-ribose, ribose-5- phosphate, ribose-5-diphosphate, ribose-5-triphosphate, 2'-0-Methyl-ribose-5-phosphate, 2'- O-Methyl-ribose-5-diphosphate, 2'-0-Methyl-ribose-5-triphosphate, deoxyribose, deoxyribose-5-phosphate, deoxyribose-5-diphosphate, deoxyribose-5-triphosphate and difluortoluene, and other nucleobase surrogates; and

wherein L is a linker as described herein.

In one aspect, the linker L as part of the modified nucleoside Y of the present invention may inter alia comprise a straight or branched hydrocarbon based moiety or a cyclic hydrocarbon based moiety. In one aspect of the present invention, the linker L may particularly comprise one or more heteroatoms. In this context, such heteroatoms may be, e.g., oxygen (O), nitrogen (N), silicium (Si) and/or sulfur (S).

In one aspect, the linker L as part of the modified nucleoside Y of the present invention may inter alia comprise one or more alkyl, cycloalkyl, alkenyl, aryl, hetero aryl and/or alkynyl groups. The linker L may also generally comprise an amide, an amidine, a disulfide, a hydrazine, a thioether and/or an ester. In this context, the amide, amidine, disulfide, hydrazine, thioether and/or ester group may be generated, e.g., by coupling of a reactive chemical group with a target functional group by methods known in the art, e.g., by treatment with activated esters, acid anhydrides, oxidizers, reagents for disulfide exchange, alkyl halides, etc. In accordance with the present invention, such target functional groups may comprise, inter alia, the Enzyme E as described herein where the cargo X is an Enzyme E and the target functional group is coupled to the pyrimidine or purine nucleoside via the linker L. Also in accordance with the present invention, such target functional group may be selected from, e.g., the group consisting of an aldehyde, a carboxylic acid, an amine, a hydroxyl, and a sulfhydryl moiety. In a specific aspect of the present invention, the target functional group is a sulfhydryl moiety. Said reactive chemical group may be selected, e.g., from the group consisting of an amine-, an aryl azide-, a carbodiimide-, a hydrazide-, an imidoester-, an iodoacetyl-, an isocyanate (PMPI)-, a maleimide-, a NHS ester-, a pyridyl disulfide, and a vinyl sulfone- reactive group. In a specific aspect of the present invention, the reactive chemical group is a maleimide-reactive group. In a further specific aspect of the present invention, the maleimide-reactive group may be coupled to the Enzyme E (for Enzyme E as cargo X as described herein) and -SH (for sulfhydryl moiety as functional group as mentioned above) may be coupled to the pyrimidine nucleoside or a purine nucleoside Y. Also in context with the present invention, said reactive chemical group may be comprised in a heterobifunctional or homobifunctional linker. In this context, examples for said heterobifunctional linkers comprise a-Maleimidoacetoxy-succinimide ester (AMAS), N(4- [p-Azidosalicylamido]butyl)- 3'-(2'-pyridyldithio) propionamide (APDP^*), (β- Maleimidopropionic acid)hydrazide^«TFA (BMPH), (3-Maleimidopropyloxy)succinimide ester (BMPS), ε-Maleimidocaproic acid (EMCA), (e-Maleimidocaproyloxy)succinimide ester (EMCS), (Y-Maleimidobutyryloxy)succinimide ester (GMBS), κ-Maleimidoundecanoic acid (KMUA), Succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 -carboxy-(6-amidocaproate) (LC-SMCC), Succinimidyl-6-(3'-[2-pyridyl-dithio]propionamido)hexanoate (LC-SPDP), m- Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), Succinimdyl-3-

(bromoacetamido)propionate (SBAP), Succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), succinimidyl iodoacetate (SIA), Succinimidyl 4-(p-maleimido-phenyl)butyrate (SMPB), NHS- PEG24-Maleimide SM(PEG24), NHS-PEG12-Maliemide (SM[PEG]12), NHS-PEG8- Maliemide (SM[PEG]8), NHS-PEG6-Maleimide (SM(PEG)6), NHS-PEG4-Maliemide (SM[PEG]4), NHS-PEG2-Maliemide (SM[PEG]2), Succinimidyl 4-(N-maleimido- methyl)cyclohexane-carboxylate (SMCC), succinimidyl iodoacetate (SIA), Succinimidyl(4- iodoacetyl)aminobenzoate (SIAB), (e-Maleimidocaproyloxy)sulfosuccinimide ester (Sulfo- EMCS),-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP), Succinimidyl-6-(3- maleimidopropionamido)hexanoate (SMPH), N-(Y-Maleimidobutryloxy)sulfosuccinimide ester (Sulfo-GMBS),-(K-Maleimidoundecanoyloxy)sulfosuccinimide ester (Sulfo-KMUS), Sulfosuccinimidyl 6-(a-methyl-a-[2-pyridyldithio]-toluamido)hexanoate (Sulfo-LC-SMPT), Sulfosuccinimidyl 6-(3'-[2-pyridyl-dithio]propionamido)hexanoate (Sulfo-LC-SPDP), Maleimidobenzoyl-hydroxysulfosuccinimide ester (Sulfo-MBS), Sulfosuccinimidyl(4-iodo- acetyl)aminobenzoate (Sulfo-SIAB), Sulfosuccinimidyl 4-(N-maleimido methyl)cyclohexane- 1 -carboxylate (Sulfo-SMCC), Sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (Sulfo-SMPB) Ν,Ν-Dicyclohexylcarbodiimide (DCC), 1 -Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), and Sulfo-NHS-(2-6-[Biotinamido]-2-(p-azidobezamido) (Sulfo-SBED). Examples for homobifunctional linkers comprise 1 ,4-bis-Maleimidobutan (BMB), Maleimidohexane (BMH), Maleimidoethane (BMOE), 1 ,8-bis-Maleimidodiethylene-glycol (BM(PEG)2), 1 ,1 1 -bis-Maleimidotriethyleneglycol (BM(PEG)3), Dimethyl suberimidate^«2HCI (DMS), Dimethyl 3,3'-dithiobispropionimidate^«2HC (DTBP), Dibenzocyclooctyne-PEG4-N- hydroxysuccinimidyl ester (DBCO-PEG₄-NHS) and (2-Maleimidoethyl)amine (Trifunctional) (TMEA^***).

The linker L of the modified nucleoside of the present invention may also comprise a carbon atom based chain comprising Ci, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C10, Cn, C12, C13, Ci₄, C15, Ci₆, C17, C18 Ci9, C₂o, C21, C22, C23, C₂₄ or C25 or more carbon atoms or a polyether based chain (e.g., a polyethylene glycol based chain with -(0-CH₂-CH₂)- repeating units). In one embodiment, the linker L of the modified nucleoside of the present invention may also comprise a carbon atom based chain comprising C15, Ci₆, Ci₇, Ci₈ C19, C₂o, C21, C22, C23, C_{24 or} C25 or more carbon atoms, preferably a carbon atom chain comprising C₇ or C15 carbon atoms.

The linker L of the modified nucleoside of the present invention may also comprise a thioether group and/or an amide group.

The linker L of the modified nucleoside of the present invention may also comprise ω- mercaptocarboxylic acid-based linkers of different lengths. For example, in context with the present invention, such ω-mercaptocarboxylic acid-based linkers may be represented by the following formula: HS-(CH₂)w-CHO-OH, wherein w is a number between 1 and 20, preferably between 1 and 15, more preferably between 2 and 10, more preferably between 4 and 10, for example between 7 and 8. It is also possible that ω-mercaptocarboxylic acid-based linkers with different lengths (i.e. where m differs between individual ω-mercaptocarboxylic acids) are mixed. In this case, it is preferred that the average number for w is within said ranges. For example, in accordance with the present invention as described herein, the ω- mercaptocarboxylic acid-based linker as shown in Figure 4 herein shows an average length of 7.15, i.e. the average w is between 7.15.

In a specific aspect of the present invention, the linker L of the modified nucleoside provided and described herein is a 5-(8-mercapto octanamido)pent-1-yn-1 -yl or a 5-(16- mercaptohexadecanamido)pent-1-yn-1 -yl.

The cargo X of the modified nucleoside as described and provided herein may be any molecule having a volume of 15,000 A³ or more (preferably at least 18,000, 21 ,000, 24,000, 27,000, 30,000, 33,000, 36,000, 39,000, 42,000, 45,000, 48,000, 51 ,000, 54,000, 57,000, 60,000, 63,000, 66,000, 69,000, 72,000, 75,000, 78,000, 81 ,000, 84,000, 87,000 or 90,000 A³). Generally, according to the present invention, there is no fix upper limit for the volume of cargo X. For example for beads as cargo X, large cargo with high volumes may be envisaged, while the volume for proteins as cargo X the volume may be lower than for beads. However, in one embodiment, there may also be an upper limit for the volume of cargo X. For example, if cargo X is a bead, the upper limit for the volume of cargo X may be 10⁶A³, 10⁷A³, 10⁸A³, 10⁹ A³, 10¹⁰ A³, 10¹¹ A³, 10¹² A³, 10¹³ A³, 10¹⁴ A³, 2,5 x 10¹⁴ A³ or 5.3 x 10¹⁴ A³. For example, if cargo X is a protein, the upper limit for the volume of cargo X may be 1 ,500,000 A³ (or 1 ,150,000, or 760,000, or 380,000, or 285,000, or 230,000, or 210,000, e.g., 76,000 or 66,500 A³. For the avoidance of doubt, the unit A means Angstrom and is equal to 0.1 nm or 10^"10 m.

In one embodiment of the present invention, cargo X of the modified nucleoside provided herein is a protein.

The volume of a given protein can be measured by methods known in the art and as also described herein. Generally, the volume of macromolecules is measured via scanning the surface with a virtual sphere. For example, as a basis, PDB (Protein Data Bank)-files of such macromolecules which provide a standard representation for macromolecular structure data are needed (for PDB-file format see http://www.wwpdb.org/documentation/file-format.php). Such macromolecular structure data needed to generate a PDB-file can be derived from, e.g., X-ray diffraction or NMR studies, or can be obtained from prediction programs where the primary structure of a given protein (i.e. the amino acid sequence) serves as a basis and the program then calculates a putative tertiary structure (i.e. folded protein). For many proteins, PDB-files already exist. Where no PDB-file exists yet, as mentioned, programs can be used to predict a putative tertiary structure (i.e. folded status) of a protein (e.g., Robetta: see http://robetta.bakerlab.org). For generating PDB-files, several programs can be used, e.g., a combination of PyMOL (The PyMOL Molecular Graphics System, Version 1 .7.4, Schrodinger LLC; see http://www.pymol.org) and COOT (Emsley et al., Features and Development of Coot Acta Cryst (2010), D66: 486-501 ). Having a PDB-file of a given protein at hand, the volume of the "folded" protein can be measured using a further calculation program such as, e.g., the 3V Voss Volume Voxelator (http://3vee.molmovdb.org; Voss and Gerstein, Nucleic Acid Res (2010), 38 (Web Server Issue): W555-W562).

The volume of a protein having a certain number of amino acids (or having a given amino acid sequence) can be calculated by methods known in the art and as also as described herein. As it is clear from the calculation methods, the volume of a given protein is primarily dependent on the number of amino acids comprised by said protein. Accordingly, in context with the present invention, alternatively to the indication of a volume of a cargo X, if said cargo X is a protein, a number of amino acids may be indicated to define the cargo X of the modified nucleoside as described and provided herein. In accordance with the present invention, it may be assumed that a protein or polypeptide comprising at least 90 amino acids also exhibits a volume of 15,000 A³ or more. That is, e.g., if the volume of a given protein or polypeptide is not available for some reason, it may be assumed in context with the present invention that if said protein or polypeptide comprises at least 90 amino acids, it exhibits a volume of 15,000 A³ or more. In this context, it may be in accordance with the present invention to indicate the size of a cargo X (being a protein) of the modified nucleoside as described and provided herein not by its volume but by its number of amino acids. Accordingly, for example, if the cargo X of the modified nucleoside as described and provided herein is a protein, said cargo X (being a protein) may comprise at least 90 amino acids, or at least 95 amino acids, at least 100 amino acids, at least 110 amino acids, at least 120 amino acids, at least 130 amino acids, at least 150 amino acids, at least 160 amino acids, at least 180 amino acids, at least 200 amino acids, or at least 250 amino acids, or at least 300 amino acids, or at least 350 amino acids or at least 400 amino acids or at least 450 amino acids, or at least 500 amino acids, or at least 550 amino acids, or at least 600 amino acids. In one embodiment, the upper limit of cargo X (being a protein) of the modified nucleoside as described and provided herein may be a polypeptide length of 8,000 amino acids, preferably of 6,000 amino acids, preferably of 4,000 amino acids, more preferably of 2,000 amino acids, for example 1 ,500 amino acids, 1 ,200 amino acids, or 1 ,100 amino acids. In one embodiment, the upper limit of cargo X (being a protein) of the modified nucleoside as described and provided herein may be a polypeptide length of 400 amino acids or 350 amino acids amino acids.

In another embodiment, the cargo X of the modified nucleoside of the present invention is a bead, e.g., a sepharose bead, polystyrene bead, glass beads, inorganic particle, that are coupled by methodology known in the art e.g., formation of amides, an amidine, a disulfide, a hydrazine, a thioether, dative bond and/or an ester. In context with the present invention, if the cargo X is a protein, in a further embodiment it may be an enzyme E or an antibody A. If the cargo X is an enzyme E, said enzyme E may comprise or consist of an amino acid sequence comprising more than 90, 95, 100, 110, 120, 130, 150, 160, 180, 200, 250, 300, 350, 400, 450, 500, 550, 600, or more amino acids. Also, in context with the present invention, for cargo X being a protein, the protein (e.g., enzyme E) coupled to the purine or pyrimidine nucleoside Y via the linker L as described herein may be, e.g., a reporter protein or a reporter enzyme. A reporter protein as used herein may be a protein which is detectable via colorimetric signals, biochemical reactions and/or by electromagnetical stimulation (e.g., (e)GFP, (e)YFP, RoGFP, HyPer, rxYFP, CFP, or others). A reporter enzyme as used herein may be an enzyme which catalyzes a biochemical reaction which is visible by naked-eye or via technical aid (e.g., colorimetric detection). For example, if cargo X is an enzyme E, particularly a reporter enzyme, the reporter enzyme may be selected from the group consisting of horseradish peroxidase (HRP, e.g., EC 1.1 1.1.7), Intestinal-type alkaline phosphatase (ALPI, e.g., EC 3.1 .3.1 ), alkaline phosphatase (AP, e.g., EC 3.1 .3.1 ), glucose-oxidase (GOX, e.g., EC 1 .1 .3.4), luciferase (e.g., EC 1.13.12.7), chloramphenicol acetyl tansferase (CAT, e.g., EC 2.3.1 .28), β-Galactosidase (β-Gal, e.g., EC 3.2.1 .23), catalase (e.g., EC 1 .1 1.1 .6), urease (e.g., EC 3.5.1.5 and soybean peroxidase, preferably HRP. Such enzymes may be pre-activated as commercially available. For example, in context with the present invention, HRP may be pre-activated with one to three maleimide groups by conversion of the lysine residues with maleimidocaproic acid N- hydroxysuccinimide ester (malHRP). Such species allow the conjugation to a target molecule via thiol-maleimide reaction. Examples for amino acid sequences for said reporter enzymes may be SEQ ID NO: 1 for HRP (Peroxidase C1A; processed protein see amino acid positions 31 to 38 of SEQ I D NO: 1 ), SEQ ID NO: 2 for AP, SEQ ID NO: 3 for GOX, SEQ ID NO: 4 for luciferase, SEQ ID NO: 5 for CAT, SEQ ID NO: 6 for β -Gal, SEQ ID NO: 7 for catalase, SEQ ID NO: 8 for urease, SEQ ID NO: 9 for soybean peroxidase (e.g., EC 1 .1 1 .1.7), SEQ ID NO: 10 for ALPI, or SEQ ID NO: 1 1 for catalase catR. Accordingly, reporter enzymes in context with the present invention also comprise enzymes which have an amino acid sequence being at least 60% or 70% (preferably at least 80%, 85%, 90%, 95%, 97%, 98%, 99%) similar or identical to one of SEQ ID NOs: 1 to 1 1 , while still being biologically active, i.e. exhibiting the same catalytic activities as the respective reporter 1 1 having an amino acid sequence being 100% identical to one of SEQ ID NOs: 1 to 1 1 , respectively. In this context, the term "similar" sequence means that a given amino acid sequence comprises identical amino acids or only conservative or highly conservative substitutions compared to either of SEQ ID NOs: 1 to 1 1 . As used herein, "conservative" substitutions mean substitutions as listed as "Exemplary Substitutions" in Table 1 below. "Highly conservative" substitutions as used herein mean substitutions as shown under the heading "Preferred Substitutions" in Table I below. "Biologically active" as used herein means exhibiting the same catalytic activities as the respective reporter enzymes having an amino acid sequence being 100% identical to one of SEQ ID NOs: 1 to 1 1 , respectively. For assessing whether a given enzyme exhibits the same catalytic activities as the respective reporter enzymes having an amino acid sequence being 100% identical to one of SEQ ID NOs: 1 to 1 1 , respectively, the same respective biochemical reporter tests which are typical for the respective enzyme type are applied to both enzymes (i.e. the enzyme having an amino acid sequence deviating from SEQ ID NOs: 1 to 1 1 , and the enzyme having an identical amino acid sequence to SEQ ID NOs: 1 to 1 1 ). If the results obtained for the enzyme having an amino acid sequence deviating from SEQ ID NOs: 1 to 1 1 does not differ by more than 25% (preferably 20%, more preferably 15%, more preferably 10%, more preferably 8%, and most preferably 5%) from the results obtained for the respective enzyme having an identical amino acid sequence to SEQ ID NOs: 1 to 1 1 , it is considered to be biologically active as used herein.

TABLE I Amino Acid Substitutions

accordance with the present invention, if the cargo X is an antibody A, this means that any recognition molecule may be linked to the purine or pyrimidine nucleoside Y as described or defined herein.

A "recognition molecule" as used herein may be a polypeptide which comprises one or more binding domains capable of binding to a target epitope. A recognition molecule, so to say, provides the scaffold for said one or more binding domains so that said binding domains can bind/interact with a given target structure/antigen/epitope. The term "binding domain" characterizes in connection with the present invention a domain of a polypeptide which specifically binds/interacts with a given target epitope. An "epitope" is antigenic and thus the term epitope is sometimes also referred to herein as "antigenic structure" or "antigenic determinant". Thus, the binding domain is an "antigen-interaction-site". The term "antigen- interaction-site" defines, in accordance with the present invention, a motif of a polypeptide, which is able to specifically interact with a specific antigen or a specific group of antigens, e.g. the identical antigen in different species. Said binding/interaction is also understood to define a "specific recognition".

The term "epitope" also refers to a site on an antigen to which the recognition molecule binds. Preferably, an epitope is a site on a molecule against which a recognition molecule, preferably an antibody will be produced and/or to which an antibody will bind.

For example, an epitope can be recognized by a recognition molecule, particularly preferably by an antibody defining the epitope. A "linear epitope" is an epitope where an amino acid primary sequence comprises the epitope recognized. A linear epitope typically includes at least 3, and more usually, at least 5, for example, about 8 to about 10 amino acids in a unique sequence.

A "conformational epitope", in contrast to a linear epitope, is an epitope wherein the primary sequence of the amino acids comprising the epitope is not the sole defining component of the epitope recognized (e.g., an epitope wherein the primary sequence of amino acids is not necessarily recognized by the antibody defining the epitope). Typically a conformational epitope comprises an increased number of amino acids relative to a linear epitope. With regard to recognition of conformational epitopes, the recognition molecule recognizes a 3- dimensional structure of the antigen, preferably a peptide or protein or fragment thereof. For example, when a protein molecule folds to form a three dimensional structure, certain amino acids and/or the polypeptide backbone forming the conformational epitope become juxtaposed enabling the antibody to recognize the epitope. Methods of determining conformation of epitopes include but are not limited to, for example, x-ray crystallography 2- dimensional nuclear magnetic resonance spectroscopy and site-directed spin labelling and electron paramagnetic resonance spectroscopy. The term "specifically recognizing" (equally used herein with "specifically binding", "directed to" or "reacting with") means in accordance with this invention that the recognition molecule is capable of specifically interacting with and/or binding to at least two, preferably at least three, more preferably at least four amino acids of an epitope as defined herein. Such binding may be exemplified by the specificity of a "lock-and-key-principle".

Thus, the term "specifically" in this context means that the recognition molecule binds to a given target epitope but does not essentially bind to another protein. The term "another protein" includes any protein including proteins closely related to or being homologous to the epitope against which the recognition molecule is directed to. However, the term "another protein" does not include that the recognition molecule cross-reacts with the epitope from a species different from that against which the recognition molecule was generated.

The term "cross-species recognition" or "interspecies specificity" as used herein means binding of a binding domain described herein to the same target molecule in humans and non-human species. Thus, "cross-species specificity" or "interspecies specificity" is to be understood as an interspecies reactivity to the same epitope expressed in different species, but not to another molecule other than X.

The term "does not essentially bind" as used herein means that the epitope recognition molecule of the present invention does not bind another protein, i.e. shows a cross-reactivity of less than 30%, preferably 20%, more preferably 10%, particularly preferably less than 9, 8, 7, 6 or 5% with another protein.

Specific binding is believed to be effected by specific motifs in the amino acid sequence of the binding domain and the antigen bind to each other as a result of their primary, secondary or tertiary structure as well as the result of secondary modifications of said structure. The specific interaction of the antigen-interaction-site with its specific antigen may result as well in a simple binding of said site to the antigen. Moreover, the specific interaction of the antigen- interaction-site with its specific antigen may alternatively result in the initiation of a signal, e.g. due to the induction of a change of the conformation of the antigen, an oligomerization of the antigen, etc. A preferred example of a binding domain in line with the present invention is an antibody.

Typically, binding is considered "specific" when the binding affinity is higher than 10^"6M. Preferably, binding is considered specific when binding affinity is about 10^"11 to 10^"8 M (K_D), preferably of about 10^"11 to 10^"9 M. If necessary, nonspecific binding can be reduced without substantially affecting specific binding by varying the binding conditions. Whether the recognition molecule specifically reacts as defined herein above can easily be tested, inter alia, by comparing the reaction of said recognition molecule with an epitope with the reaction of said recognition molecule with (an) other protein(s). The term "polypeptide" is equally used herein with the term "protein". Proteins (including fragments thereof, preferably biologically active fragments, and peptides, usually having less than 30 amino acids) comprise one or more amino acids coupled to each other via a covalent peptide bond (resulting in a chain of amino acids). The term "polypeptide" as used herein describes a group of molecules which typically comprise more than 15 amino acids. Polypeptides may further form multimers such as dimers, trimers and higher oligomers, i.e. consisting of more than one polypeptide molecule. Polypeptide molecules forming such dimers, trimers etc. may be identical or non-identical. The corresponding higher order structures of such multimers are, consequently, termed homo- or heterodimers, homo- or heterotrimers etc. An example for a heteromultimer is an antibody molecule, which, in its naturally occurring form, consists of two identical light polypeptide chains and two identical heavy polypeptide chains. The terms "polypeptide" and "protein" also refer to naturally modified polypeptides/proteins wherein the modification is effected e.g. by post-translational modifications like glycosylation, acetylation, phosphorylation and the like. Such modifications are well known in the art.

A preferred recognition molecule to be employed in context with the present invention is an antibody. An "antibody" when used herein is a protein comprising one or more polypeptides (comprising one or more binding domains, preferably antigen binding domains) substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein.

The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes.

In particular, an "antibody" when used herein, is typically tetrameric glycosylated proteins composed of two light (L) chains of approximately 25 kDa each and two heavy (H) chains of approximately 50 kDa each. Two types of light chain, termed lambda and kappa, may be found in antibodies. Depending on the amino acid sequence of the constant domain of heavy chains, immunoglobulins can be assigned to five major classes: A, D, E, G, and M, and several of these may be further divided into subclasses (isotypes), e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 , and lgA2. An IgM antibody consists of 5 of the basic heterotetramer unit along with an additional polypeptide called a J chain, and contains 10 antigen binding sites, while IgA antibodies comprise from 2-5 of the basic 4-chain units which can polymerize to form polyvalent assemblages in combination with the J chain. In the case of IgGs, the 4-chain unit is generally about 150,000 daltons. Binding of linker L to an antibody A in accordance with the present invention may be performed by methods known in the art; see, e.g., Drake et al., Curr Opin Chem Biol (2015), 28: 174-180. Each light chain includes an N-terminal variable (V) domain (VL) and a constant (C) domain (CL). Each heavy chain includes an N-terminal V domain (VH), three or four C domains (CHs), and a hinge region. The constant domains are not involved directly in binding an antibody to an antigen. The pairing of a VH and VL together forms a single antigen-binding site. The CH domain most proximal to VH is designated as CH 1. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. The VH and VL domains consist of four regions of relatively conserved sequences called framework regions (FR1 , FR2, FR3, and FR4), which form a scaffold for three regions of hypervariable sequences (complementarity determining regions, CDRs). The CDRs contain most of the residues responsible for specific interactions of the antibody with the antigen. CDRs are referred to as CDR 1 , CDR2, and CDR3. Accordingly, CDR constituents on the heavy chain are referred to as H1 , H2, and H3, while CDR constituents on the light chain are referred to as L1 , L2, and L3.

The term "variable" refers to the portions of the immunoglobulin domains that exhibit variability in their sequence and that are involved in determining the specificity and binding affinity of a particular antibody (i.e. the "variable domain(s)"). Variability is not evenly distributed throughout the variable domains of antibodies; it is concentrated in sub-domains of each of the heavy and light chain variable regions. These sub-domains are called "hypervariable" regions or "complementarity determining regions" (CDRs).

The more conserved (i.e. non-hypervariable) portions of the variable domains are called the "framework" regions (FRM). The variable domains of naturally occurring heavy and light chains each comprise four FRM regions, largely adopting a β- sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the β -sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRM and, with the hypervariable regions from the other chain, contribute to the formation of the antigen- binding site (after Kabat et al.). The constant domains are not directly involved in antigen binding, but exhibit various effector functions, such as, for example, antibody- dependent, cell-mediated cytotoxicity and complement activation.

The terms "CDR", and its plural "CDRs", refer to a complementarity determining region (CDR) of which three make up the binding character of a light chain variable region (CDRL1 , CDRL2 and CDRL3) and three make up the binding character of a heavy chain variable region (CDRH1 , CDRH2 and CDRH3). CDRs contribute to the functional activity of an antibody molecule and are separated by amino acid sequences that comprise scaffolding or framework regions. The exact definitional CDR boundaries and lengths are subject to different classification and numbering systems. CDRs may therefore be referred to by Kabat, Chothia, contact or any other boundary definitions, including the numbering system described herein. Despite differing boundaries, each of these systems has some degree of overlap in what constitutes the so called "hypervariable regions" within the variable sequences. CDR definitions according to these systems may therefore differ in length and boundary areas with respect to the adjacent framework region. See for example Kabat, Chothia, and/or MacCallum (Kabat et al., loc. cit; Chothia et al., J Mol Biol (1987), 196: 901 ; and MacCallum et al., J Mol Biol (1996), 262: 732). However, the numbering in accordance with the so-called Kabat system is preferred. The term "amino acid" or "amino acid residue" as used herein typically refers to an amino acid having its art recognized definition such as an amino acid selected from the group consisting of: alanine (Ala or A); arginine (Arg or R); asparagine (Asn or N); aspartic acid (Asp or D); cysteine (Cys or C); glutamine (Gin or Q); glutamic acid (Glu or E); glycine (Gly or G); histidine (His or H); isoleucine (He or I): leucine (Leu or L); lysine (Lys or K); methionine (Met or M); phenylalanine (Phe or F); pro line (Pro or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp or W); tyrosine (Tyr or Y); and valine (Val or V), although modified, synthetic, or rare amino acids may be used as desired. Generally, amino acids can be grouped as having a nonpolar side chain (e.g., Ala, Cys, He, Leu, Met, Phe, Pro, Val); a negatively charged side chain (e.g., Asp, Glu); a positively charged sidechain (e.g., Arg, His, Lys); or an uncharged polar side chain (e.g., Asn, Cys, Gin, Gly, His, Met, Phe, Ser, Thr, Trp, and Tyr).

The term "hypervariable region" (also known as "complementarity determining regions" or CDRs) when used herein refers to the amino acid residues of an antibody which are (usually three or four short regions of extreme sequence variability) within the V-region domain of an immunoglobulin which form the antigen-binding site and are the main determinants of antigen specificity. There are at least two methods for identifying the CDR residues: (1 ) An approach based on cross-species sequence variability (i.e. Kabat et al., loc. cit); and (2) An approach based on crystallographic studies of antigen-antibody complexes (Chothia, C. et al., J Mol Biol (1987), 196: 901 -917). However, to the extent that two residue identification techniques define regions of overlapping, but not identical regions, they can be combined to define a hybrid CDR. However, in general, the CDR residues are preferably identified in accordance with the so-called Kabat (numbering) system.

The term "framework region" refers to the art-recognized portions of an antibody variable region that exist between the more divergent (i.e. hypervariable) CDRs. Such framework regions are typically referred to as frameworks 1 through 4 (FR1 , FR2, FR3, and FR4) and provide a scaffold for the presentation of the six CDRs (three from the heavy chain and three from the light chain) in three dimensional space, to form an antigen-binding surface. The term "canonical structure" refers to the main chain conformation that is adopted by the antigen binding (CDR) loops. From comparative structural studies, it has been found that five of the six antigen binding loops have only a limited repertoire of available conformations. Each canonical structure can be characterized by the torsion angles of the polypeptide backbone. Correspondent loops between antibodies may, therefore, have very similar three dimensional structures, despite high amino acid sequence variability in most parts of the loops (Chothia and Lesk, J Mol Biol (1987), 196: 901 ; Chothia et al., Nature (1989), 342: 877; Martin and Thornton, J Mol Biol (1996), 263: 800, each of which is incorporated by reference in its entirety). Furthermore, there is a relationship between the adopted loop structure and the amino acid sequences surrounding it. The conformation of a particular canonical class is determined by the length of the loop and the amino acid residues residing at key positions within the loop, as well as within the conserved framework (i.e. outside of the loop). Assignment to a particular canonical class can therefore be made based on the presence of these key amino acid residues. The term "canonical structure" may also include considerations as to the linear sequence of the antibody, for example, as catalogued by Kabat (Kabat et al., loc. cit). The Kabat numbering scheme (system) is a widely adopted standard for numbering the amino acid residues of an antibody variable domain in a consistent manner and is the preferred scheme applied in the present invention as also mentioned elsewhere herein. Additional structural considerations can also be used to determine the canonical structure of an antibody. For example, those differences not fully reflected by Kabat numbering can be described by the numbering system of Chothia et al and/or revealed by other techniques, for example, crystallography and two or three- dimensional computational modeling. Accordingly, a given antibody sequence may be placed into a canonical class which allows for, among other things, identifying appropriate chassis sequences (e.g., based on a desire to include a variety of canonical structures in a library). Kabat numbering of antibody amino acid sequences and structural considerations as described by Chothia and their implications for construing canonical aspects of antibody structure, are described in the literature. CDR3 is typically the greatest source of molecular diversity within the antibody-binding site. H3, for example, can be as short as two amino acid residues or greater than 26 amino acids. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known in the art. For a review of the antibody structure, see Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, eds. Harlow et al., 1988. One of skill in the art will recognize that each subunit structure, e.g., a CH, VH, CL, VL, CDR, FR structure, comprises active fragments, e.g., the portion of the VH, VL, or CDR subunit the binds to the antigen, i.e., the antigen-binding fragment, or, e.g., the portion of the CH subunit that binds to and/or activates, e.g., an Fc receptor and/or complement. The CDRs typically refer to the Kabat CDRs, as described in Sequences of Proteins of immunological Interest, US Department of Health and Human Services (1991 ), eds. Kabat et al. Another standard for characterizing the antigen binding site is to refer to the hypervariable loops as described by Chothia. See, e.g., Chothia et al., J Mol Biol (1992), 227:799-817; and Tomlinson et al., EMBO J (1995), 14: 4628-4638. Still another standard is the AbM definition used by Oxford Molecular's AbM antibody modelling software. See, generally, e.g., Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg). Embodiments described with respect to Kabat CDRs can alternatively be implemented using similar described relationships with respect to Chothia hypervariable loops or to the AbM-defined loops.

The sequence of antibody genes after assembly and somatic mutation is highly varied, and these varied genes are estimated to encode 10¹⁰ different antibody molecules (Immunoglobulin Genes, 2nd ed., eds. Jonio et al., Academic Press, San Diego, CA, 1995). Accordingly, the immune system provides a repertoire of immunoglobulins. The term "repertoire" refers to at least one nucleotide sequence derived wholly or partially from at least one sequence encoding at least one immunoglobulin. The sequence(s) may be generated by rearrangement in vivo of the V, D, and J segments of heavy chains, and the V and J segments of light chains. Alternatively, the sequence(s) can be generated from a cell in response to which rearrangement occurs, e.g., in vitro stimulation. Alternatively, part or all of the sequence(s) may be obtained by DNA splicing, nucleotide synthesis, mutagenesis, and other methods, see, e.g., U.S. Patent 5,565,332. A repertoire may include only one sequence or may include a plurality of sequences, including ones in a genetically diverse collection.

When used herein the term "antibody" does not only refer to an immunoglobulin (or intact antibody), but also to a fragment thereof, and encompasses any polypeptide comprising an antigen-binding fragment or an antigen-binding domain. Preferably, the fragment such as Fab, F(ab')₂, Fv, scFv, Fd, dAb, and other antibody fragments that retain antigen-binding function. Typically, such fragments would comprise an antigen-binding domain and have the same properties as the antibodies described herein.

The term "antibody" as used herein includes antibodies that compete for binding to the same epitope as the epitope bound by the antibodies of the present invention, preferably obtainable by the methods for the generation of an antibody as described herein elsewhere. To determine if a test antibody can compete for binding to the same epitope, a cross- blocking assay e.g., a competitive ELISA assay can be performed. In an exemplary competitive ELISA assay, epitope-coated wells of a microtiter plate, or epitope-coated sepharose beads, are pre-incubated with or without candidate competing antibody and then a biotin-labeled antibody of the invention is added. The amount of labeled antibody bound to the epitope in the wells or on the beads is measured using avidin-peroxidase conjugate and appropriate substrate.

Alternatively, the antibody can be labeled, e.g., with a radioactive or fluorescent label or some other detectable and measurable label. The amount of labeled antibody that binds to the antigen will have an inverse correlation to the ability of the candidate competing antibody (test antibody) to compete for binding to the same epitope on the antigen, i.e. the greater the affinity of the test antibody for the same epitope, the less labeled antibody will be bound to the antigen-coated wells. A candidate competing antibody is considered an antibody that binds substantially to the same epitope or that competes for binding to the same epitope as an antibody of the invention if the candidate competing antibody can block binding of the antibody by at least 20%, preferably by at least 20-50%, even more preferably, by at least 50% as compared to a control performed in parallel in the absence of the candidate competing antibody (but may be in the presence of a known noncompeting antibody). It will be understood that variations of this assay can be performed to arrive at the same quantitative value.

The term "antibody" also includes but is not limited to polyclonal, monoclonal, monospecific, polyspecific such as bispecific, non-specific, humanized, human, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitro generated antibodies, with a polyclonal antibody being preferred. Said term also includes domain antibodies (dAbs) and nanobodies. Accordingly, the term "antibody" also relates to a purified serum, i.e. a purified polyclonal serum. Accordingly, said term preferably relates to a serum, more preferably a polyclonal serum and most preferably to a purified (polyclonal) serum. The antibody/serum is obtainable, and preferably obtained, for example, by the method or use described herein and illustrated in the appended Examples.

"Polyclonal antibodies" or "polyclonal antisera" refer to immune serum containing a mixture of antibodies specific for one (monovalent or specific antisera) or more (polyvalent antisera) antigens which may be prepared from the blood of animals immunized with the antigen or antigens.

Furthermore, the term "antibody" as employed in the invention also relates to derivatives or variants of the antibodies described herein which display the same specificity as the described antibodies. Examples of "antibody variants" include humanized variants of non- human antibodies, "affinity matured" antibodies (see, e.g., Hawkins et al., J Mol Biol (1992), 254, 889-896; and Lowman et al., Biochemistry (1991 ), 30: 10832- 10837) and antibody mutants with altered effector function (s) (see, e.g., US Patent 5, 648, 260). The terms "antigen-binding domain", "antigen-binding fragment" and "antibody binding region" when used herein refer to a part of an antibody molecule that comprises amino acids responsible for the specific binding between antibody and antigen. The part of the antigen that is specifically recognized and bound by the antibody is referred to as the "epitope" as described herein above. As mentioned above, an antigen-binding domain may typically comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH); however, it does not have to comprise both. Fd fragments, for example, have two VH regions and often retain some antigen-binding function of the intact antigen-binding domain. Examples of antigen-binding fragments of an antibody include (1 ) a Fab fragment, a monovalent fragment having the VL, VH, CL and CH1 domains; (2) a F(ab')2 fragment, a bivalent fragment having two Fab fragments linked by a disulfide bridge at the hinge region; (3) a Fd fragment having the two VH and CH 1 domains; (4) a Fv fragment having the VL and VH domains of a single arm of an antibody, (5) a dAb fragment (Ward et al., (1989) Nature 341 :544-546), which has a VH domain; (6) an isolated complementarity determining region (CDR), and (7) a single chain Fv (scFv). Although the two domains of the Fv fragment, VL and VH> are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al., (1988) Science (1988), 242: 423-426; and Huston ef a/., (1988) PNAS USA (1988), 85: 5879-5883). These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are evaluated for function in the same manner as are intact antibodies.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post- translation modifications (e.g., isomerizations, amidations) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature (1975), 256: 495, or may be made by recombinant DNA methods (see, e.g., U. S. Patent No. 4,816, 567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature (1991 ), 352: 624- 628; and Marks et al., J Mol Biol (1991 ), 222: 581 -597, for example. The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain (s) is (are) identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U. S. Patent No. 4,816, 567; Morrison et al., PNAS USA (1984), 81 : 6851 -6855). Chimeric antibodies of interest herein include "primitized" antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape etc.) and human constant region sequences. "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F (ab') 2 or other antigen-binding subsequences of antibodies) of mostly human sequences, which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (also CDR) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, "humanized antibodies" as used herein may also comprise residues which are found neither in the recipient antibody nor the donor antibody. These modifications are made to further refine and optimize antibody performance. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature (1986), 321 : 522-525; Reichmann et al., Nature (1988), 332: 323-329; and Presta, Curr. Op. Struct Biol (1992), 2: 593-596.

The term "human antibody" includes antibodies having variable and constant regions corresponding substantially to human germline immunoglobulin sequences known in the art, including, for example, those described by Kabat et al. (See Kabat et al., loc. cit). The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs, and in particular, CDR3. The human antibody can have at least one, two, three, four, five, or more positions replaced with an amino acid residue that is not encoded by the human germline immunoglobulin sequence.

As used herein, "in vitro generated antibody" refers to an antibody where all or part of the variable region (e.g., at least one CDR) is generated in a non-immune cell selection (e.g., an in vitro phage display, protein chip or any other method in which candidate sequences can be tested for their ability to bind to an antigen). This term thus preferably excludes sequences generated by genomic rearrangement in an immune cell.

A "bispecific" or "bifunctional antibody" is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin Exp Immunol (1990), 79: 315-321 ; Kostelny et al., J Immunol (1992), 148: 1547-1553. In one embodiment, the bispecific antibody comprises a first binding domain polypeptide, such as a Fab' fragment, linked via an immunoglobulin constant region to a second binding domain polypeptide. Numerous methods known to those skilled in the art are available for obtaining antibodies or antigen-binding fragments thereof. For example, antibodies can be produced using recombinant DNA methods (U.S. Patent 4,816,567). Monoclonal antibodies may also be produced by generation of hybridomas (see e.g., Kohler and Milstein, Nature (1975), 256: 495-499) in accordance with known methods. Hybridomas formed in this manner are then screened using standard methods, such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (BIACORE™) analysis, to identify one or more hybridomas that produce an antibody that specifically binds with a specified antigen. Any form of the specified antigen may be used as the immunogen, e.g., recombinant antigen, naturally occurring forms, any variants or fragments thereof, as well as antigenic peptide thereof.

One exemplary method of making antibodies includes screening protein expression libraries, e.g., phage or ribosome display libraries. Phage display is described, for example, in U.S. Patent No. 5,223,409; Smith, Science (1985), 228: 1315-1317; Clackson et al., Nature (1991 ), 352: 624-628; Marks et al., J Mol Biol (1991 ), 222: 581 -597WO 92/18619; WO 91/17271 ; WO 92/20791 ; WO 92/15679; WO 93/01288; WO 92/01047; WO 92/09690; and WO 90/02809.

In another embodiment, a monoclonal antibody is obtained from the non-human animal, and then modified, e.g., humanized, deimmunized, chimeric, may be produced using recombinant DNA techniques known in the art. A variety of approaches for making chimeric antibodies have been described. See, e.g., Morrison et al., PNAS USA (1985), 81 : 6851 ; Takeda et al., Nature (1985), 314: 452; U.S. Patent No. 4,816,567; U.S. Patent No. 4,816,397; EP 171496; EP 173494, GB 2177096. Humanized antibodies may also be produced, for example, using transgenic mice that express human heavy and light chain genes, but are incapable of expressing the endogenous mouse immunoglobulin heavy and light chain genes. Winter describes an exemplary CDR-grafting method that may be used to prepare the humanized antibodies described herein (U.S. Patent No. 5,225,539). All of the CDRs of a particular human antibody may be replaced with at least a portion of a non-human CDR, or only some of the CDRs may be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for binding of the humanized antibody to a predetermined antigen. Humanized antibodies or fragments thereof can be generated by replacing sequences of the Fv variable domain that are not directly involved in antigen binding with equivalent sequences from human Fv variable domains. Exemplary methods for generating humanized antibodies or fragments thereof are provided by Morrison, Science(1985), 229: 1202-1207; Oi et al., BioTechniques (1986), 4: 214; US 5,585,089; US 5,693,761 ; US 5,693,762; US 5,859,205; and US 6,407,213. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable domains from at least one of a heavy or light chain. Such nucleic acids may be obtained from a hybridoma producing an antibody against a predetermined target, as described above, as well as from other sources. The recombinant DNA encoding the humanized antibody molecule can then be cloned into an appropriate expression vector.

In certain embodiments, a humanized antibody is optimized by the introduction of conservative substitutions, consensus sequence substitutions, germline substitutions and/or backmutations. Such altered immunoglobulin molecules can be made by any of several techniques known in the art, (e.g., Teng et al., PNAS USA (1983), 80: 7308-731 ; Kozbor et al., Immunology Today (1983), 4: 7279; Olsson et al., Meth Enzymol (1982), 92: 3-16), and may be made according to the teachings of WO 92/06193 or EP 239400).

Techniques for the production of antibodies, including polyclonal, monoclonal, humanized, bispecific and heteroconjugate antibodies follow.

1 ) Polyclonal antibodies.

Polyclonal antibodies may be raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, using a bifunctional or derivatizing agent, e.g., maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysien residues), glutaraldehyde, succinic anhydride. Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.

For example, the animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, the animals are boosted with 1/5 to 1/10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to fourteen days later, the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitable used to enhance the immune response.

The term "immunizing" refers to the step or steps of administering one or more antigens to a non-human animal so that antibodies can be raised in the animal.

The terms "antigen "and "immunogen" are used interchangeably herein to refer to a molecule or substance which induces an immune response (preferably an antibody response) in an animal, preferably a non-human animal immunized therewith (i.e. the antigen is "immunogenic" in the animal).

Preferably, the antigen used for immunizing a non-human animal is a purified antigen. A "purified" antigen is one which has been subjected to one or more purification procedures. The purified antigen may be "homogeneous", which is used herein to refer to a composition comprising at least about 70% to about 100% by weight of the antigen of interest, based on total weight of the composition, preferably at least about 80% to about 100% by weight of the antigen of interest.

Generally, immunizing comprises injecting the antigen or antigens into the non-human animal. Immunization may involve one or more administrations of the antigen or antigens.

Specifically, the non-human animal is preferably immunized at least two, more preferably three times with said polypeptide (antigen), optionally in admixture with an adjuvant. An "adjuvant" is a nonspecific stimulant of the immune response.

The adjuvant may be in the form of a composition comprising either or both of the following components: (a) a substance designed to form a deposit protecting the antigen (s) from rapid catabolism (e.g. mineral oil, alum, aluminium hydroxide, liposome or surfactant (e.g. pluronic polyol) and (b) a substance that nonspecifically stimulates the immune response of the immunized host animal (e.g. by increasing lymphokine levels therein).

Exemplary molecules for increasing lymphokine levels include lipopolysaccaride (LPS) or a Lipid A portion thereof; Bordetalla pertussis; pertussis toxin; Mycobacterium tuberculosis; and muramyl dipeptide (MDP). Examples of adjuvants include Freund's adjuvant (optionally comprising killed M. tuberculosis; complete Freund's adjuvant); aluminium hydroxide adjuvant; and monophosphoryl Lipid A-synthetic trehalose dicorynomylcolate (MPL-TDM). The "non-human animal" to be immunized herein is preferably a rodent. A "rodent" is an animal belonging to the rodentia order of placental mammals. Exemplary rodents include mice, rats, guinea pigs, squirrels, hamsters, ferrets etc, with mice being the preferred rodent for immunizing according to the method herein.

Other non-human animals which can be immunized herein include non-human primates such as Old World monkey (e.g. baboon or macaque, including Rhesus monkey and cynomolgus monkey ; see US Patent 5, 658, 570) ; birds (e.g. chickens); rabbits; goats; sheep; cows; horses; pigs; donkeys; dogs etc).

2) Monoclonal antibodies.

Monoclonal antibodies can be obtained from a population of substantially homogeneous antibodies, i.e. the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translational modifications (e.g., isomerizations, amidations) that may be present in minor amounts. Thus, the modifier "monoclonal"indicates the character of the antibody as not being a mixture of discrete antibodies.

For example, the monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature (1975), 256: 495, or may be made by recombinant DNA methods (U. S. Patent No. 4,816, 567).

In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized as hereinabove described to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization.

Alternatively, lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986).

The immunizing agent will typically include the antigenic protein or a fusion variant thereof. Generally either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphoctyes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell. Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103. Immortalized cell lines are usually transformed mammalian cell, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells. Preferred immortalized myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, preferred are murine myeloma lines, such as those derived from MOPC-21 and MPC-1 1 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California USA, and SP-2 cells (and derivatives thereof, e.g. , X63-Ag8-653) available from the American Type Culture Collection, Manassus, Virginia USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J Immunol (1984), 133: 3001 ; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, 51 - 63 (Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).

The culture medium in which the hybridoma cells are cultured can be assayed for the presence of monoclonal antibodies directed again desired antigen. Preferably, the binding affinity and specificity of the monoclonal antibody can be determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked assay (ELISA). Such techniques and assays are known in the in art. For example, binding affinity may be determined by the Scatchard analysis of Munson et al., Anal Biochem, (1980), 107: 220. After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in a mammal.

The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

Monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U. S. Patent No. 4,816, 567, and as described above. DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, in order to synthesize monoclonal antibodies in such recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr Opinion in Immunol (1993), 5: 256-262; and Pluckthun, Immunol Revs (1992), 130: 151 -188.

In a further embodiment, antibodies can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature (1990), 348: 552-554. Clackson et al., Nature (1991 ), 352: 624-628 and Marks et al., J Mol Biol (1991 ), 222: 581 -597 describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al., Biotechnology (1992), 10: 779-783), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al., Nucl. Acids Res., 21 : 2265-2266 (1993)). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies.

The DNA also may be modified, for example, by substituting the coding sequence for human heavy-and light-chain constant domains in place of the homologous murine sequences (U. S. Patent No. 4,816, 567; Morrison, et al., Proc. Natl Acad. Sci. USA, 81 : 6851 (1984)), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Typically such non-immunoglobulin polypeptides are substituted for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigen- combining site having specificity for a different antigen.

The monoclonal antibodies described herein may by monovalent, the preparation of which is well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and a modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues may be substituted with another amino acid residue or are deleted so as to prevent crosslinking. In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly Fab fragments, can be accomplished using routine techniques known in the art. Chimeric or hybrid antibodies also may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins may be constructed using a disulfide-exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.

3) Humanized antibodies

The antibodies of the invention may further comprise humanized or human antibodies. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F (ab') 2 or other antigen- binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.

Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domain, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Jones et al., Nature 321 : 522-525 (1986); Riechmann et al., Nature 332: 323-329 (1988) and Presta, Curr. Opin. Struct. Biol. 2 : 593-596 (1992).

Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be essentially performed following the method of Winter and co-workers, Jones et al., Nature 321 : 522-525 (1986); Riechmann et al., Nature 332: 323-327 (1988); Verhoeyen et al., Science 239: 1534-1536 (1988), or through substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U. S. Patent No. 4, 816, 567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity. According to the so-called "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody. Sims et al., J. Immunol.) 151 : 2296 (1993); Chothia et al., J. Mol. Biol., 196: 901 (1987). Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies. Carter et al., Proc. Natl. Acad. Sci. USA, 89 : 4285 (1992); Presta et al., J. Immunol., 151 : 2623 (1993).

It is further important that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e. the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen (s), is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.

Various forms of the humanized antibody are contemplated. For example, the humanized antibody may be an antibody fragment, such as an Fab, which is optionally conjugated with one or more cytotoxic agent (s) in order to generate an immunoconjugate.

Alternatively, the humanized antibody may be an intact antibody, such as an intact IgGI antibody.

4) Human antibodies

As an alternative to humanization, human antibodies can be generated. For example, it is now possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ- line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255-258 (1993); Bruggermann et al., Year in Immun., 7: 33 (1993); U. S. Patent Nos. 5,591 , 669 and WO 97/17852.

Alternatively, phage display technology can be used to produce human antiobdies and antibody fragments in vitro, from immunoglublin variable (V) domain gene repertoires from unimmunized donors. McCafferty et al., Nature 348: 552-553 (1990); Hoogenboom and Winter, J. Mol. Biol. 227: 381 (1991 ). According to this technique, antibody V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in seletion of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of the B-cell. Phage display can be performed in a variety of formats, reviewed in, e.g., Johnson, Kevin S. and Chiswell, David J., Curr. Opin Struct. Biol. 3: 564-571 (1993). Several sources of V-gene segments can be used for phage display. Clackson et al., Nature 352: 624-628 (1991 ) isolated a diverse array of anti- oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. A repertoire of V genes from unimmunized hman donors can be constructed and antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially following the technqieus described by Marks et al. , J. Mol. Biol. 222: 581 -597 (1991 ), or Griffith et al. , EMBO J. 12 : 725-734 (1993). See also, U. S. Patent. Nos. 5,565,332 and 5,573,905.

The techniques of Cole et al., and Boerner et al., are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol. 147 (1 ) : 86-95 (1991 ). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resemble that seen in human in all respects, including gene rearrangement, assembly and antibody repertoire. This approach is described, for example, in U. S. Patent Nos. 5,545, 807; 5,545, 806,5, 569,825, 5,625, 126,5, 633,425, 5,661 , 016 and in the following scientific publications: Marks et al., Bio/Technology 10: 779-783 (1992); Lonberg et al., Nature 368 : 856-859 (1994); Morrison, Nature 368: 812-13 (1994), Fishwild et al., Nature Biotechnology 14 : 845- 51 (1996), Neuberger, Nature Biotechnology 14: 826 (1996) and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995). Finally, human antibodies may also be generated in vitro by activated B cells (see U. S. Patent Nos 5,567, 610 and 5,229, 275).

5) Antibody Fragments

Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., J Biochem Biophys. Method. 24: 107-1 17 (1992); and Brennan et al., Science 229: 81 (1985)). However, these fragments can now be produced direclty by recombinant host cells. Fab, Fv and scFv antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of these fragments.

Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F (ab') 2 fragments (Carter et al_, BiolTechnology 10 : 163-167 (1992)).

According to another approach, F (ab') 2 fragments can be isolated directly from recombinant host cell culture. Fab and F (ab') 2 with increase in vivo half-life is described in U. S. Patent No. 5,869, 046. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv); see WO 93/16185; U. S. Patent No. 5,571 , 894 and U. S. Patent No. 5,587, 458. The antibody fragment may also be a"linear antibody", e.g., as described in U. S. Patent 5,641 , 870. Such linear antibody fragments may be monospecific or bispecific.

The present invention further relates to the use of the modified nucleoside as described and provided herein as a substrate for a DNA polymerase, a Reverse Transcriptase or an RNA polymerase. For example, the modified nucleoside as described and provided herein may be used items in an oligonucleotide/nucleic acid molecule amplification method. Generally, in context with the present invention, oligonucleotide/nucleic acid molecule amplification methods are known in the art and described, e.g., in MQIIer, PCR, Spektrum Akademischer Verlag, Heidelberg-Berlin (2001 ); Schrimpf, Gentechnische Methode, 3. Auflage, Spektrum Akademischer Verlag, Heidelberg-Berlin (2002). In one embodiment of the present invention, the modified nucleoside as described and provided herein is a substrate for a DNA polymerase or an RNA polymerase, preferably a DNA polymerase. In this context, examples for DNA polymerases comprise DNA polymerase I, DNA polymerase II, DNA polymerase III holoenzyme, and DNA polymerase IV. In a specific aspect of the present invention, the DNA polymerase may be KlenTaq DNA polymerase (KTq as represented, e.g., by SEQ ID NO: 12 or the exonuclease deficient Klenow fragment of E. coli DNA polymerase I (KF exo as represented, e.g., by SEQ ID NO: 13. Also, in context with the present invention, the DNA polymerase may comprise reverse transcriptase activity. Accordingly in one embodiment of the present invention, the modified nucleoside as described and provided herein is a substrate for RT-KTq2 DNA polymerase (as represented, e.g., by SEQ ID NO: 14. If the modified nucleoside as described and provided herein is a substrate for an RNA polymerase, such RNA polymerase may be selected from the group consisting of RNA polymerase I, RNA polymerase II, RNA polymerase III, and T7 RNA polymerase. As already mentioned, the modified nucleoside as described and provided herein is suitable to be incorporated by DNA or RNA polymerase in strand synthesis. Accordingly, it is in accordance with the present invention that 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more modified nucleosides as described and provided herein may be incorporated into a nucleic acid molecule. That is, the present invention also relates to nucleic acid molecules comprising 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more the modified nucleosides as described and provided herein. The present invention also relates to the use of the modified nucleoside as described and provided herein in the in vitro diagnosis or prognosis of a disease or disorder of a subject, which disease is associated with a target sequence. Examples for such diseases or disorders comprise in this context viral infections, cancer, cardiovascular diseases, bacterial infections, autoimmune diseases, hereditary diseases. For example, the modified nucleoside as described and provided herein may be used for detection of at least one single nucleotide variation/polymorphism (SNP) or of epigenetic markers, which is comprised in a target sequence. The present invention also relates to the use of the modified nucleoside as described and provided herein for the detection of target sequences, such as pathogenic target sequences, such as those from bacteria, viruses including retroviruses, fungi, or unicellular organisms. In one embodiment of the present invention, the target sequence may be comprised in a subject, e.g., a bacterium, virus or vertebrate, preferably the subject is a mammal, more preferably the subject is a human being. In a particular embodiment of the present invention, said target sequence is selected from the group consisting of APC, AR, BRCA1 , BRAF, CDH1 , CDH1 1 , CDH13, CDKN2A, CDKN2B, DAPK1 , EMP3, ESR1 , GSTP1 , IGFBP3, LGAL3, MASPIN, MGMT, MLH1 , NORE1A, NSD1 , PYCARD, RARB, RASSF1A, RBP1 , IZ1 , S100P, SFRP1 , SFRP2, SNCG, SOCS1 , TFPI2, TIG1 , TMP2, TP73, TSHR, VHL, WIF, WRN, and SEPT9, preferably is BRAF. Said target sequence may inter alia be comprised in a test sample of a subject, e.g., a test sample derived from tissue biopsy, blood, saliva, stool or urine. In order to detect said target sequence in a given sample by using the modified nucleoside in accordance with the present invention, said target sequence is preferably present in said test sample in an amount of less than 1000 fmol, 500 fmol, 400 fmol, 300 fmol, 200 fmol, 100 fmol, 50 fmol, 20 fmol, 10 fmol, 5 fmol, 2 fmol or less than 1 fmol in said test sample. In accordance with the present invention, the modified nucleoside as described and provided herein may also be used for the discrimination of a matched primer and a mismatched primer, wherein said primers hybridize to a target sequence and wherein the mismatched primer comprises a non-canonical nucleotide at its 3' end in relation the target sequence to which it hybridizes. The present invention also relates to the use of the modified nucleoside as described and provided herein for the discrimination of a matched nucleotide and a mismatched nucleotide, wherein said nucleotide incorporates opposite a matched target sequence and wherein the mismatched nucleotide is discriminated. In this context, it is envisaged that further a DNA polymerase, Reverse Transcriptases, or RNA polymerase may be used. Also, appropriate substrates for enzymes E (for cargo X being an enzyme E) may additionally be employed in accordance with the present invention. Such substrate may be used, e.g., for colorimetric detection or electrochemical detection of a matched or mismatched primer, an SNP, or a target sequence as defined and described herein. Such use may preferably be applied at room temperature and does not employ the use of a thermocycler. In a preferred embodiment of the present invention, said detection of a matched or mismatched primer, an SNP, or a target sequence as defined and described herein can be achieved by naked-eye detection (e.g., by choosing enzymes E which catalyze conversion of corresponding substrates resulting in colorimetric signals which are detectable by naked-eye such as, e.g., HRP). In context with the present invention, for the discrimination of a matched primer and a mismatched primer, wherein said primers hybridize to a target sequence and wherein the mismatched primer comprises a non-canonical nucleotide at its 3' end in relation the target sequence to which it hybridizes, it is possible to employ a primer binding to a conserved sequence within a target sequence. In one embodiment, the target sequence may be known (e.g., 16S rRNA for detecting E. coli).

Generally, for detecting SNP in context with the present invention, a primer may be used which ends immediately before the SNP site. In one embodiment, the target sequence containing the SNP may be known except for the SNP itself. For discrimination, the different efficiency of incorporation of the modified nucleoside of the present invention compared to the matched (i.e. canonical) nucleotide/the mismatched (i.e. non-canonical) nucleotide via the (DNA) polymerase at the SNP-site can be used. The primer may be same for both cases at is does not cover the SNP site.

The present invention further relates to the modified nucleoside as described and provided herein for use in an in vitro method of diagnosis or prognosis of a disease or disorder of a subject, said disease is preferably caused by pathogens such as bacteria, viruses including retroviruses, fungi, and unicellular organisms. Said disease may also be associated with a target sequence as described and defined herein, wherein said target sequence is comprised in the genome or in the transcribed RNA or DNA of the subject. As mentioned, in accordance with the present invention, said subject is preferably mammal, more preferably a human being. Furthermore, in an aspect of the present invention, said disease or disorder is associated with an SNP or mutation in a target sequence as described and defined herein, wherein said target sequence is comprised in the genome of the subject. As used herein, the genome may be, e.g., DNA or RNA. In context with the present invention, with regard to an SNP to be detected by using the modified nucleoside of the present invention or an SNP associated with a disease or disorder as described and defined herein, said SNP may be inter alia selected form the group consisting of SNPs associated with cancer, factor II protrombin, factor V Leiden, ApoB, coeliac disease, alpha-1 -antitrypsin-deficiency, lactose intolerance, fructose-intolerance, hemochromatosis, chronic myeloproliferative disorders, methylenetetrahydrofolate reductase, cystic fibrosis, APOE genotypes, glutathione S transferase M (GSTM1 ) genotyping, GSTT1 genotyping, human leucocyte antigen (HLA) subtypes, and interleukin- 28B.

The present invention further relates to a method for producing a modified nucleoside as defined and provided herein, said method comprising

(a) providing a pyrimidine or purine nucleoside Y,

(b) conjugating the pyrimidine or purine nucleoside Y to a linker L as described and defined herein above and below; and

(c) conjugating the linker L to a cargo X having a volume of 15000 A³ or more as described and defined herein above and below (see, e.g., Hermanson, Bioconjugate Techniques (2013) Academic Press; Mark, Bioconjugation Protocols, Methods Mol. Biol. 751 (201 1 ), Humana Press),

wherein said cargo X is preferably an enzyme E as described and defined herein above and below,

thereby obtaining the modified nucleoside.

Additionally or alternatively the present invention further relates to a method for producing a modified nucleoside as defined and provided herein, said method comprising

(a) providing a pyrimidine or purine nucleoside Y,

(c) conjugating the linker L to a cargo X having at least 90 amino acids for cargo X being a protein as described and defined herein above and below (see, e.g., Hermanson,

Bioconjugate Techniques (2013) Academic Press; Mark, Bioconjugation Protocols, Methods Mol. Biol. 751 (201 1 ), Humana Press),

thereby obtaining the modified nucleoside. In this context, steps (b) and (c) may take place simultaneously or sequentially.

The present invention also relates to modified nucleosides obtainable or directly obtained by the producing method provided herein.

The present invention also relates to a preparation comprising the modified nucleoside described and provided herein and/or comprising a nucleic acid molecule containing said modified nucleoside. The present invention further relates to a kit or kit-of-parts comprising the modified nucleoside described and provided herein and/or comprising a nucleic acid molecule containing said modified nucleoside. In one embodiment of the present invention, kit or kit-of- parts contains a modified nucleoside of the present invention which comprises an enzyme E as cargo X as described herein and the kit or kit-of-parts further comprises a substrate for said enzyme E. The kit or kit-of-parts of the present invention may also comprise suitable means to conduct an oligonucleotide/nucleic acid molecule amplification, e.g., DNA and/or RNA polymerase(s) and/or Reverse Transcriptases, buffer(s), and/or one or more primer(s).

The present invention further relates to an in vitro method for diagnosing or predicting a disease or disorder associated with a target sequence as described and defined herein, or a pathogen associated with a target sequence as described and defined herein, in a subject as described and defined herein, said method comprising:

(A) contacting the target sequence obtained from a subject's sample with a modified nucleoside as defined and provided in accordance with the present invention.

The present invention further relates to an in vitro method for detecting a target sequence in a sample, said method comprising:

(A) contacting said target sequence with a modified nucleoside as defined and provided in accordance with the present invention.

Said methods for diagnosing, predicting, or detecting may further comprise a step (B) of contacting the target sequence with a DNA or RNA polymerase, and/or a step (C) of performing an amplification of the target sequence. In one embodiment, the method of detecting a target sequence in a sample is achievable by naked-eye.

The present invention further relates to a modified nucleoside as defined and provided herein or a nucleic acid molecule comprising said modified nucleoside for use in treating a disease or disorder in a subject. In one embodiment, said disease or disorder to be treated by using the modified nucleoside of the present invention is a disease or disorder associated with a target sequence of a pathogen. For example, in accordance with the present invention, the modified nucleoside of the present invention for use in treating a disease or disorder in a subject may comprise an antibody A as cargo X as described herein. In this context, the modified nucleoside may further be comprised in a nucleic acid molecule as provided herein, wherein said nucleic acid molecule has a sequence complementary to a target sequence of a pathogen. In one embodiment of the present invention, said target sequence is a nucleic acid molecule of a pathogen, e.g., of a bacterium or a virus, preferably a virus. In a further embodiment, said nucleic acid molecule of a pathogen (e.g., a virus) is DNA or RNA, e.g., RNA. For example, said RNA is RNA derived from Hepatitis C Virus (HCV), Hepatitis A Virus (HCV), human immunodeficiency virus (HIV), human influenza, avian influence, Ebola virus, Dengue virus, Hanta virus, Lassa virus, noro virus, Middle East respiratory syndrome coronavirus, SARS coronavirus, swine-origin influenza virus, yellow fever virus. West Nile virus, polio virus, measles virus, Rubella virus, Marburg virus, Mumps virus, rabies virus, rotavirus. The modified nucleoside as defined and provided herein or a nucleic acid molecule comprising said modified nucleoside for use in treating a disease or disorder in a subject may be applied to said subject together with a pharmaceutically acceptable carrier in a suitable manner. In one embodiment, the modified nucleoside as defined and provided herein or a nucleic acid molecule comprising said modified nucleoside is applied to the blood stream of said subject, e.g., intravenously (i.V.).

Particularly in context of to a modified nucleoside as defined and provided herein or a nucleic acid molecule comprising said modified nucleoside for use in treating a disease or disorder in a subject, it is further in accordance with the present invention that said modified nucleoside or said nucleic acid molecule comprising the same further contains an additional compound which protects said modified nucleoside or said nucleic acid molecule comprising the same from being degraded after being applied to said subject. For example, said modified nucleoside or said nucleic acid molecule comprising the same may further be PEGylated, i.e. contain one or more polyethylenglycol (PEG) additions.

As used herein, unless specifically defined otherwise, the term "nucleic acid" or "nucleic acid molecule" is used synonymously with "oligonucleotide", "nucleic acid strand", or the like, and means a polymer comprising one, two, or more nucleotides. In this context, also the term "target sequence" as used herein comprises nucleic acid molecules.

The embodiments which characterize the present invention are described herein, shown in the Figures, illustrated in the Examples, and reflected in the claims. It must be noted that as used herein, the singular forms "a", "an", and "the", include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "a reagent" includes one or more of such different reagents and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

The term "and/or" wherever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term". The term "about" or "approximately" as used herein means within 20%, preferably within 10%, and more preferably within 5% of a given value or range.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term "comprising" can be substituted with the term "containing" or "including" or sometimes when used herein with the term "having".

When used herein "consisting of" excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

In each instance herein any of the terms "comprising", "consisting essentially of" and "consisting of may be replaced with either of the other two terms.

It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

All publications and patents cited throughout the text of this specification (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.

The present invention also relates to the following items:

A modified nucleoside, which comprises a structure represented by formula (I) below

Y-L-X (I)

wherein

Y is a pyrimidine or purine nucleoside, wherein

L is a linker, and

X is a cargo having a volume of 15000 A³ or more or a cargo having at least 90 amino acids for cargo X being a protein,

The modified nucleoside of item 1 , wherein Y-L-X are covalently linked to each other. The modified nucleoside of item 1 or 2, wherein said cargo is a protein. The modified nucleoside of item 3, wherein said protein is an enzyme E or antibody A. The modified nucleoside of item 1 or 2, wherein said cargo is a bead.

The modified nucleoside of any one of the preceding items, wherein the nucleoside is a (ribosyl)nucleosid, a desoxy(ribosyl)nucleosid, an arabinosylnucleosid or an (methylribosyl)nucleosid.

7. The modified nucleoside of any one of the preceding items comprising a structure represented by formula (II) below

R¹-Y-L-X (II)

wherein R¹ is H, or a (poly)phosphate represented by , with n being an integer from 1 to 20, and Z being selected from the group consisting of H, free electron, and ribose or a desoxyribose. 8. The modified nucleoside of any one of the preceding items, wherein the purine nucleoside is selected from the group consisting of (desoxy)adenosine, inosine, and (desoxy)guanosine.

The modified nucleoside of any one of the preceding items, wherein the purine

nucleoside has a purine selected from the group the group consisting of a deazapurine, an azidopurine, an alkylpurine, a thiopurine, a bromopurine, an O- alkylpurine, and an isopurine.

10. The modified nucleoside of any one of the preceding items, wherein the purine nucleoside is selected from the group consisting of

1 -methyl(deoxy)adenosine, 2-methyl-(deoxy)adenosine, N⁶-methyl(deoxy)adenosine, N⁶,N⁶-dimethyl(deoxy)adenosine, 7-deaza(deoxy)adenosine, 7-deaza-8- aza(deoxy)adenosine, 7-deaza-7-bromo(deoxy)adenosine, 7-deaza-7- iodo(deoxy)adenosine, 8-azido(deoxy)adenosine, 8-bromo(deoxy)adenosine, 8- iodo(deoxy)adenosine, 8-bromo-2'-deoxy(deoxy)adenosine, 2'-0-methyladenosin, inosin, 1 -methylinosin, 2'-0-methylinosin, 1 -methyl(deoxy)guanosine, 7- methyl(deoxy)guanosine, N²-methyl(deoxy)guanosine, N²,N² dimethyl-guanosine, isoguanosine, 7-deaza(deoxy)guanosine, 7-deaza-8-aza(deoxy)guanosine, 7-deaza- 7-bromo(deoxy)guanosine, 7-deaza-7-iodo(deoxy)guanosine, 6- thio(deoxy)guanosine, 06-methyl(deoxy)guanosine, 8-azido(deoxy)guanosine, 8- bromo(deoxy)guanosine, 8-iodo(deoxy)guanosine, 2'-0-methylguanosine, 8- azidoinosine, 7-azainosine,8-bromoinosine, 8- iodoinosine, 1 -methylinosine, and 4- methylinosine. 1 1 . The modified nucleoside of any one of the preceding items, wherein the purine nucleoside is selected from the group consisting of a queuosine, an archaeosine, a wyosine and a N⁶-threonylcarbamoyladenosine.

12. The modified nucleoside of any one of the preceding items, wherein the pyrimidine nucleoside is selected from the group consisting of (desoxy)cytidine, (desoxy)thymidine, and (desoxy)uridine.

The modified nucleoside of any one of the preceding items, wherein the pyrimidine nucleoside is selected from the group consisting of a alkylpyrimidine, a thiopyrimidine, a bromopyrimidine, an O-alkylpyrimidine, an isopyrimidine, an acetylpyrimidine hydropyrimidine, and a pseudopyrimidine.

The modified nucleoside of any one of the preceding items, wherein the pyrimidine nucleoside is selected from the group consisting of 3-methyl-(deoxy)cytidine, N⁴- methyl(deoxy)cytidine, N⁴,N⁴-dimethyl(deoxy)cytidine, iso(deoxy)cytidine, pseudo(deoxy)cytidine, pseudoiso(deoxy)cytidine, 2-thio(deoxy)cytidine, N⁴- acetyl(deoxy)cytidine, 3-methyl(deoxy)uridine, pseudo(deoxy)uridine, 1 -methyl- pseudo(deoxy)uridine, 5,6-dihydro(deoxy)uridine, 2-thio(deoxy)uridine, 4- thio(deoxy)uridine, 5-bromodeoxy(deoxy)uridine, (5-iodo-)2'-deoxyuridine, 4- thio(deoxy)thymidine, 5, 6-dihydro(deoxy thymidine, 04-methylthymidine, difluortolune, and other nucleobase surrogates,

wherein the nucleoside is preferably a 2'-deoxyuridine.

The modified nucleoside of any one of any one of the preceding items, which comprises a structure represented by formula (III) below

wherein B is a purine, a purine derivative, a pyrimidine or a pyrimidine derivative; wherein L is a linker; and

wherein X is a cargo having a volume of 15000 A³ or more or alternatively at least 90 amino acids for cargo X being a protein, and wherein X is preferably an enzyme E.

The modified nucleoside of any one of items 1 to 1 1 or 15, wherein the purine or purine nucleoside is comprised by a structure represented by formula (IV) below

wherein R⁴ is -H, a free electron pair, or -CH₃;

wherein R⁵ is H, -CH₃, -NH₂, -N(CH₃)₂ or =0;

wherein R⁶ is a free electron pair or H;

wherein R⁷ is -H, -NH₂, -CH₃, -N(CH₃)₂, =0 or L;

wherein R⁸ is a free electron pair or -CH₃ or L;

wherein R⁹ is L or -H;

wherein R¹⁰ is selected from the group consisting of ribose, ribose-5- phosphate, ribose-5-diphosphate, ribose-5-triphosphate, 2'-0-Methyl-ribose, 2'-0-Methyl-ribose-5-phosphate, 2'-0-Methyl-ribose-5-diphosphate, 2'-0- Methyl-ribose-5-triphosphate, deoxyribose, deoxyribose-5-phosphate, deoxyribose-5-diphosphate, and deoxyribose-5-triphosphate; and

wherein at least one L is present in the purine or purine nucleoside.

The modified nucleoside of any one of items 1 to 1 1 , 15 or 16, wherein the purine or purine nucleoside is comprised by a structure represented by formulas (V) or (VI) below

(V) or

wherein R³ is H, or ,with n being an integer from 1 to 20, and Z being selected from the group consisting of H, free electron, and ribose or a desoxyribose; wherein R⁵ is H, -CH₃, -NH₂, -N(CH₃)₂ or =0;

wherein R⁸ is L or a free electron pair;

wherein R⁹ is L or -H; and

wherein L is a linker and wherein at least one of R⁸ or R⁹ is L. The modified nucleoside of item 17, wherein R⁵ is H. The modified nucleoside of item 17, wherein R⁵ is NH₂.

The modified nucleoside of any one of items 1 to 1 1 or 15 to 19, wherein the purine nucleoside is represented formula (VII) below

wherein R³ is H, or , with n being an integer from 1 to 20, and Z being selected from the group consisting of H, free electron, and ribose or a desoxyribose; wherein R¹¹ is -H, or a heteroatom containing group;

wherein R¹² is -NH₂, or -H; and

wherein L is a linker. The modified nucleoside of item 20, wherein the heteroatom containing group is selected from the group consisting of a nitrogen containing moiety, an oxygen containing moiety, or a halogen containing moiety, wherein the halogen is selected from the group consisting of a fluorine (F), a chlorine (CI), a bromine (Br), and an iodine (I). The modified nucleoside of any one of items 1 to 1 1 or 15 to 21 , wherein the purine nucleoside is represented by formula (VIII) below

wherein R² is -OH, -H or -0(CH2)n-CH₃, with n being an integer from 0 to 20;

wherein R is H or , with n being an integer from 1 to 20, and Z being selected from the group consisting of H, free electron, and ribose or a desoxyribose; and

wherein L is a linker.

The modified nucleoside of any one of items 1 to 7 or 15, wherein the pyrimidine or pyrimidine nucleoside is represented by formula (IX) below

wherein R¹³ is selected from the group consisting of ribose, ribose-5-phosphate, ribose-5-diphosphate, ribose-5-triphosphate, 2'-0-Methyl-ribose, 2'-0-Methyl-ribose-

5-phosphate, 2'-0-Methyl-ribose-5-diphosphate, 2'-0-Methyl-ribose-5-triphosphate, deoxyribose, deoxyribose-5-phosphate, deoxyribose-5-diphosphate, and deoxyribose-5-triphosphate;

wherein R¹⁴ is a free electron pair, =0, -NH₂, or =S;

wherein R¹⁵ is a free electron pair, -H or -CH₃;

wherein R¹⁶ is -NH₂, -CH₃, -N(CH₃)₂, =0 or -NH-CO-CH₃; and

wherein R¹⁷ is L.

The modified nucleoside of any one of items 1 to 7, 15 or 23, wherein the pyrimidine or pyrimidine nucleoside is represented by formula (X) below

wherein R¹³ is selected from the group consisting of ribose, ribose-5-phosphate, ribose-5-diphosphate, ribose-5-triphosphate, 2'-0-Methyl-ribose, 2'-0-Methyl-ribose- 5-phosphate, 2'-0-Methyl-ribose-5-diphosphate, 2'-0-Methyl-ribose-5-triphosphate, deoxyribose, deoxyribose-5-phosphate, deoxyribose-5-diphosphate, and deoxyribose-5-triphosphate; and

wherein L is a linker.

The modified nucleoside of any one of items 1 to 7, 15, 23 or 24, wherein the pyrimidine or pyrimidine nucleoside is represented by formula (XI) below

wherein L is a linker.

The modified nucleoside of any one of items 1 to 7, 15 or 23 to 25, wherein pyrimidine or pyrimidine nucleoside is represented by formula (XII) below

wherein R¹³ is selected from the group consisting of ribose, 2'-0-Methyl-ribose, ribose-5-phosphate, ribose-5-diphosphate, ribose-5-triphosphate, 2'-0-Methyl-ribose- 5-phosphate, 2'-0-Methyl-ribose-5-diphosphate, 2'-0-Methyl-ribose-5-triphosphate, deoxyribose, deoxyribose-5-phosphate, deoxyribose-5-diphosphate, deoxyribose-5- triphosphate and difluortoluene, and other nucleobase surrogates; and

wherein L is a linker.

The modified nucleoside of any one of the preceding items, wherein the linker does not comprise an amino acid. 28. The modified nucleoside of any one of the preceding items, wherein the linker comprises a straight or branched hydrocarbon based moiety or a cyclic hydrocarbon based moiety. The modified nucleoside of item 27 or 28, wherein the linker comprises one or more heteroatoms.

The modified nucleoside of item 29, wherein the one or more heteroatom is oxygen (O), nitrogen (N), silicium (Si) and/or sulfur (S).

The modified nucleoside of any one of the preceding items, wherein the linker comprises one or more alkyl, alkenyl and/or alkynyl groups.

The modified nucleoside of any one of the preceding items, wherein the linker comprises an amide, an amidine, a disulfide, a hydrazine, a thioether and/or an ester.

The modified nucleoside of item 32, wherein the amide, disulfide, hydrazine, thioether and/or ester group is generated by coupling of a reactive chemical group with a target functional group.

The modified nucleoside of item 33, wherein the reactive chemical group is coupled to the Enzyme E where X is an Enzyme E and the target functional group is coupled to the pyrimidine or purine nucleoside.

The modified nucleoside of item 33 or 34, wherein the reactive chemical group is selected from the group consisting of an amine-, an aryl azide-, a carbodiimide-, a hydrazide-, an imidoester-, an iodoacetyl-, an isocyanate (PMPI)-, a maleimide-, a NHS ester-, a pyridyl disulfide, and a vinyl sulfone- reactive group,

wherein the reactive chemical group preferably is a maleimide-reactive group.

The modified nucleoside of item 33 or 34, wherein the target functional group is selected from the group consisting of an aldehyde, a carboxylic acid, an amine, a hydroxyl, and a sulfhydryl moiety,

wherein the target functional group preferably is a sulfhydryl moiety.

The modified nucleoside of any one of the preceding items, wherein the linker is a 5- (8-mercapto octanamido)pent-1 -yn-1-yl or a 5-(16-mercaptohexadecanamido)pent-1- yn-1-yl.

The modified nucleoside of any one of items 34-36, wherein reactive chemical group is comprised in a heterobifunctional or homobifunctional linker. The modified nucleoside of item 38, wherein heterobifunctional linker is selected from the group consisting of a-Maleimidoacetoxy-succinimide ester (AMAS), N(4-[p- Azidosalicylamido]butyl)- 3'-(2'-pyridyldithio) propionamide (APDP^*), (β- Maleimidopropionic acid)hydrazide^«TFA (BMPH), (3-Maleimidopropyloxy)succinimide ester (BMPS), ε-Maleimidocaproic acid (EMCA), (e-Maleimidocaproyloxy)succinimide ester (EMCS), (Y-Maleimidobutyryloxy)succinimide ester (GMBS), κ- Maleimidoundecanoic acid (KMUA), Succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 -carboxy-(6-amidocaproate) (LC-SMCC), Succinimidyl-6-(3'-[2-pyridyl- dithio]propionamido)hexanoate (LC-SPDP), m-Maleimidobenzoyl-N- hydroxysuccinimide ester (MBS), Succinimdyl-3-(bromoacetamido)propionate (SBAP), Succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), succinimidyl iodoacetate (SIA), Succinimidyl 4-(p-maleimido-phenyl)butyrate (SMPB), NHS-PEG24-Maleimide SM(PEG24), NHS-PEG12-Maliemide (SM[PEG]12), NHS-PEG8-Maliemide (SM[PEG]8), NHS-PEG6-Maleimide (SM(PEG)6), NHS-PEG4-Maliemide (SM[PEG]4), NHS-PEG2-Maliemide (SM[PEG]2), Succinimidyl 4-(N-maleimido- methyl)cyclohexane-carboxylate (SMCC), succinimidyl iodoacetate (SIA), Succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), (ε-

Maleimidocaproyloxy)sulfosuccinimide ester (Sulfo-EMCS),-Succinimidyl-3-(2- pyridyldithio)propionate (SPDP), Succinimidyl-6-(3- maleimidopropionamido)hexanoate (SMPH), Ν-(γ-

Maleimidobutryloxy)sulfosuccinimide ester (Sulfo-GMBS),-(K-

Maleimidoundecanoyloxy)sulfosuccinimide ester (Sulfo-KMUS), Sulfosuccinimidyl 6- (a-methyl-a-[2-pyridyldithio]-toluamido)hexanoate (Sulfo-LC-SMPT), Sulfosuccinimidyl 6-(3'-[2-pyridyl-dithio]propionamido)hexanoate (Sulfo-LC-SPDP), Maleimidobenzoyl- hydroxysulfosuccinimide ester (Sulfo-MBS), Sulfosuccinimidyl(4-iodo- acetyl)aminobenzoate (Sulfo-SIAB), Sulfosuccinimidyl 4-(N-maleimido methyl)cyclohexane-1 -carboxylate (Sulfo-SMCC), Sulfosuccinimidyl 4-(p- maleimidophenyl)butyrate (Sulfo-SMPB) Ν,Ν-Dicyclohexylcarbodiimide (DCC), 1 - Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDO), and Sulfo-NHS- (2-6-[Biotinamido]-2-(p-azidobezamido) (Sulfo-SBED).

The modified nucleoside of item 38, wherein homobifunctional linker is selected from the group consisting of 1 ,4-bis-Maleimidobutan (BMB), Maleimidohexane (BMH), Maleimidoethane (BMOE), 1 ,8-bis-Maleimidodiethylene-glycol (BM(PEG)2), 1 ,1 1 -bis- Maleimidotriethyleneglycol (BM(PEG)3), Dimethyl suberimidate^«2HCI (DMS), Dimethyl 3,3'-dithiobispropionimidate^«2HC (DTBP), and (2-Maleimidoethyl)amine (Trifunctional) (TMEA^***). The modified nucleoside of item 35, wherein maleimide is coupled to Enzyme E and - SH is coupled to the pyrimidine nucleoside or a purine nucleoside. The modified nucleoside of any one of the preceding items, wherein the linker comprises a carbon atom based chain comprising Ci, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, Ci₀, On, C-I2, Ci3, Ci₄, Ci5, C-I6, C17, C18 C19, C20, C21, C22, C23, C2₄ or C25 or more carbon atoms or a polyether based chain.

The modified nucleoside of any one of the preceding items, wherein the linker comprises a carbon atom based chain comprising Ci₅, Ci₆, Ci₇, Ci₈ C19, C₂o, C₂i, C22, C23, C₂₄ or C25 or more carbon atoms.

The modified nucleoside of item 43, wherein the linker comprises a carbon atom chain comprising C₇ or C15 carbon atoms.

The modified nucleoside of any one of the preceding items, wherein the linker comprises a thioether group and/or an amide group.

The modified nucleoside of item 43, wherein the polyether based chain is a polyethylene glycol based chain with -(0-CH₂-CH₂)- repeating units. The modified nucleoside of any one of the preceding items, wherein X is an enzyme E and wherein said enzyme E has an amino acid sequence comprising more than 5, 10,

20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or 2000 or more amino acids. The modified nucleoside of the preceding items, wherein the X is an enzyme E, and wherein enzyme E is a reporter enzyme. The modified nucleoside of item 48, wherein the reporter enzyme is selected from the group consisting of horseradish peroxidase (HRP; SEQ ID NO: 1 ), alkaline phosphatase (AP; SEQ ID NO: 2), glucose-oxidase (GOX; SEQ ID NO:3), luciferase (SEQ ID NO: 4), chloramphenicol acetyl tansferase, (CAT; SEQ ID NO: 5), β- Galactosidase (β-Gal; SEQ IS NO: 6), catalase (SEQ ID NO: 7), urease (SEQ ID NO:8), and soybean peroxidase (SEQ ID NO: 9).

50. The modified nucleoside of item 48, wherein the reporter enzyme has an amino acid sequence having a sequence similarity or identity of at least 60%, 70%, 80% 90%,

95%, 98%, 99% or 100% with any one of SEQ ID NOs: 1-9 and is biologically active.

51 . Use of the modified nucleoside as defined in any one of the preceding items as a substrate for a DNA polymerase, a Reverse Transcriptase or an RNA polymerase.

52. Use of the modified nucleoside as defined in any one of the preceding items in an oligonucleotide/nucleic acid molecule amplification method.

53. The use of item 52, wherein the oligonucleotide amplification method comprises the use of a DNA polymerase or an RNA polymerase.

54. The use of item 51 or 53, wherein the DNA polymerase is selected from the group consisting of DNA polymerase I, DNA polymerase II, DNA polymerase III holoenzyme, and DNA polymerase IV.

55. The use of any one of items 51 , 53 or 54, wherein the DNA polymerase is KlenTaq DNA polymerase (KTq; SEQ ID NO: 10) or the exonuclease deficient Klenow fragment from E.coli DNA polymerase I (KF exo; SEQ ID NO: 1 1 ). 56. The use of any one of items 51 , 53, 54 or 55, wherein the DNA polymerase comprises reverse transcriptase activity.

57. The use of item 56, wherein the DNA polymerase with reverse transcriptase activity is RT-KTq2 DNA polymerase (SEQ ID NO: 12).

58. The use of item 51 or 53, wherein the RNA polymerase is selected from the group consisting of RNA polymerase I, RNA polymerase II, RNA polymerase III, and T7 RNA polymerase. 59. The use of any one of items 51 -58 for incorporation of modified nucleosides as defined in any one of items 1 -50 into a nucleic acid molecule. 60. The use of item 59, wherein 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modified nucleosides are incorporated into a nucleic acid molecule.

61 . Use of the modified nucleoside as defined in any one of items 1 -50 in the in vitro diagnosis or prognosis of a disease or disorder of a subject, which disease is associated with a target sequence.

62. Use of the modified nucleoside as defined in any one of items 1 -50 for detection of at least one single nucleotide variation/polymorphism (SNP), which is comprised in a target sequence.

63. Use of the modified nucleoside as defined in any one of items 1 -50 for the detection of epigenetic markers in a target sequence, which is comprised in said target sequence.

64. Use of the modified nucleoside as defined in any one of items 1 -50 for the detection of target sequences, such as pathogenic target sequences, such as those from bacteria, viruses including retroviruses, fungi, or unicellular organisms. 65. The use of item 63, wherein the target sequence comprises a mutation or a SNP.

66. The use of any one of items 63-65, wherein the target sequence is comprised in a subject. 67. The use of item 66, wherein the subject is a bacterium or vertebrate, preferably the subject is a mammal, more preferably the subject is a human being.

68. The use of any one of items 63-67, wherein the target sequence is selected from the group consisting of APC, AR, BRCA1 , BRAF, CDH 1 , CDH1 1 , CDH13, CDKN2A, CDKN2B, DAPK1 , EMP3, ESR1 , GSTP1 , IGFBP3, LGAL3, MASPIN, MGMT, MLH1 ,

NORE1A, NSD1 , PYCARD, RARB, RASSF1A, RBP1 , IZ1 , S100P, SFRP1 , SFRP2, SNCG, SOCS1 , TFPI2, TIG1 , TMP2, TP73, TSHR, VHL, WIF, WRN, and SEPT9, wherein the target sequence preferably is BRAF. 69. The use of any one of items 63-68, wherein the target sequence is comprised in a test sample. The use of item 69, wherein the test sample is selected from tissue biopsy, blood, saliva, stool or urine.

The use of any one of items 63-70, wherein the target sequence is present in an amount of less than 1000 fmol, 500 fmol, 400 fmol, 300 fmol, 200 fmol, 100 fmol, 50 fmol, 20 fmol, 10 fmol, 5 fmol, 2 fmol or less than 1 fmol in said test sample.

Use of the modified nucleoside as defined in any one of items 1 -50 for detection of a target sequence, which target sequence is present in an amount of less than 1000 fmol in a test sample.

Use of the modified nucleoside as defined in any one of items 1 -50 for the discrimination of a matched primer and a mismatched primer, wherein said primers hybridize to a target sequence and wherein the mismatched primer comprises a non- canonical nucleotide at its 3' end in relation the target sequence to which it hybridizes.

Use of the modified nucleoside as defined in any one of items 1 -50 for the discrimination of a matched nucleotide and a mismatched nucleotide, wherein said nucleotide incorporates opposite a matched target sequence and wherein the mismatched nucleotide is discriminated.

The use of any one of items 72-74, wherein the use further comprises the use of a DNA polymerase, Reverse Transcriptases, or RNA polymerase.

The use of any one of items 63-75, wherein the use further comprises the use of substrate for Enzyme E.

The use of item 76, wherein the substrate is used for colorimetric detection or electrochemical detection of a target sequence.

The use of item 76, wherein the substrate is used for colorimetric detection or electrochemical detection of a SNP.

The use of item 76, wherein the substrate is used for colorimetric detection or electrochemical detection of a matched or mismatched primer.

The use of any one of items 63-7579 wherein the use is performed at room temperature.

The use of any one of items 63-7680 wherein the use does not comprise utilization of a thermocycler.

The use of any one of items 62-80, wherein detection is achieved by a naked eye assay.

A modified nucleoside of any one of items 1 -50 for use in an in vitro method of diagnosis or prognosis of a disease or disorder of a subject, said disease is preferably caused by pathogens such as bacteria, viruses including retroviruses, fungi, unicellular organisms.

The modified nucleoside for use of item 83, wherein the disease or disorder is associated with a target sequence, which target sequence is comprised in the genome or in the transcribed RNA or DNA of the subject.

The modified nucleoside for use of item 83 or 84, wherein the disease or disorder is associated with an SNP or mutation in a target sequence, which target sequence is comprised in the genome of the subject.

The modified nucleoside for use of any one of items 83-85, wherein the genome is DNA or RNA.

The modified nucleoside for use of item 85 or 86, wherein the SNP is selected form the group consisting of SNPs associated with cancer, factor II protrombin, with factor V Leiden, with ApoB, with coeliac disease, with alpha-1 -antitrypsin-deficiency, with lactose intolerance, with fructose-intolerance, with hemochromatosis, with chronic myeloproliferative disorders, with the methylenetetrahydrofolate reductase; with cystic fibrosis, with APOE genotypes, with glutathione S transferase M (GSTM1 ) genotyping, with GSTT1 genotyping, with human leucocyte antigen (HLA) subtypes, and with interleukin-28B.

A nucleic acid molecule comprising a modified nucleoside as defined in any one of items 1 -50.

A method for producing a modified nucleoside as defined in any one of items 1 -50, comprising

a) providing a pyrimidine or purine nucleoside Y,

b) conjugating the pyrimidine or purine nucleoside Y to a linker L; and

c) conjugating the linker L to a cargo X having a volume of 15000 A³ or more or to a cargo having at least 90 amino acids for cargo X being a protein, wherein said cargo X is preferably an enzyme E,

thereby obtaining the modified nucleoside.

90. The method of item 89, wherein steps b) and c) take place simultaneously or sequentially.

91 . A preparation comprising a modified nucleoside of any one of items 1 -50 or a nucleic acid molecule of item 88. 92. A kit or kit-of-parts comprising a modified nucleoside of any one of items 1 -50 or a nucleic acid molecule of item 88. 93. The kit or kit-of-parts of item 92, wherein cargo X is an enzyme E and wherein the kit further comprises a substrate for enzyme E.

94. The kit or kit-of-parts of item 92 or 93, wherein the kit further comprises means to conduct an oligonucleotide/nucleic acid molecule amplification. 95. An in vitro method for diagnosing or predicting a disease or disorder associated with a target sequence, or a pathogen associated with a target sequence, in a subject, the method comprising.

a) contacting the target sequence obtained from a subject's sample with a modified nucleoside as defined in any one of items 1 -50. 96. An in vitro method for detecting a target sequence in a sample, the method comprising.

a) contacting said target sequence with a modified nucleoside as defined in any one of items 1 -50. 97. The method of item 95 or 96, wherein the method further comprises

b) contacting the target sequence with a DNA or RNA polymerase. 98. The method of any one of items 95-96, wherein the method further comprises c) performing an amplification of the target sequence.

99. The method of any one of items 96-98, wherein detection is achievable by naked-eye.

100. A modified nucleoside of any one of items 1 -50 or a nucleic acid molecule comprising said modified nucleoside for use in treating a disease or disorder in a subject.

101 . The modified nucleoside or the nucleic acid molecule of item 100, which further comprises PEG.

102. The nucleic acid molecule of items 100 or 101 , which is complementary to a target sequence associated with said disease or disorder. 103. The nucleic acid molecule of item 102, wherein said target sequence is from a bacterium or a virus, preferably from a virus.

104. The nucleic acid molecule of item 103, wherein said virus is Hepatitis C Virus (HCV). Figures

The Figures show:

Figure 1 Scheme of the conjugation strategy and the proportions of structures

a - Size comparison of KTq DNA Polymerase (PDB: 1 KTQ), HRP C1 A (PDB: 1 HCH), dTTP and a modified dTTP bearing a C15-thiol-linker (dT^i5SHTP) to scale.

b - Coupling of dT"^SHTP and malHRP (derived from HRP C1A) via thiol-maleimide reaction.

c - ESI-MS analysis of the conjugates. The shifts obtained for dT^7SHTP (m/z calc. 707) and dT^i5SHTP (m/z calc. 819) suggest the successful monofuncationalization of malHRP with the corresponding nucleotide. Figure 2 PAGE of primer extension reactions employing the synthesized conjugates a - Partial sequence of the primer and the template at the incorporation site.

b - Left: Autoradiography of a Primer Extension Reaction employing natural dTTP (Lane 1 ), the two thiol-modified nucleotides (dT^7SHTP and dT^i5SHTP, Lane 2 and 3) and the two conjugates dT^7HRPTP & dT^i5HRPTP

(Lanes 4 and 5) and the KTq DNA polymerase on a template containing the BRAF T1796A point mutation site. Right: Primer extension with RT-KTq2 DNA polymerase on a RNA version template of the BRAF point mutation sequence using dT^i5HRPTP (Lane 6) and in the absence of the template (Lane 7). P: Primer, M: Marker, Nt:

Nucleotides.

c - Incorporation competition experiments between the dT"^HRPTP and dTTP with the KTq DNA polymerase. Ratios applied for dT^7HRPTP (left) were 1 : 0/1, 2: 1/1, 3: 3/1, 4: 19/1, 5: 49/1, 6: 99/1 and 7: 1/0. Ratios applied for dT^i5HRPTP were 1 : 0/1, 2: 1/1, 3: 3/1, 4: 4/1, 5: 9/1, 6: 19/1,

7: 99/1 and 8: 1/0.

Figure 3 A possible application for the conjugates in a naked-eye detection assay

a - Scheme of the naked-eye detection assay employing the enzyme- labeled nucleotides. The primer was immobilized on a solid support via Biotin-Streptavidin interaction. After annealing of the template sequence, DNA polymerase and conjugates were added. After the incubation period, the unbound conjugate was removed by repeated filtration and a dye-solution was added giving a colorimetric read-out visible by naked eye.

b - Results of the assay depicted in A with KTq and KF exo^~ DNA polymerase on a matched or mismatched ssDNA template and RT- KTq2 DNA polymerase on an E. Coli ribosomal rRNA mixture with and without the presence of an excess of human RNA.

Figure 4 Synthesis of thiol-modified dTTP derivatives

a) Cul, Pd(PPh₃)₄, Et₃N, DMF, rt, ovn.; b) proton sponge, POCI₃, PO(OMe)₃, (Bu₃NH)₂H₂P207, nBu₃N, TEAB buffer, ammonium hydroxide; c) HATU, DIPEA, DMF, rt, ovn. Figure 5 Coomassie-stained SDS-PAGE of a primer extension reaction with the conjugates

Unconjugated malHRP (1 ), conjugates dT^7HRPTP (2) and dT^i5HRPTP (3) and the conjugates after the primer extension reaction (4 and 5) with the BRAF template. The reaction mixture contained 10 μΜ BRAF DNA template, 10 μΜ unlabeled BRAF primer, 100 nM KTq DNA polymerase and 12 μΜ dT^i5HRPTP. After 1 .5 h of incubation at 55 °C, the reaction was quenched by addition of SDS loading buffer and denaturation at 95 °C for 5 min. SDS-PAGE was performed in a 15% gel with unstained protein ladder (NEB) as molecular weight marker.

Figure 6 Multiple incorporation of the conjugates into a primer strand

A primer extension reaction with KTq DNA polymerase was performed according to the general procedure either with a template encoding for one insertion (left) or a template containing eleven consecutive adenosine residues (right). Samples were taken after 1 and 20 min. SDS-PAGE was performed in a 12.5% gel and the bands visualized by autoradiography. P: Primer.

Legend top: 5 -..TA TC 5 -..TA TC

3 -..AT AGA GGG. 4 -..AT AGA AAA..

Bold and underlined letters represent sites where the conjugate may be incorporated complementarily.

Figure 7 Competition experiment between dT"^HRPTP and dTTP with KTq

DNA polymerase

A primer extension was carried out at 55 °C with KTq DNA polymerase (100 nM) in presence of the conjugates dT^7HRPTP (·, a) and dT^i5HRPTP (·, b) in defined ratios with dTTP (■, a and b). Samples were taken after 3 min. Ratios applied for dT^7HRPTP were 0/1 , 1/1 , 3/1 , 19/1 , 49/1 , 99/1 and 1/0. Ratios applied for dT^i5HRPTP were 0/1 , 1/1 , 3/1 , 4/1 , 9/1 ,

19/1 , 99/1 and 1/0. To evaluate the incorporation efficiencies, the product bands and the background were quantified using the Bio-Rad ImageLab 5.2 software. The conversion in % was plotted versus the concentration using the program OriginPro 9.1 32Bit. The vertical line indicates the approximate ratio where both nucleotides are equally incorporated. Figure 8 Competition experiment between dT"^HRPTP and dTTP with KF exo^'

DNA polymerase

A primer extension was carried out at 37 °C with KF exo^' DNA polymerase (0.5 U) in presence of the conjugates dT^7HRPTP (·, a) and dT^i5HRPTP (·, b) in defined ratios with dTTP (■. a and b). Samples were taken after 3 min. Ratios applied for dT^7HRPTP were 0/1, 1/1, 3/1, 19/1, 49/1, 99/1 and 1/0. Ratios applied for dT^i5HRPTP were 0/1, 1/1, 3/1, 4/1, 9/1, 19/1, 99/1 and 1/0. To evaluate the incorporation efficiencies, the product bands and the background were quantified using the Bio-Rad ImageLab 5.2 software. The conversion in % was plotted versus the concentration using the program OriginPro 9.1 32Bit. The vertical line indicates the approximate ratio where both nucleotides are equally incorporated. Figure 9 Determination of the lower detection limit of the assay with RT-

KTq2 DNA polymerase on the BRAF RNA template

Assay was carried out according to the procedure mentioned afore. The reaction mixture was incubated for 5 min at 55 °C with 333 nM RT- KTq2 DNA polymerase.

Figure 10 NMR

¹H and ³¹P NMR spectra of dT^7SHTP. L = Linker

Figure 11 NMR

1H and ³¹P NMR spectra of dT^i5SHTP. L = Linker

Figure 12 ESI-MS spectra recorded of malHRP

(a) , dT^7SHTP

(b) and dT^i5SHTP

(c) The conjugates were purified as described above without 2- mercaptoethanol blocking.

Figure 13 Multiple incorporation of the conjugates into a primer strand

A primer extension reaction with different DNA polymerases was performed according to the general procedure with a template encoding for eleven consecutive adenosine residues (1 1 A). Samples were taken after 50 min. SDS-PAGE was performed in a 12.5% gel without loading buffer and the bands visualized by autoradiography. P:

Primer.

Figure 14 Application for the conjugates in a naked-eye detection assay

a - Scheme of the naked-eye detection assay of epigenetic markers in

RNA employing the enzyme-labeled nucleotides. The primer is immobilized on a solid support via Biotin-Streptavidin interaction. After annealing of the respective target sequence, a DNA polymerase and conjugates are added. After the incubation period, the unbound conjugate is removed by repeated filtration and a dye-solution is added giving a colorimetric read-out visible by naked eye.

b - Results of the assay depicted in a) depending on the respective target sequence. 2'OMe: light red; m⁶A: light red; A: deep red; -: clear. Figure 15 Scheme showing synthesis of the azide- and amino-modified nucleotide (dC^c16N3TP and dC^c16NH2TP).

Figure 16 Scheme showing synthesis of the primary antibody-labeled nucleotide

_{(d c}Ab-EMCS_{T p )} Figure 17 Scheme showing synthesis of the primary antibody-labeled nucleotide

_{(d c}Ab-DBCO_{T p )}

Figure 18 PAGE of PEX single incorporations of employing the synthesized conjugated nucleotides (dC^Ab"EMCSTP, dC^^JP); a) Partial sequence of the primer and the template at the incorporation site; b)

Autoradiography of a primer extension reaction employing natural dCTP (Lane 2) and one of the two conjugates (dC^Ab_EMCSTP, dC^- ^DBCOTP) with the KTq and KOD DNA polymerase on a template containing the BRAF T1796A point mutation site; Samples were collected after 5 min (Lane 3), 10 min (Lane 4), 15 min (Lane 5), 20 min (Lane 6). Lane 1 : Primer, L: Marker.

Figure 19 Scheme showing naked-eye detection assay employing dC^^'^^TP. Figure 20 NMR spectra ¹ H and ¹³C NMR of 16-azidohexadecanoic acid.

Figure 21 NMR spectra ¹ H NMR and ³¹ P spectra of dC^c16N3TP. Figure 22 NMR spectra ¹H NMR and ³¹P spectra of dC^c16NH2TP.

The present invention is further illustrated by the following examples. Yet, the examples and specific embodiments described therein must not be construed as limiting the invention to such specific embodiments.

Examples All materials, methods and sequence data are described below. Briefly, the conjugates were synthesized by incubation of the thiol-modified nucleotide with the maleimide-activated HRP in PBS buffer (pH 7) over night. After anion exchange FPLC (20 mM TRIS to 1 M NaCI in 20 mM TRIS, pH 9), the conjugate containing fractions (dPAGE) were concentrated via Vivaspin, blocked with β-mercaptoethanol (1.5 mM in PBS buffer ovn. pH 7) and again concentrated. Concentration of the thusly generated solutions was measured spectrophotometrically with ε(403 nm) = 102000 M^"1 cm^"1 (cf. Ohlsson et al., Acta Chemica Scandinavica Series B-Organic Chemistry and Biochemistry (1976), 30(4): 373-375).

Primer extension reactions in solution and on beads were performed as described in Verga et al., Chem Comm (2015), 51 (34): 7379-7381 .

Example 1

Synthesis of C5-thiol functionalized deoxythymidine derivatives (dT"^SHTP) and HRP- conjugates (dT"^HRPTP)

Horseradish peroxidase (HRP) activated with maleimide groups by conversion of the lysine residues with maleimidocaproic acid /V-hydroxysuccinimide ester (malHRP) was used. This species allows the conjugation to a target molecule via thiol-maleimide reaction.

Two thymidine analogues bearing ω-mercaptocarboxylic acid-based linkers of different lengths in C5-position were synthesized (Figure 4, dT"^SHTP). To do so, the synthetic pathway that has already been reported for hydroxyl-functionalized nucleotides was followed (Baccaro et al., Angew Chem Int Ed (2012), 51 (1 ): 254-257), starting with a Sonogashira coupling of 5- trifluoroacetamidopentyne with 5-lodo-2 -deoxyuridine (Figure 4, a) to yield the TFA protected, amino-functionalized nucleoside 1 of Figure 4. The nucleoside was then converted to the triphosphate (Figure 4, b) and deprotected in an ammonium hydroxide solution to yield compound 2 of Figure 4. The ω-mercaptocarboxylic acid was finally introduced using the coupling reagent HATU together with DIPEA in DMF to yield the final products dT^7SHTP and _dTi5SH_Tp (_{Fig u re 4 j C})

Conjugation to the enzyme was performed by simple incubation with malHRP in PBS buffer (Figure 1 b). Subsequently, the conjugates (dT TP) were purified via anion exchange FPLC and the conjugation evaluated. A shift in mass-to-charge ratio in ESI-MS experiments (Figure 1 c and Figure 12) indicated successful conjugation. For unconjugated malHRP, two major peaks at m/z 43370 and m/z 43564 were observed representing a singly and doubly maleimidocaproic acid activated enzyme species (ΔΓΤΙ/Ζ 194, calcd. 193). After the conjugation, the doubly activated species is removed during the FPLC purification. The obtained peaks for dT^7HRPTP and dT^i5HRPTP demonstrated shifts fitting the used nucleotides (left: dT^7HRPTP Am/z 712, calcd. 707; right: dT^7HRPTP Am/z 822, calcd. 819) within the limit of accuracy. Furthermore, for both conjugates we found peaks around -80 m/z and -160 m/z of the main peak which might correspond to the loss of one or respectively two phosphate groups of the triphosphate during the analysis by mass spectrometry.

Example 2

Primer Extension Studies

To examine whether the synthesized conjugates are substrates for DNA polymerases, single nucleotide primer extension reactions were performed on a template containing the B type raf kinase (BRAF) T1796A point mutation, which is strongly associated with carcinogenesis (Figure 2a, see Table II for sequences) (Davies et al., Nature (2002), 417(6892): 949-954). The 21 nt 5'-radioactively labeled primer was designed to end directly 5' of the mutation site so that a single deoxythymidine analogue could be inserted. This insertion was then visualized by the retarded migration of the band corresponding to the primer in analysis by denaturing polyacrylamide gel electrophoresis (PAGE) and subsequent autoradiography. When employing the unconjugated nucleotides, dT^7SHTP and dT^i5SHTP, a band of retarded mobility was observed, confirming the successful insertion of the bulky nucleotides by KlenTaq DNA polymerase (KTg) (Figure 2a, Lanes 2 and 3). The ill-shaped appearance of the band compared to the one of natural dTTP (Lane 1 ) might be caused by interaction of the free thiol with the gel matrix.

When employing the conjugates dT^7HRPTP and dT^i5HRPTP (Lanes 4 and 5), a vastly retarded migration of the band corresponding to the primer (approximately 250 nt) was detected, indicating a successful incorporation of the nucleotide with its protein 'cargo'. Therefore, it was shown that enzyme-labeled nucleotides, despite their bulky size, are indeed accepted as DNA polymerase substrates. This is also supported by non-radioactive SDS-PAGE of a primer extension reaction (Figure 5) where a shift of the HRP band towards higher molecular weights is observed. Here it was detected that the conjugate connected via the C15-linker is better accepted as the one with the shorter linker. It was further observed a second, stronger shifted band, possibly representing a second incorporation. Therefore, a primer extension on a template encoding for eleven consecutive dTMP insertions was conducted to evaluate the possibility of a multiple incorporation of the conjugates (Figure 8). It was found that the conjugate bearing the longer linker can be successively incorporated into a primer strand. To further investigate the incorporation efficiency and the influence of the length of the linkers, competition assays with the conjugates and natural dTTP (Figure 2b for Gel, Figure 6) were carried out. Single nucleotide incorporation experiments were performed in which the modified nucleotides directly compete for incorporation with their natural counterparts. This experimental setup was previously used for the same purpose (Obeid et al., PNAS USA (2010), 107(50): 21327-21331 ). Furthermore, it was also possible to incorporate the conjugates using the exonuclease deficient Klenow Fragment from E. Coli DNA Polymerase I (KF exo^") resulting in a 68-fold diminished efficiency for dT^7HRPTP and 21 -fold for dT^i5HRPTP (Figure 7).

Finally, also RT-KTq2 DNA polymerase was tested, a KTq mutant with reverse transcriptase activity. This polymerase, is able to elongate a DNA primer strand on the basis of both DNA and RNA templates (Blatter et al., Angew Chem Int Ed (2013), 52(45): 1 1935-1 1939). Therefore, the same primer extension experiment was conducted in the same RNA sequence context (Figure 2a, Lanes 6 and 7, see Table II for sequence). As the competition experiments disclosed the higher incorporation efficiency, only dT^i5HRPTP was employed exclusively. The same characteristic shift of primer mobility by PAGE analysis as described above were found, showing the applicability of HRP-modified nucleotides in reverse transcription as well.

Example 3

Naked-eye nucleic acids detection systems at single-nucleotide resolution

As was shown herein, the herein introduced enzyme-modified nucleotides of the present invention are able to combine the naked-eye colorimetric read-out of peroxidase-based assays with the versatility and fidelity of a DNA polymerase reaction.

In order to explore this potential of protein-modified nucleotides an assay design exploiting primer extensions of solid-phase immobilized primer strand was composed (Figure 3a). It was envisioned that due to the template dependency of DNA polymerases, only primers that bind sequence-selectively to a target sequence will be extended by elongation with a HRP- modified nucleotide. Thereby, single nucleotide variations in the target should be detectable (Figure 3a, match and absence). After removal of excess of the nucleotide protein, conjugate and addition of the substrate of HRP resulted in a colorimetric read-out (cf. Verga et al., Chem Comm (2015), 51 (34): 7379-7381 ).

The assay was carried out using a 5'-biotin-immobilized version of the BRAF sequence context used for the primer extension in solution. Employing dT^i5HRPTP, it was possible to reliably detect as little as 1 fmol of DNA with KTq DNA polymerase-promoted incorporation and subsequent HRP reaction (Figure 3b).

Aside from the detection of the presence or absence of a target sequence, the intrinsic fidelity of the DNA polymerase to produce Watson-Crick base pairs has the potential to be exploited to discriminate single nucleotide variations at the insertion site (Figure 3a, match and mismtach). This application was tested by employing a mismatch template sequence bearing a thymidine residue at the insertion site leading to a T:T mismatch. A strong discrimination between the matched and mismatched template was observed with KTq DNA polymerase (Figure 3b, KTq match/mismatch) which made it possible to distinguish between 5 fmol of the matched and a 20 fold excess (100 fmol) of the mismatched template.

Next, the assay was carried out for the detection of single nucleotide variations with KF exo^' DNA polymerase as it is active at room temperature, which is beneficial for the applicability of point-of-care testing (POCT). In addition, the incubation at lower temperature allows using longer incubation times of 15 min as the unspecific binding to the beads is decreased. A detection limit of 1 fmol was found when KF exo^" DNA polymerase was used. Also this enzyme was able to distinguish between a match and mismatch template (see Figure 3b, KF exo^'), demonstrating the selectivity of the herein applied approach.

In order to extend the naked-eye detection system to RNA diagnostics, the assay using RT- KTq2 DNA polymerase that has significant reverse transcriptase activity was performed. Therefore, a primer complementary to a sequence in the E. Coli 16S rRNA was employed and the assay was carried out with and without the presence of a 6-fold excess of human total RNA. Doing so, it was possible to reliably detect 0.5 μg of a E. Coli rRNA mixture in less than 10 minutes without any interference of the human RNA (Figure 3b, RT-KTq2). The experiment was carried out without any annealing step although the primer was designed to bind to a double stranded segment of the 16S rRNA. The amount of template sequence bound to the primer (and detected) might therefore be considerably lower than the amount added. Also the lower detection limit for the RT-KTq2 DNA polymerase-promoted reaction was investigated, yielding a similar detection of 1 fmol as for the other DNA polymerases (Figure 9).

Example 4

Materials

5-lodo-2^'-deoxyuridine was purchased from Carbosynth. 2,2,2-trifluoro-/V-(pent-4- ynyl)acetamide¹, and 5-(aminopentynyl)-2'-deoxyuridinetriphosphate² were prepared according to literature (see, e.g., Labbe et al., Tetrahedron (1993), 49(20): 4439-4446; Baccaro et al., Angew Chem Int Ed (2012), 51 (1 ): 254-257). Dry solvents, o-dianisidine dihydrochloride, 8-mercaptooctanoic acid and 16-mercaptohexadecanoic acid were obtained from Sigma-Aldrich and used without further purification. All synthetic reactions were performed under an inert atmosphere. Flash chromatography was performed using Merck silica gel G60 (230-400 mesh) and Merck precoated plates (silica gel 60 F254) were used for TLC. Anion-exchange chromatography was performed on an AktaPurifier (GE Healthcare) with a DEAE Sephadex™ A-25 (GEHealthcare Bio-SciencesAB) column using a linear gradient (0.1 M - 1 .0 M) of triethylammonium bicarbonate buffer (TEAB, pH 7.5). Reversed phase high pressure liquid chromatography (RP-HPLC) for the purification of compounds was performed using a Shimadzu system having LC8a pumps and a Dynamax UV-1 detector. A VP 250/16 NUCLEODUR C18 HTec, 5 m (Macherey-Nagel) column and a gradient of acetonitrile in 50 mM TEAA buffer were used. All compounds purified by RP- HPLC were obtained as their triethylammonium salts after repeated freeze-drying. NMR spectra were recorded on Bruker Avance III 400 (1 H: 400 MHz, 13 C: 101 MHz, 31 P: 162 MHz) or Bruker Avance III 600 (1 H: 600 Mhz, 13 C: 150 Mhz, 31 P: 243 Mhz) spectrometer. The solvent signals were used as references and the chemical shifts converted to the TMS scale and are given in ppm (δ). HR-ESI-MS spectra were recorded on a Bruker Daltronics microTOF II. KF (Klenow Fragment) exo^" DNA polymerase and the respective reaction buffer were purchased from Thermo Scientific. RT-KTq2 DNA polymerase was provided by myPOLS biotec. KlenTaq (KTq) DNA polymerase was expressed and purified as described before (Betz et al., Angew Chem Int Ed (2010), 49(30): 5181 -5184; Summerer et al., Angew Chem INt Ed (2005), 44(30): 4712-4715). T4 polynucleotide kinase PNK was purchased from New England BioLabs. Oligonucleotides were purchased from Metabion and Biomers.net. [v- ³²P]ATP was purchased from Hartmann Analytics and natural dNTPs from Thermo Scientific. Streptavidin sepharose high performance was purchased from GE Healthcare (Matrix: highly cross-linked agarose, 6%; binding capacity/ml: >300 nmol biotin/ml medium; average particle size: 34 μηη). 16S- and 23S-ribosomal RNA was purchased from Roche Life Science, Human Brain Total RNA from Life Science Technologies.

Buffers and solutions

TEAA - 50 mM triethylammonium acetate, pH 7

Conjugation buffer - 0.15 M sodium chloride, 0.1 M sodium phosphate, pH 7

10x KTq reaction buffer - 500 mM Tris-HCI, 160 mM (NH₄)S0₄, 25 mM MgCI₂, 1 %

Tween 20, pH 9.2

Stopping solution - 80% v/v formamide, 20 mM EDTA, 0.025% w/v bromphenol blue, 0.025% w/v xylene cyanol

Binding buffer - 200 mM Na₃P0₄, 1.5 M NaCI

Detection buffer - 100 mM Sodium citrate, pH 5 Synthesis of dT"^¾MTP

To a solution of 25.0 μηιοΙ 5-(aminopentynyl)-2'-deoxyuridinetriphosphate tetrabutylammonium salt² in 1 mL DMF, 37.5 μηηοΙ (1.5 eq., 9.7 mg) of DIPEA were added. In parallel, 1.1 eq. of the mercaptocarboxylic acid and 1.5 eq. DIPEA were dissolved in 1 mL DMF and stirred for 30 min. Both mixtures were then combined and stirred and room temperature for another 12 h. The solvent was removed under reduced pressure and the residual oil was subjected to Ci₈- P-HPLC (95% 50 mM triethylammonium acetate (TEAA) buffer to 100% MeCN). The yield was determined by Nanodrop ND1000 Spectrophotometer with ε(290 nm) = 13300 M^"1-cm^"1. The compounds were diluted in milliQ and kept as a stock solution at -20 °C.

5-(5-(8-mercaptooctanamido)pent-1 -yn-1 -yl)deoxyuridine (dT^7SHTP)

Yield: 70% of dT^7SHTP as triethylammonium salt

¹H NMR (400 MHz, Methanol-d₄): 5 7.99 (s, 1 H, H-C(6)), 6.24 (t, ³ J = 6.8 Hz, 1 H, H-C(1 ')), 4.62 - 4.57 (m, 1 H, H-C(3')), 4.32 - 4.27 (m, 1 H, H-(C5') a)), 4.21 -4.15 (m, 1 H, H-(C^'5)b),

4.08- 4.05 (m, 1 H, H-C(4')), 3.20 (q, ³ J = 7.3 Hz, 18H, -CH₂CH₂CH₂NH-, Et₃N), 2.49 (t, ³ J = 7.3 Hz, 2H, H-C(L8)), 2.44 (t, ³J = 6.8 Hz, 2H, -CH₂CH₂CH₂NH-), 2.29-2.25 (m, 2H, H-2'), 2.20 (t, ³J = 7.3 Hz, 2H, H-C(L2)), 1 .77 (p, ³ = 6.8 Hz, 2H, -CH₂CH₂CH₂NH-), 1 .63-1.55 (m, 4H, H-C(L3+7)), 1 .43-1 .36 (m, 2H, H-C(L4/6)), 1 .31 (t, ³ = 7.1 Hz, 25H, H-C(L4-5/5-6), Et₃N). ³¹P NMR (162 MHz, Methanol-d₄) δ -10.20 (d, ² J = 20.9 Hz), -1 1.15 (d, ² J = 20.8 Hz), - 23.41 (t, ²J = 21 .1 Hz). HR-ESI-MS (m/z): [M-H]^" = calcd: 706.0996; found: 706.0991

5-(5-(16-mercaptohexadecanamido)pent-1 -yn-1 -yl)deoxyuridine (dT^i5SHTP)

Yield: 19% of dT^i5SHTP as triethylammonium salt

1H NMR (600 MHz, Methanol-d₄) 5 7.99 (s, 1 H, H-C(6)), 6.24 (t, ³ J = 6.8 Hz, 1 H, H-C(1 ')), 4.62-4.58 (m, 1 H, H-C(3')), 4.32 - 4.26 (m, 1 H, H-C(5'a)), 4.21 - 4.16 (m, 1 H, H-C(5'b)),

4.09- 4.05 (m,1 H, H-C(4')), 3.19 (q, ³ = 7.2, 24H, Et₃N, -CH₂CH₂CH₂NH-), 2.49 (t, ³ = 7.3 Hz, 2H, H-C(L16)), 2.44 (t, ³ J = 6.8 Hz, 2H, -CH₂CH₂CH₂NH-), 2.29-2.24 (m, 2H, H-C(2')), 2.20 (t, ³ = 7.6 Hz, 2H, H-C(L2)), 1 .77 (p, ³ = 6.8 Hz, 2H, -CH₂CH₂CH₂NH-), 1 .59 (p, ³ = 7.3 Hz, 4H, H-C(L3+15)), 1.44-1 .36 (m, 4H, H-C(L4/14)), 1 .33-1.28 (m, 51 H, H-C(5-13), Et₃N). ³¹P NMR (243 MHz, Methanol-d₄): δ = -10.48 (d, ² = 21 .2 Hz), -1 1.39 (d, ² = 21 .6 Hz), -23.84 (t, ²J = 21 .8 Hz). HR-ESI-MS (m/z): [M-H]^" = calcd: 818.2259; found: 818.2279.

Example 5

Preparation of dT"^HRPTP conjugates

2 mg of maleimide-activated HRP (malHRP, Sigma) were reconstituted in 250 μί conjugation buffer. The concentration was determined by Nanodrop Spectrometer with ε(403 nm) = 102000 M^"1 cm^"1.⁵ 75 μΙ_ of the solution were then mixed with 5 eq. of the thiol-modified nucleotide and incubated overnight in a thermo-shaker at 30 °C. The solution was subjected to anion exchange FPLC (HiTrap Q HP, GE Life Science) with a gradient from 20 mM TRIS- HCI buffer (pH 9) to 1 M NaCI in 20 mM TRIS-HCI (pH 9). The fractions containing the conjugate were identified by PAGE. To remove the FPLC buffer, the pooled fractions were purified via Vivaspin 6 (10,000 MWCO, Sartorius) and subsequently washed with conjugation buffer. The concentrated conjugate was then incubated with 1.5 mM 2-mercaptoethanol in conjugation buffer overnight to block remaining maleimide groups. The excess 2- mercaptoethanol was removed again by Vivaspin concentration and repeated washing with milliQ. The final concentration of the obtained conjugate was again determined via Nanodrop spectrometry with ε(403 nm) = 102000 M^"1 cm^"1.

Example 6

Oligonucleotide sequences

Names and sequences of the oligonucleotides used herein. The incorporation site is marked as a bold and underlined letter in the template sequences. TEG = Triethylene glycol

Table II

Name Sequence

BRAF Primer 5^'-d(GAC CCA CTC CAT CGA GAT TTC) (SEQ ID NO:

15)

Biotinylated BRAF Primer 5^'-biotin-d(TTT TTT TTT TTT TTT TTT TGA CCC ACT

CCA TCG AGA I M C) (SEQ ID NO: 16)

Biotinylated 16S rRNA Primer⁶ 5'-biotin-TEG-d(GCA GTT TCC CAG ACA TTA C) (SEQ

ID NO: 17)

BRAF Template (DNA) 5^'-d(TGC CTG GTG TTT GGG AGA AAT CTC GAT

GGA GTG GGT C) (SEQ ID NO: 18)

BRAF Template (RNA) 5^'-(UGC CUG GUG UUU GGG AGA AAU CUC GAU

GGA GUG GGU C) (SEQ ID NO: 19)

Match template 5^'-d(GGT CTA GCT ACA GAG AAA TCT CGA TGG

AGT GGG TC) (SEQ ID NO: 20)

Mismatch template 5^'-d(GGT CTA GCT ACA GTG AAA TCT CGA TGG

AGT GGG TC) (SEQ ID NO: 21 )

Multiple incorporation template 5^'-d(T GCC TGG TGT TTG GGA AAA AAA AAA AGA

AAT CTC GAT GGA GTG GGT C) (SEQ ID NO: 22) 5'-Radioactive labeling of ODNs

DNA oligonucleotide primers were radioactively labeled at the 5' terminus with a ³²P containing phosphate group using T4 PNK (NEB) and [γ-³²Ρ]ΑΤΡ. The reaction contained primer (0.4 μΜ), PNK reaction buffer (1 x), [γ-³²Ρ]ΑΤΡ (0.8 μθϊ/μΐ.) and T4 PNK (0.4 U/μί) in a total volume of 50 μΙ_ and was incubated for 1 h at 37 °C. The reaction was stopped by denaturing the T4 PNK for 2 min at 95 °C and buffers and excess [γ-³²Ρ]ΑΤΡ were removed by gel filtration (MicroSpin Sephadex G-25). Addition of unlabeled primer (20 μΙ_, 10 μΜ) led to a final concentration of 3 μΜ of diluted radioactive labeled primer.

Primer Extension (PEx) in solution and dPAGE of dT^nHRPTP conjugates

To 1 x polymerase buffer, 150 nM primer (100 nM for competition experiments) and 200 nM template were added. The mixture was annealed at 95 °C for 5 min. Subsequently, DNA polymerase was added (KF exo^" 0.5 U, KTq 100 nM and RT-KTq2 500 nM) and the reaction was started by addition of 10 μΜ of the dNTP (100 μΜ dNTP mixture for competition experiments). Time points were collected by quenching 2 μΙ_ of the reaction mixture with 10 μΙ_ stopping solution (80% v/v formamide, 20 mM EDTA, 0.025% w/v bromphenol blue, 0.025% w/v xylene cyanol).

Denaturing polyacrylamide gels (9 %) were prepared by polymerization of a solution of urea (8.3 M) and bisacrylamide/acrylamide (9 %) in TBE buffer using ammonium peroxodisulfate (APS, 0.08 %) and Ν,Ν,Ν',Ν'-tetramethylethylene-diamine (TEMED, 0.04 %). Immediately after addition of APS and TEMED, the solution was filled in a sequencing gel chamber (Bio- Rad) and left for polymerization for at least 45 min. After addition of TBE buffer (1 x) to the electrophoresis unit, gels were pre-warmed by electrophoresis at 100 W for 30 min and samples were added and separated during electrophoresis (100 W) for approx. 1 .5 h. The gel was transferred to Whatman filter paper, dried at 80 °C in vacuo using a gel dryer (model 583, Bio-Rad) and exposed to a imager screen. Readout was performed with a molecular imager (FX, Bio-Rad). Primer Extension on streptavidin-coated sepharose beads (Verga et al., Chem Comm (2015), 51 (34): 7379-7381 )

10 μΙ_ of a streptavidin-coated sepharose bead slurry (GE Life Science) were spun down (2400 x g) and the supernatant was discharged carefully. The beads were then washed two times with 40 μΙ_ 1 x binding buffer before 9.5 μΙ_ binding buffer and 0.5 μΙ_ 5'-biotinylated primer (100 μΜ were added. Following 10 min of incubation with gentle mixing, 5.5 μΙ_ of 1 mM (D)-+-biotin in binding buffer were added to block the remaining streptavidin moieties. After further 5 min, the beads were spun down again and washed one time with 40 μΙ_ binding buffer and two times with the polymerase buffer. For the primer extension reaction, 22.4 μΙ_ milliQ, 3 μΙ_ 10x polymerase buffer and 1 μΙ_ template were added to the beads and the mixture was mixed every minute for 10 min to prevent the beads from settling down in the tube. Subsequently, 0.6 μΙ_ DNA polymerase solution (KF exo- 0.5 U, KTq 100 nM, RT-KTq2 1 μΜ) and 3 μΙ_ of 10 μΜ dT^nHRPTP were added. The reaction mixture was incubated at room temperature (KF exo^", 15 min) or at 55°C (RT-KTq2/KTq DNA polymerase, 5 min) before the beads were spun down and transferred to an empty spin column cartridge. After removing the reaction mixture by short spin centrifugation in a table top centrifuge, the beads were washed four times with 100 μΙ_ detection buffer in the same way. Subsequently, they were transferred back to reaction tubes, the supernatant was discharged and 20 μΙ_ of the developing solution were applied (0.5 mM o-dianisidine/hydrogen peroxide in detection buffer). The reaction was quenched by the addition of an equal volume of 10 N sulfuric acid after 1 min. Pictures were taken with a Canon PowerShot A620 digital camera. Mass spectrometry of the conjugates

Samples were analyzed using an Agilent 1200 Series HPLC and a Nucleodur 300-5 150/2 C4ec column (Machery-Nagel) with a flow of 0.3 ml/min. 1 .5 mM ammonium acetate was co- injected post-column with a flow of 10 μΙ_/η"ΐίη. The data was acquired on a Bruker micrOTOFII spectrometer. The resulting spectra were deconvoluted using the maximum entropy algorithm provided by Bruker (Dataanalysis 4.1 User Manual, page 191 , Maximum Entropy Deconvolution: Copyright 1991 -2004 Spectrum Square Associates Inc.).

Example 7

Primer Extension (PEx) in solution of dT"^HRPTP conjugates

To 1 x polymerase buffer, 150 nM primer and 200 nM template were added. The mixture was annealed at 95 °C for 5 min. Subsequently, DNA polymerase was added ( Tq 100 nM (myPOLS Biotec), RT-KTq2 100 nM, Vulcano 2x (myPOLS Biotec), Therminator 100 nM (NEB), Dpo 4 2x, Isotherm DNA polymerase ("SD-Pol", myPOLS Biotec) 100 nM, KF exo- 0.1 U) and the reaction was started by addition of 1 μΜ of the conjugate. Additionally, a primer extension reaction with Dpo4 DNA polymerase with 4 mM Mn(ll) chloride present in the reaction solution was performed. Reactions were incubated at 55°C (KTq, RT-KTq2, Vulcano, Therminator, Dpo4, "SD-Pol") or 37°C (KF exo-).

Time points were collected by quenching 2 μΙ_ of the reaction mixture with 10 μΙ_ stopping solution (80% v/v formamide, 20 mM EDTA, 0.025% w/v bromphenol blue, 0.025% w/v xylene cyanol). Samples were analyzed by SDS-PAGE without prior denaturation in SDS loading buffer. Sequences:

Primer sequence (BRAF Primer): 5^'-d(GAC CCA CTC CAT CGA GAT TTC) (SEQ ID NO: 15)

1 1 A Template (Multiple incorporation template): 5^'-d(T GCC TGG TGT TTG GGA AAA AAA AAA AGA AAT CTC GAT GGA GTG GGT C) (SEQ ID NO: 22)

Example 8

Multiple Incorporation with different DNA polymerases

To examine whether the conjugates can also be (multiply) incorporated by different DNA polymerases, a primer extension reaction was performed with SDS-PAGE. As can be seen by the vastly shifted primer bands in Figure 13 (all Lanes except P), all DNA polymerases were able to incorporate the conjugates into the primer strand. Additional bands observed at approximately half the height might be caused by formation of a primer template duplex not found with only the primer (Lane P).

Example 9

Synthesis and evaluation of primary antibody-labeled nucleotides as substrates of DNA polymerases

In order to increase the signal and reduce nonspecific background in the naked-eye detection assay, we explored a new concept. The protein, in this case 146 kDa primary antibody (Ab), that is approximately 3x larger than HRP, is sequence-specifically incorporated into the DNA chain. An enzyme-conjugated secondary antibody with specificity against the primary antibody converts a chromogenic substrate to a detectable product which allow direct visualization. Such developed method was exploited for colorimetric read-out of the nucleotide incorporation that is detectable by the naked eye.

9.1 Synthesis of functionalized dNTPs

Synthesis of azide/amino-modified nucleotides

The most commonly employed method for covalently crosslinking monoclonal antibodies to other molecules is the use of crosslinking reagents (N. Kotagiri, Z. Li, X. Xu, S. Mondal, A. Nehorai, S. Achilefu, Bioconjug. Chem., 2014, 25, 1272-1281 ; b) A. M. Sochaj, K. W. Swiderska, J. Otlewski, Biotechnol. Adv., 2015, 33, 775-784; c) N. J. Alves, N. Mustafaoglu, B. Bilgicer, Bioconjug. Chem., 2014, 25, 1 198-1202; d) D. Zeng, Y. Guo, A. G. White, Z. Cai, J. Modi, R. Ferdani, C. J. Anderson, Mol. Pharmaceutics, 2014, 1 1 , 3980-3987; e) P. L. Ross, J. L. Wolfe, Journal of Pharmaceutical Sciences, 2016, 105, 391 -397; f) H. Gong, I. Holcomb, A. Ooi, X. Wang, D. Majonis, M. A. Unger, R. Ramakrishnan, Bioconjug. Chem., 2016, 27, 217-225; g) P. Akkapeddi, S.- A. Azizi, A. M. Freedy, P. M. S. D. Cal, P. M. P. Gois, G. J. L. Bernardes, Chem. Sci., 2016, 7, 2954-2963). The conjugation approach itself should be (cost-)efficient and applicable to all antibodies. The considerable attention should be focused on developing methods to accurately control the number of sites of conjugation. To get primary antibody-labeled dNTPs, the direct bioconjugation of functionalized dCTPs to primary antibody (mouse anti-GAPDH monoclonal Ab) has been apllied and the conceptual design is illustrated in Fig. 16 and Fig. 17. Both approaches of synthesis of antibody-labeled nucleotides were implemented.

An amino- and azido-functionalized nucleotides have been envisioned for conjugation to the primary antibody. The amino-modified nucleotide (dC^c16NH2TP) was synthesized by using HATU-promoted coupling of 5-(aminopentynyl)-2'-deoxycytidinetriphosphate (A. Baccaro, A.- L. Steck, A. Marx, Angew. Chem. Int. Ed., 2012, 51 , 254-257) with 16-azidehexadecanoic acid to get azide-modified nucleotide (dC^c16N3TP) followed by reduction of the azide-modified nucleotide (dC^c16N3TP) to the amino-modified nucleotide (dC^c16NH2TP) by the Staudinger reaction using mild conditions (Fig. 15).

Synthesis of primary antibody-labeled dNTP (dCAb-EMCSTP)

The first strategy for the synthesis of antibody-labeled nucleotides consists of three steps. First, the primary antibody is functionalized with Traut's reagent. The number of sulfhydryl groups per antibody (4 sulfhydryl groups per primary antibody) was determined with Ellman's reagent. Subsequently, amino-modified nucleotide (dC^c16NH2TP) is functionalized with the EMCS (6-maleimidohexanoic acid N-hydroxysuccinimide ester) linker to get the maleimide- modified dNTP. Finally, the conjugate is obtained by mixing the functionalized primary antibody and maleimide-modified dNTP (dC^^TP, Fig. 16). Purification of the primary antibody-labeled nucleotide (dC^^'^^TP) was performed using anion-change protein liquid chromatography (FPLC) (22 %).

Synthesis of primary antibody-labeled dNTP (dCAb-DBCOTP)

The second approach of the bioconjugation process is based on strain-promoted alkyne- azide cycloaddition, in which the primary antibody is activated with a dibenzocyclooctyne (DBCO) moiety and subsequently linked covalently with an azide-modified dNTP (Fig. 17). For DBCO functionalization, DBCO-PEG₄-NHS has been used to react with NH₂ groups of lysine on the primary antibody. The degree of DBCO incorporation on the primary antibody (4 DBCO per primary antibody) was calculated by dividing the molar concentration of DBCO by the molar concentration of the antibody (see Example 10). The DBCO-functionalized primary antibody was conjugated to azide-labeled nucleotide (dC^c16N3TP) to obtain the antibody- labeled nucleotide (άΟ^^ΤΡ). Purification of antibody-labeled nucleotide (dC^-^TP) was performed using anion-change protein liquid chromatography (FPLC) (15 %).

9.2 Processing of primary-antibody labelled dNTPs by DNA polymerases

In order to validate whether DNA polymerases are capable to incorporate these primary antibody-labeled dNTPs (dC^-^TP, dC^Ab-^DBCOTP) into DNA, single-nucleotide incorporation experiments were performed. In brief, enzymatic incorporations of primary antibody-labeled nucleotides (dC^-^TP, dC^Ab-^DBCOTP) into DNA was studied using a primer extension (PEX) assay with the primary antibody-labeled nucleotides (dC^^'^^TP,

_gs substrates, a 5'-³²P labeled primer, a template in the sequence context of the B-type Raf kinase (BRAF) T1796A point mutation that is strongly associated with carcinogenesis (H. Davies, G. R. Bignell, C. Cox, P. Stephens, S. Edkins, S. Clegg, J. Teague, H. Woffendin, M. J. Garnett, W. Bottomley, et al., Nature, 2002, 417, 949-954, see for sequences Table III) and diverse DNA polymerases (KlenTaq {KTq) and KOD DNA polymerase). The insertion of primary antibody-labeled nucleotides (dC^^'^^TP, dC"^3' ^DBCOTP) into DNA was subsequently analyzed by denaturing polyacrylamide gel electrophoresis (PAGE) and autoradiography. We found, that the modified nucleotides (dC*^15" EMCS_{TPj dC}Ab-DBco_TP) _were successfully used by both KOD and KTq DNA polymerase as substrates despite their size (Figure 18).

Table III. Primers and templates used for PEX experiments.

^[a| In the template, segments that form a duplex with the primer are in bold. 9.3 Naked-eye detection assay

The employed approach for the development of a DNA sequence-selective naked-eye detection system is shown in Fig. 19. This naked-eye assay offers major advantages: First, each primary antibody contains several epitopes that can be bound by the labeled secondary antibody, allowing for signal amplification. Secondly, different visualization markers (fluorescent tags and enzymes such as horseradish peroxidase and alkaline phosphatase) bound to the secondary antibody can be used with the same primary antibody.

For the naked eye detection system, the biotinylated primer strand is immobilized on the streptavidin-coated beads. Due to the template-dependency of DNA polymerases, primer that bind sequence-selectively to a target sequence are extended by elongation with a primary antibody-modified nucleotide. An enzyme-conjugated secondary antibody (goat Anti- Mouse IgG H+L, HRP) with specificity against the primary antibody converts the chromogenic substrate to a detectable product which allow direct visualization (Fig. 19). The negative control reactions (Fig. 19: absence of template and absence of dC^^'^^TP) have been performed to reveal false positive signals caused by deficient washing or unspecific binding of the primary antibody-modified nucleotides. This developed method can be exploited for colorimetric read-out of the nucleotide incorporation that is detectable by the naked eye.

10. Experimental section

This section further describes how data as provided in Example 9 have been obtained. 5- (aminopentynyl)-2'-deoxycytidinetriphosphate (A. Baccaro, A.-L. Steck, A. Marx, Angew. Chem. Int. Ed. , 2012, 51, 254-257) was prepared according to the literature procedure. 6- Maleimidohexanoic acid /V-hydroxysuccinimide ester, DBCO-PEG₄-NHS were purchased from Sigma Aldrich. Other chemicals were purchased from commercial suppliers and were used as received. Flash chromatography was performed using Merck silica gel G60 (230-400 mesh) and Merck precoated plates (silica gel 60 F254) were used for TLC. Reversed phase high pressure liquid chromatography (RP-HPLC) for the purification of compounds was performed using a Shimadzu system having LC8a pumps and a Dynamax UV-1 detector. A VP 250/16 NUCLEODUR C18 HTec, 5 μηι (Macherey-Nagel) column and a gradient of acetonitrile in 50 mM TEAA buffer were used. All nucleotides purified by RP-HPLC were obtained as their triethylammonium salts after repeated freeze-drying. NMR spectra were recorded on Bruker Avance II I 400 (1 H: 400 MHz, 13 C: 101 MHz, 31 P: 162 MHz) or Bruker Avance III 600 (1 H: 600 MHz, 13 C: 150 MHz, 31 P: 243 MHz) spectrometer. The solvent signals were used as references and the chemical shifts converted to the TMS scale and are given in ppm (δ). HR-ESI-MS spectra were recorded on a Bruker Daltronics microTOF II.

KTq DNA polymerase was expressed and purified as described before (D. Summerer, N. Z. Rudinger, I. Detmer, A. Marx, Angew. Chem. Int. Ed., 2005, 44, 4712; b) K. Betz, F. Streckenbach, A. Schnur, T. Exner, W. Welte, K. Diederichs, A. Marx, Angew. Chem. Int. Ed., 2010, 49, 5181 ). T4 polynucleotide kinase PNK was purchased from New England BioLabs. Oligonucleotides were purchased from Biomers.net. [γ-³²Ρ]ΑΤΡ was purchased from Hartmann Analytics and natural dNTPs from Thermo Scientific. Streptavidin sepharose high performance was purchased from GE Healthcare (Matrix: highly cross-linked agarose, 6%; binding capacity/ml: >300 nmol biotin/ml medium; average particle size: 34 μηη). Primary and secondary antibodies were purchased from Abeam.

Table IV. Buffer and Solutions

10.1 Synthesis of modified nucleotides

Synthesis of 16-azidohexadecanoic acid

To a stirred solution of 16-bromohexadecanoic acid (1 eq.) in DMF was added NaN₃ (2 eq.). Then the reaction mixture was warmed to 100 °C and refluxed for 16 h, then cooled to room temperature. The mixture was extracted with ethyl acetate (3x50 mL) and the combined organic phase was washed with water (3x50 mL) and brine (50 mL) and dried over MgS0₄ and then concentrated to get 16-azidohexadecanoic acid. ¹H NMR (400 MHz, CDCI₃): ¹ H NMR (400 MHz, CDCI₃): δ 3.25 (t, J=7.0 Hz, 2H, H-16), 2.35 (t, J=7.5 Hz, 2H, H-2), 1 .69- 1 .54 (m, 4H, H-3, H-15), 1 .42-1 .21 (m, 22H, H-4-14). ¹³C NMR (101 MHz, CDCI₃): δ 179.75, 51.66, 34.12, 29.76, 29.72, 29.68, 29.63, 29.57, 29.38, 29.30, 29.21 , 28.99, 26.87, 24.84 (NMR spectra are shown in Fig. 20).

Synthesis of

5-[5-(16-azidohexadecanamido)pent-1-yn-1-yl]-2'-deoxycytidinetriphosphate

(dCC16N3TP)

To a solution of 16-bromohexadecanoic acid (2 eq.) in DMF was added Et₃N (5 eq.) followed by HATU (2 eq.). The mixture was stirred for 15 min at rt and then 5-(aminopentynyl)-2'- deoxycytidinetriphosphate (0.05 g, 1 eq.) was added and mixture was stirred for 16 h at rt. The solvent was removed and the product was purified from the crude reaction mixture by RP-HPLC on a C18 column (95% 50 mM triethylammonium acetate (TEAA) buffer to 100% MeCN). The product was isolated as a white solid (35 %). ¹H NMR (400 MHz, Methanol-d₄): δ 8.15 (s, 1 H, H-6), 6.29 (t, J=6.4 Hz, 1 H, H-1 '), 4.69-4.59 (m, 1 H, H-3'), 4.44-4.33 (m, 1 H, H- 5'a), 4.32-4.21 (m, 1 H, H-5'b), 4.20-4.06 (m, 1 H, H-4'), 3.37 (p, J=1 .6 Hz, 22H, H-L-16), 3.25 (q, J=7.2 Hz, 17H, Et₃N), 2.54 (t, J=6.8 Hz, 2H, -CH₂CH₂CH₂NH-), 2.45-2.36 (m, 1 H, H-2'b), 2.30-2.18 (m, 3H, H-L-2, H-2'a), 1 .84 (p, J=6.7 Hz, 2H, -CH₂CH₂CH₂NH-), 1 .64 (p, J=7.3 Hz, 4H, H-L-3, H-L-15), 1 .48-1 .26 (m, 48H, H-L-4-14, Et₃N). ³¹P NMR (162 MHz, Methanol-d₄): δ -10.48 (d, J=20.8 Hz, P), -1 1.43 (d, J=21 .5 Hz, P), -23.83 (d, J=20.7 Hz, P). HRMS (ESI-): calcd. for C₃oH₅₁N₇0₁₄P₃: 826.2707; found 826.2729 (NMR spectra are shown in Fig. 21 ).

Synthesis of 5-[5-(16-aminohexadecanamido)pent-1-yn-1-yl]- 2'-deoxycytidinetri- phosphate (dC^c16NH2TP)

The azide-modified triphosphate (0.03g, 1 eq.) was dissolved in water/methanol/triethylamine (2:2:1 ) and tris-(2-carboxyethyl)-phosphine hydrochloride (7 eq.) was added and stirred for 3 hours at room temperature until complete conversion. The solvents were evaporated and the product was purified by RP-HPLC on a C18 column (95% 50 mM triethylammonium acetate (TEAA) buffer to 100% MeCN). ). The product was isolated as a white solid (20 %). ¹H NMR (400 MHz, Methanol-d₄): δ 8.12 (s, 1 H, H-6), 6.29 (t, J=6.5 Hz, 1 H, H-1 '), 4.65-4.58 (m, 1 H, H-3'), 4.39-4.30 (m, 1 H, H-5'a), 4.30-4.22 (m, 1 H, H-5'b), 4.17-4.1 1 (m, 1 H, H-4'), 3.26 (q, J=7.3 Hz, 1 1 H, Et₃N), 2.99 (t, J=7.7 Hz, 2H, H-L-16), 2.55 (t, J=6.8 Hz, 2H, -CH₂CH₂CH₂NH- ), 2.48-2.35 (m, 1 H, H-2'b), 2.31 -2.15 (m, 3H, H-L-2, H-2'a), 1 .85 (p, J=6.7 Hz, 2H, - CH₂CH₂CH₂NH-), 1 .80-1 .61 (m, 4H, H-L-3, H-L-15), 1.52-1.23 (m, 36H, H-L-4-14, Et₃N). ³¹P NMR (162 MHz, Methanol-d₄): δ -10.15 (d, J=20.7 Hz, P), -10.98 (d, J=20.2 Hz, P), -23.12 (t, J=20.6 Hz, P). HRMS (ESI-): calcd. for C₃oH₅3N₅0₁₄P3: 800.2802; found 800.2825. (NMR spectra are shown in Fig. 22).

Synthesis of primary antibody-labeled nucleotide (dC ^^TP)

The antibody-labeled dNTP has been prepared by reacting of amino-modified nucleotide (dC^c16NH2TP) with EMCS linker (6-maleimidohexanoic acid N-hydroxysuccinimide ester) followed by addition of sulfhydryl-functionalized antibody. The amino-modified nucleotide (dC^c16NH2TP,10 eq.) was mixed with EMCS linker (6-maleimidohexanoic acid N- hydroxysuccinimide ester, 10 eq.) in PBS buffer (pH 7.2) for 2 h at room temperature. The primary antibody (mouse anti-GAPDH monoclonal antibody, 100 μg) was mixed with Traut's reagent (10 eq.) in PBS buffer (pH 8) to get sulfhydryl-functionalized primary antibody. Afterwards, excess reagent was removed using the Amicon Ultra Centrifugal Filter. The number of sulfhydryl groups per antibody was determined with Ellman's reagent. The sulfhydryl-functionalized primary antibody was added to the solution of amino-modified nucleotide with EMCS linker and was mixed for 16 h at 4°C. The solution was subjected to anion exchange FPLC (HiTrap Q HP, GE Life Science) with a gradient from Buffer A (20 mM TRIS, pH 9) to Buffer B (20 mM TRIS, 1 M NaCI, pH 9). To remove the FPLC buffer, the pooled fractions were purified via Vivaspin 6 (30,000 MWCO, Sartorius) and subsequently washed with PBS buffer. The final concentration of the obtained conjugate was determined using their respective molar extinction coefficient (204 000 M^"1cm^"1 at 280 nm). Synthesis of primary antibody-labeled nucleotide (d( ^{b DBCO}TP)

The antibody was first reacted with DBCO-PEG₄-NHS following a standard procedure for antibody conjugation through the NH₂ group. In brief, primary antibody (mouse anti-GAPDH monoclonal antibody, 100 \ig) was incubated with DBCO-PEG₄-NHS (10 eq.) in PBS buffer (pH 7.4) for 2 h at room temperature. After reaction, the free DBCO-PEG₄-NHS was removed using the Amicon Ultra Centrifugal Filter. To determine the number of DBCO molecules on the antibody, the absorbance at 309 and 280 nm was measured. The molar concentrations of DBCO and antibody were determined using their respective molar extinction coefficient (12 000 M^"1cm^"1 for DBCO at 309 nm and 204 000 M^"1cm^"1 for antibody at 280 nm). The number of DBCO molecules per antibody was calculated by dividing the molar concentration of DBCO by the molar concentration of antibody. The DBCO-functionalized antibody reacted with azide-modified nucleotide (dC^c16N3TP, 10 eq.). The Cu-free click reaction was conducted at 4 °C for 16 h. The solution was subjected to anion exchange FPLC (HiTrap Q HP, GE Life Science) with a gradient from Buffer A (20 mM TRIS, pH 9) to Buffer B (20 mM TRIS, 1 M NaCI, pH 9). To remove the FPLC buffer, the pooled fractions were purified via Vivaspin 6 (30,000 MWCO, Sartorius) and subsequently washed with PBS buffer. The final concentration of the obtained conjugate was determined using their respective molar extinction coefficient (204 000 M^"1cm^"1 at 280 nm).

10.2 Enzymatic incorporation of modified dNTPs

Primer extension experiment

The reaction mixture (20 μί) contained DNA polymerase (KOD 200 nM, KTq 100 nM), template (0.2 μΜ), primer (150 nM) [γ³²Ρ]ΑΤΡ, natural or modified dNTPs (10 μΜ) in 1 x polymerase buffer. Primer was labeled by use of [γ³²Ρ]ΑΤΡ according to standard techniques. Reaction mixtures were incubated for time points at 55 °C and analysed by PAGE electrophoresis.

PEX on streptavidin-coated sepharose beads

PEX on streptavidin-coated sepharose beads (dC^Ab'EMCSTP)

10 L of a streptavidin-coated sepharose bead slurry (GE Life Science) were spun down (2400 x g) and the supernatant was discharged carefully. The beads were then washed three times with 100 μΙ_ 1 x binding buffer and 9.5 μΙ_ binding buffer and 0.5 μΙ_ 5'-biotinylated primer (100 μΜ) were added. Following 30 min of incubation with gentle mixing, 5.5 μΙ_ of 1 mM (D)-+-biotin in binding buffer were added to block the remaining streptavidin moieties. After further 10 min, the beads were spun down again and washed three times with 100 μΙ_ binding buffer and three times with the polymerase buffer. For the primer extension reaction, 22.4 μΙ_ milHQ, 3 μΙ_ 10x polymerase buffer and 1 μΙ_ template were added to the beads and the mixture was mixed every minute for 10 min to prevent the beads from settling down in the tube. Subsequently, DNA polymerase and 3 μΙ_ of 10 μΜ dC^Ab"EMCSTP were added. The reaction mixture was incubated at 55°C for 5 min, then the beads were spun down and transferred to an empty spin column cartridge. After removing the reaction mixture by short spin centrifugation in a table top centrifuge, the beads were washed three times with 100 μΙ_ binding buffer and the secondary antibody (goat anti-mouse IgG H and L, HRP) was added to the reaction mixture and incubated for 1 h and then beads were washed two times with binding buffer and three times with 100 μΙ_ detection buffer. Subsequently, the supernatant was discharged and 20 μΙ_ of the developing solution were applied (1 mM o-dianisidine/H₂0₂ in detection buffer). The reaction was quenched by the addition of 5 μΙ_ of 10 N sulfuric acid after 1 min. Pictures were taken with a digital camera.

Claims

1 . A modified nucleoside, which comprises a structure represented by formula (I) below

Y-L-X (I)

wherein

Y is a pyrimidine or purine nucleoside, wherein

L is a linker, and

2. The modified nucleoside of claim 1 , wherein said cargo is a protein.

3. The modified nucleoside of claim 2, wherein said protein is an enzyme E or antibody A.

4. The modified nucleoside of any one of the preceding claims, wherein the nucleoside is a (ribosyl)nucleosid, a desoxy(ribosyl)nucleosid, an arabinosylnucleosid or an (methylribosyl)nucleosid.

5. The modified nucleoside of any one of the preceding claims comprising a structure represented by formula (II) below

R¹-Y-L-X (II)

wherein R¹ is H, or a (poly)phosphate represented by , with n being an integer from 1 to 20, and Z being selected from the group consisting of H, free electron, and ribose or a desoxyribose.

6. The modified nucleoside of any one of the preceding claims, wherein the purine nucleoside has a purine selected from the group consisting of a deazapurine, an azidopurine, an alkylpurine, a thiopurine, a bromopurine, an O-alkylpurine, and an isopurine.

7. The modified nucleoside of any one of the preceding claims, wherein the pyrimidine nucleoside is selected from the group consisting of (desoxy)cytidine, (desoxy)thymidine, and (desoxy)uridine.

8. The modified nucleoside of any one of any one of the preceding claims, which comprises a structure represented by formula (III) below

wherein R³ is H, or , with n being an integer from 1 to 20 (preferably

1 , 2 or 3; more preferably 3), and Z being selected from the group consisting of H, free electron, and ribose or desoxyribose (preferably H);

wherein X is a cargo having a volume of 15000 A³ or more or alternatively at least 90 amino acids for cargo X being a protein.

9. The modified nucleoside of any one of the preceding claims, wherein the linker comprises an amide, an amidine, a disulfide, a hydrazine, a thioether and/or an ester.

10. The modified nucleoside of claim 9, wherein the amide, disulfide, hydrazine, thioether and/or ester group is generated by coupling of a reactive chemical group with a target functional group.

1 1 . The modified nucleoside of claim 10, wherein the reactive chemical group is a maleimide-reactive group.

12. The modified nucleoside of claim 10, wherein the target functional group is a sulfhydryl moiety.

13. The modified nucleoside of any one of claims 3 to 12, wherein said enzyme is a reporter enzyme.

14. The modified nucleoside of claim 13, wherein said reporter enzyme is selected from the group consisting of horseradish peroxidase (HRP; SEQ ID NO: 1 ), alkaline phosphatase (AP; SEQ ID NO: 2), glucose-oxidase (GOX; SEQ ID NO:3), luciferase (SEQ ID NO: 4), chloramphenicol acetyl transferase, (CAT; SEQ ID NO: 5), β- Galactosidase (β-Gal; SEQ IS NO: 6), catalase (SEQ ID NO: 7), urease (SEQ ID NO:8), and soybean peroxidase (SEQ ID NO: 9).

15. Use of the modified nucleoside of any one of claims 1 to 14 as a substrate for a DNA polymerase, a Reverse Transcriptase or an RNA polymerase.

16. Use of the modified nucleoside of any one of claims 1 to 14 in an oligonucleotide/nucleic acid molecule amplification method.

17. Use of the modified nucleoside of any one of claims 1 to 14 in the in vitro diagnosis or prognosis of a disease or disorder of a subject, which disease is associated with a target sequence.

18. Use of the modified nucleoside of any one of claims 1 to 14 for detection of at least one single nucleotide variation/polymorphism (SNP), which is comprised in a target sequence.

19. Use of the modified nucleoside of any one of claims 1 to 14 for the detection of target sequences, such as pathogenic target sequences, such as those from bacteria, viruses including retroviruses, fungi, or unicellular organisms.

20. Use of the modified nucleoside of any one of claims 1 to 14 for the discrimination of a matched primer and a mismatched primer, wherein said primers hybridize to a target sequence and wherein the mismatched primer comprises a non-canonical nucleotide at its 3' end in relation the target sequence to which it hybridizes.

21 . A modified nucleoside of any one of claims 1 to 14 for use in an in vitro method of diagnosis or prognosis of a disease or disorder of a subject.

22. A nucleic acid molecule comprising a modified nucleoside of any one of claims 1 to

14.

23. A method for producing a modified nucleoside of any one of claims 1 to 14, comprising

a) providing a pyrimidine or purine nucleoside Y,

b) conjugating the pyrimidine or purine nucleoside Y to a linker L; and

c) conjugating the linker L to a cargo X having a volume of 15000 A³ or more or to a cargo having at least 90 amino acids for cargo X being a protein,

thereby obtaining the modified nucleoside.

24. A preparation comprising a modified nucleoside of any one of claims 1 to 14 or a nucleic acid molecule of claim 22.

25. A kit or kit-of-parts comprising a modified nucleoside of any one of claims 1 to 14 or a nucleic acid molecule of claim 22.

26. An in vitro method for diagnosing or predicting a disease or disorder associated with a target sequence, or a pathogen associated with a target sequence, in a subject, the method comprising

a) contacting the target sequence obtained from a subject's sample with a modified nucleoside of any one of claims 1 to 14.

27. An in vitro method for detecting a target sequence in a sample, the method comprising

a) contacting said target sequence with a modified nucleoside of any one of claims 1 to 14.

28. A modified nucleoside of any one of claims 1 to 14 or a nucleic acid molecule comprising said modified nucleoside for use in treating a disease or disorder in a subject.