Macromolecular nucleotide compounds and methods for using the same
The invention describes new structures of the nucleotide conjugates (nuc-macromolecules) comprising at lease one nucleotide moiety coupled to at least one macromolecular compound via a short linker. These conjugates can be used as substrates for various kinds of polymerizing enzymes in the enzymatic synthesis of nucleic acids. In particular, these compounds can be used for labeling nucleic acids.
1. A nucleotide conjugate (nuc-macromolecule) having the structure
wherein:
Base is selected independently from the group consisting of purine nucleobases, pyrimidine nucleobases and analogs of purine or pyrimidine nucleobases capable of forming Watson-Crick-base pairing,
R 1 —is H,
R 2 —is selected independently from the group consisting of H, OH, halogen, NH 2 and SH,
R 3 —is selected independently from the group consisting of H, OH, halogen,—O—PO 3 H, SH, N 3 , NH 2 , O—CH 3 , O—CH 2 —O—CH 3 and O—CH 2 —CH═CH 2 ,
R 4 —is H or OH,
R 5 —is selected independently from the group consisting of a monophosphate group, a triphosphate group, a tetraphosphate group, and an alpha thiotriphosphate group; and
a pharmaceutically acceptable salt thereof, and
Linker is a linker component, which connects said Base and Polynucleotide 1,
wherein the length of the Linker is in the range of from 5 to 100 chain atoms, and said Linker is attached covalently to the Base at one of the positions N 4 or C-5 when the Base is a pyrimidine nucleobase, or at one of the positions N 6 or C-8 when the Base is a purine nucleobase, or at position C-7 when the Base is a 7-deaza-purine nucleobase, and is attached covalently to said Polynucleotide 1, and
wherein said Polynucleotide 1 is selected from the group consisting of at least one nucleic acid chain having a length of at least 10 nucleotide monomers, and at least one nucleic acid chain having a length of at least 10 nucleotide monomers wherein said nucleic acid chain is labeled with at least one Detectable Marker Unit.
2. The nucleotide conjugate (nuc-macromolecule) according to claim 1 wherein the purine nucleobase or its analog is selected from the group consisting of adenin-9-yl; 7-deazaadenin-9-yl; guanin-9-yl; 7-deaza-guanin-9-yl;
wherein:
R 1 —is a sugar moiety of the nucleotide conjugate, and
R 2 —is the linker moiety of the nucleotide conjugate.
3. The nucleotide conjugate (nuc-macromolecule) according to claim 1 wherein the pyrimidine nucleobase or its analog is uracil-1-yl; or cytosin-1-yl;
wherein:
R 1 —is a sugar moiety of the nucleotide conjugate, and
R 2 —is the linker moiety of the nucleotide conjugate.
4. The nucleotide conjugate (nuc-macromolecule) according to claim 1 , wherein the Linker has at least one cleavable bond.
5. The nucleotide conjugate (nuc-macromolecule) according to claim 4 , wherein the cleavable bond is a disulfide bond.
6. The nucleotide conjugate (nuc-macromolecule) according to claim 1 , wherein the length of the Linker ranges from 5 to 50 chain atoms.
7. The nucleotide conjugate (nuc-macromolecule) according to claim 1 , wherein the length of the Linker ranges from 20 to 50 chain atoms.
8. The nucleotide conjugate (nuc-macromolecule) according to claim 1 , wherein the length of the Linker ranges from 20 to 100 chain atoms.
9. The nucleotide conjugate (nuc-macromolecule) according to claim 1 , wherein the length of the linker ranges from 30 to 100 chain atoms.
10. The nucleotide conjugate (nuc-macromolecule) according to claim 1 , wherein the linker is in the range of from 5 to 100 chain atoms and has the structure:
L-Polymer-T,
wherein (L) is a linkage moiety attached covalently at one end to the nucleobase at position N 4 or C-5 when the Base is a pyrimidine nucleobase, at position N 6 or C-8 when the Base is a purine nucleobase, or at position C-7 when the Base is a 7-deaza-purine nucleobase, and is attached covalently on the other end to the Polymer, and linkage moiety (T) has two ends, one of which is attached covalently to the Polymer and the other end of which is attached covalently to the Polynucleotide, and wherein the Polymer is a water soluble polymer.
11. The nucleotide conjugate (nuc-macromolecule) according to claim 10 , having a linker which is bound to the nucleobase of the nuc-component via linkage moiety (L) selected from the group: 3-amino-allyl and 3-amino-propargyl, wherein remaining linker is bound to amino-group of the said linkage moiety (L).
12. The nucleotide conjugate (nuc-macromolecule) according to claim 10 wherein the structure of the Base and linkage moiety (L) is selected from the group consisting of:
R 6 —NH—R 7 , R 6 —O—R 7 , R 6 —S—R 7 , R 6 —SS—R 7 , R 6 —CO—NH—R 7 , R 6 —NH—CO—R 7 , R 6 —CO—O—R 7 , R 6 —O—CO—R 7 , R 6 —CO—S—R 7 , R 6 —S—CO—R 7 , R 6 —P(O) 2 —R 7 , R 6 —Si—R 7 , R 6 —(CH 2 ) n —R 7 ,
R 6 —(CH 2 ) n —R 7 , R 6 -A-(CH 2 ) n —R 7 , R 6 —(CH 2 ) n —B—R 7 ,
R 6 —(CH═CH—) n —R 7 , R 6 -(A-CH═CH—) n —R 7 , R 6 —(CH═CH—B—) n —R 7 ,
R 6 -A-CH═CH—(CH 2 —) n —R 7 , R 6 —(—CH═CH—CH 2 ) n —B—R 7 ,
R 6 —(C≡C—) n —R 7 , R 6 -(A-C≡C—) n —R 7 , R 6 —(C≡C—B—) n —R 7 ,
R 6 -A-C≡C—(CH 2 —) n —R 7 , R 6 —(—C≡C—CH 2 ) n —B—R 7 , and R 6 —(—C≡C—CH 2 —CH 2 —) n —B—R 7 ; and
R 6 is the N 4 or C-5 position when the Base is a pyrimidine nucleobase, or the N 6 or C-8 position when the Base is a purine nucleobase, or the C-7 position when the Base is a 7-deaza-purine nucleobase, and
R 7 is the remaining Linker connected to the polynucleotide 1 moiety, and A and B are selected from the group consisting of —NH—, —O—, —S—, —SS—, —CO—NH—,
—NH—CO—, —CO—O—, —O—CO—, —CO—S—, —S—CO—, —P(O) 2 —, —Si(CH 3 ) 2 —and —(CH 2 ) n —, wherein n ranges from 1 to 5.
13. The nucleotide conjugate (nuc-macromolecule) according to claim 10 , wherein said Polymer is selected independently from the group consisting of:
polyethylene glycol (PEG), polysaccharides, dextran, polyamides, polypeptides, polyphosphates, polyalkylene glycols, copolymers from ethylene glycol and propylene glycol, polyolefinic alcohols, polyvinyl pyrrolidones, poly(hydroxyalkylmethacrylamides), polyhydroxyalkylmethacrylates, polyacrylic acid, polyacrylamide, and polyvinyl alcohol.
14. The nucleotide conjugate (nuc-macromolecule) according to claim 10 , wherein said Polymer consists of identical monomers.
15. The nucleotide conjugate (nuc-macromolecule) according to claim 10 , wherein said polymer consists of two different kinds of monomers.
16. The nucleotide conjugate (nuc-macromolecule) according to claim 10 , linkage moiety (T) is bound to the Polynucleotide 1 and wherein linkage moiety (T) is selected from the group consisting of —O—, —NH—, —CONH—, —NHCO—, and —S—.
17. The nucleotide conjugate (nuc-macromolecule) according to claim 1 wherein the Polynucleotide 1 is an oligonucleotide with a length of from about 10 to about 100 nucleotides.
18. The nucleotide conjugate (nuc-macromolecule) according to claim 1 wherein the Polynucleotide 1 has a length of about 20 to 100000 nucleotides.
19. The nucleotide conjugate (nuc-macromolecule) according to claim 1 wherein the Polynucleotide 1 is single stranded.
20. The nucleotide conjugate (nuc-macromolecule) according to claim 1 wherein the Polynucleotide 1 is double stranded.
21. The nucleotide conjugate (nuc-macromolecule) according to claim 1 wherein the Polynucleotide 1 is selected from the group consisting of DNA, RNA, modified DNA, modified RNA and PNA.
22. The nucleotide conjugate (nuc-macromolecule) according to claim 1 , wherein the Base is coupled via said Linker to the 3′-position of said Polynucleotide 1.
23. The nucleotide conjugate (nuc-macromolecule) according to claim 1 , wherein the Base is coupled via said linker to the 5′-position of said Polynucleotide 1.
24. The nucleotide conjugate (nuc-macromolecule) according to claim 1 , wherein the Base is coupled via said Linker to an internal position of said Polynucleotide 1.
25. A nucleotide conjugate (nuc-macromolecule) according to claim 1 , wherein Polynucleotide 1 is labeled with at least one Detectable Marker Unit.
26. The nucleotide conjugate (nuc-macromolecule) according to claim 25 , wherein the said Detactable Marker Unit is selected from the group consisting of a dye, fluorescent dye, protein, polynucleotide, dendrimer, quantum dot, nanoparticle, microparticle and water soluble polymer.
27. The nucleotide conjugate (nuc-macromolecule) according to claim 25 consisting of multiple Detectable Marker Units of number (N), wherein (N) is a positive integer of from 2 to 100.
28. A composition comprising a compound having the structure of claim 1 and at least one further chemical ingredient selected from the group consisting of buffer, at least one DNA polymerase, at least one primer, and at least one 2′-deoxynucleoside 5′-triphosphate.
29. A polynucleotide (polynucleotide 4) consisting of at least one nucleotide conjugate having the structure according to claim 1 , wherein said nucleotide conjugate is connected via its 5′-monophosphate diester group to the 3′-OH— terminal group of said polynucleotide 4
and wherein:
Base is selected independently from the group consisting of purine nucleobases, pyrimidine nucleobases and analogs of purine or pyrimidine nucleobases capable of forming Watson-Crick-base pairing,
R 1 —is H,
R 2 —is selected independently from the group consisting of H, OH, halogen, NH 2 and SH,
R 3 —is selected independently from the group consisting of H, OH, halogen, —O—PO 3 H, SH, N 3 , NH 2 , O—CH 3 , O—CH 2 —O—CH 3 and O—CH 2 —CH═CH 2 ,
R 4 —is H,
R 5 —is a monophosphate diester group or a pharmaceutically acceptable salt thereof, and
Linker is a linker component, which connects said Base and Polynucleotide 1, and
wherein the length of the Linker is in the range of from 5 to 100 chain atoms, and said Linker is attached covalently to the Base at one of the positions N 4 or C-5 when the Base is a pyrimidine nucleobase, or at one of the positions N 6 or C-8 when the Base is a purine nucleobase, or at position C-7 when the Base is a 7-deaza-purine nucleobase, and is attached covalently to said Polynucleotide 1, and
wherein said Polynucleotide 1 consists of at least one nucleic acid chain having a length of at least 10 nucleotides, and Polynucleotide 4 is a nucleic acid.
30. A polynucleotide (polynucleotide 4) consisting of at least one nucleotide conjugate having the structure according to claim 1 , wherein said nucleotide conjugate is connected via its 5′-monophosphate diester group to the 3′-OH— terminal group of said Polynucleotide 4, and a further Polynucleotide 5 connected via its 5′-monophosphate diester group to the 3′-position of the said nucleotide conjugate
and wherein:
Base is selected independently from the group consisting of purine nucleobases, pyrimidine nucleobases and analogs of purine or pyrimidine nucleobases capable of forming Watson-Crick-base pairing,
R 1 —is H,
R 2 —is selected independently from the group consisting of H, —OH, halogen, —NH 2 and —SH,
R 3 —is selected independently from the group consisting of —O— and —O—(PO 2 )—O—; wherein Polynucleotide 5 is a nucleic acid,
R 4 —is H,
R 5 —is a monophosphate diester group or a pharmaceutically acceptable salt thereof, and
Linker is a linker component, which connects said Base and Polynucleotide 1, and
wherein the length of the Linker is in the range of from 5 to 100 chain atoms, and said Linker is attached covalently to the Base at one of the positions N 4 or C-5 when the Base is a pyrimidine nucleobase, or at one of the positions N 6 or C-8 when the Base is a purine nucleobase, or at position C-7 when the Base is a 7-deaza-purine nucleobase, and is attached covalently to said Polynucleotide 1, and
wherein said Polynucleotide 1 consists of at least one nucleic acid chain having a length of at least 10 nucleotides, and Polynucleotide 4 is a nucleic acid.
31. A nucleotide conjugate (nuc-macromolecule) having the structure:
wherein:
Polynucleotide 1 is selected from the group consisting of at least one nucleic acid chain having a length of at least 10 nucleotides, and at least one nucleic acid chain having a length of at least 10 nucleotide monomers wherein said nucleic acid chain is labeled with at least one Detectable Marker Unit, and
Polynucleotide 2 is selected from the group consisting of at least one nucleic acid chain having a length of between 10 and 10000 nucleotide monomers, at least one nucleic acid chain having a length between 10 and 10000 nucleotide monomers and said nucleic acid chain is labeled with at least one Detectable Marker Unit, and
Polynucleotide 1 is attached to Polynucleotide 2 via complementary base pairing, and
Base is selected independently from the group consisting of purine nucleobases, pyrimidine nucleobases and analogs of purine or pyrimidine nucleobases capable of forming Watson-Crick-base pairing,
R 1 —is H,
R 2 —is selected independently from the group consisting of H, OH, halogen, NH 2 and SH,
R 3 —is selected independently from the group consisting of H, OH, halogen, —O—PO 3 H, SH, N 3 , NH 2 , O—CH 3 , O—CH 2 —O—CH 3 and O—CH 2 —CH═CH 2 ,
R 4 —is H or OH,
R 5 —is selected independently from the group consisting of a triphosphate group, a tetraphosphate group, and an alpha thiotriphosphate group; or a pharmaceutically acceptable salt thereof, and
Linker is a linker component, which connects said Base and Polynucleotide 1, and
wherein the length of the Linker is in the range of from 5 to 100 chain atoms, and said Linker is attached covalently to the Base at one of the positions N 4 or C-5 when the Base is a pyrimidine nucleobase, or at one of the positions N 6 or C-8 when the Base is a purine nucleobase, or at position C-7 when the Base is a 7-deaza-purine nucleobase, and is attached covalently to said Polynucleotide 1, and
(N) is a positive integer of 1 to 1000.
32. The nucleotide conjugate (nuc-macromolecule) according to claim 31 wherein (N) is a positive integer ranging from 2 to 10.
33. The nucleotide conjugate (nuc-macromolecule) according to claim 31 wherein (N) is a positive integer ranging from 10 to 1000.
34. The nucleotide conjugate (nuc-macromolecule) according to claim 31 wherein (N) is 1.
35. The nucleotide conjugate (nuc-macromolecule) according to claim 31 wherein the Polynucleotide 2 is selected from the group consisting of DNA, RNA, and PNA.
36. The nucleotide conjugate (nuc-macromolecule) according to claim 31 wherein said Polynucleotide 2 is labeled with 1 to 1000 Detectable Marker Units.
37. The nucleotide conjugate (nuc-macromolecule) according to claim 35 , wherein the Detectable Marker Unit is selected from the group consisting of dye, fluorescent dye, protein, polynucleotide, dendrimers, quantum dot, nanoparticles and microparticles.
38. A nucleotide conjugate (nuc-macromolecule) having the structure:
wherein:
Nuc-component consists of a 2′-deoxyuridine-5 ′-triphosphate substituent attached at its 5-position to the coupling unit; or a pharmaceutically acceptable salt thereof, and
Linker consists of Coupling unit L, Polymer 1 and Coupling unit T and connects the nucleobase of the nuc-component and a Polynucleotide 1, and
the length of the Linker is in the range of from 10 to 100 chain atoms, and said Linker is attached covalently to the nucleobase at the C-5 position of the uracil-1-yl nucleobase, and is attached covalently to said Polynucleotide 1, and
Polynucleotide 1 is a nucleic acid chain with the length ranging from 10 to 100000 nucleotides, and
Coupling unit L consists of R1 which is selected from the group consisting of:
R 6 —NH—R 7 , R 6 —O—R 7 , R 6 —S—R 7 , R 6 —SS—R 7 , R 6 —CO—NH—R 7 , R 6 —NH—CO—R 7 , R 6 —CO—O—R 7 , R 6 —O—CO—R 7 , R 6 —CO—S—R 7 , R 6 —S—CO—R 7 , R 6 —P(O) 2 —R 7 , R 6 —Si—R 7 , R 6 —(CH 2 ) n —R 7 ,
R 6 —(CH 2 ) n —R 7 , R 6 -A-(CH 2 ) n —R 7 , R 6 —(CH 2 ) n —B—R 7 ,
R 6 —(CH═CH—) n —R 7 , R 6 -(A-CH═CH—) n —R 7 , R 6 —(CH═CH—B—) n —R 7 ,
R 6 -A-CH═CH—(CH 2 —) n —R 7 , R 6 —(—CH═CH—CH 2 ) n —B—R 7 ,
R 6 —(C≡C—) n —R 7 , R 6 -(A-C≡C—) n —R 7 , R 6 —(C≡C—B—) n —R 7 ,
R 6 -A-C≡C—(CH 2 —) n —R 7 , R 6 —(—C≡C—CH 2 ) n —B—R 7 ; and R 6 —(—C≡C—CH 2 —CH 2 —) n —B—R 7 ; and
R 6 represents the attachment point at the C-5 position of a pyrimidine nucleobase, and
R 7 is the remaining Linker connected to the Polynucleotide 1 moiety, and A and B are selected from the group consisting of —NH—, —O—, —S—, —SS—, —CO—NH—,
—NH—CO—, —CO—O—, —O—CO—, —CO—S—, —S—CO—, —P(O) 2 —,—Si(CH 3 ) 2 —and —(CH 2 ) n —, wherein n ranges from 1 to 5, and
said Polymer 1 is selected independently from the group consisting of:
polyethylene glycol (PEG), polysaccharides, dextran, polyamides, polypeptides, polyphosphates, polyalkylene glycols, copolymers from ethylene glycol and propylene glycol, polyolefinic alcohols, polyvinyl pyrrolidones, poly(hydroxyalkylmethacrylamides), polyhydroxyalkylmethacrylates, polyacrylic acid, polyacrylamide, and polyvinyl alcohol, and
coupling unit T connects the Polymer 1 and Polynucleotide 1 and consists of R3, which is a linkage selected from the group consisting of —O—, —NH—, —CONH—, —NHCO—, and —S—, and
R2 is selected independently from the group consisting of H, OH, halogen, —O—PO 3 H, SH, N 3 , NH 2 , O—CH 3 , O—CH 2 —O—CH 3 and O—CH 2 —CH═CH 2 .
39. A nucleotide conjugate (nuc-macromolecule) having the structure:
wherein:
Nuc-component consists of a 2′-deoxyuridine-5′-triphosphate substituent attached at its 5-position to the coupling unit; or a pharmaceutically acceptable salt thereof, and
Linker consists of Coupling unit L, Polymer 1 and Coupling unit T and connects the nucleobase of the nuc-component and a Polynucleotide 1, and
the length of the Linker is in the range of from 10 to 100 chain atoms, and said Linker is attached covalently to the nucleobase at the C-5 position of the uracil-1-yl nucleobase, and is attached covalently to said Polynucleotide 1, and
Polynucleotide 1 is a nucleic acid chain with the length ranging from 10 to 100 nucleotides, and
Coupling unit L consists of R1 which is selected from the group consisting of:
R 6 —NH—R 7 , R 6 —O—R 7 , R 6 —S—R 7 , R 6 —SS—R 7 , R 6 —CO—NH—R 7 , R 6 —NH—CO—R 7 , R 6 —CO—O—R 7 , R 6 —O—CO—R 7 , R 6 —CO—S—R 7 , R 6 —S—CO—R 7 , R 6 —P(O) 2 —R 7 , R 6 —Si—R 7 , R 6 —(CH 2 ) n —R 7 ,
R 6 —(CH 2 ) n —R 7 , R 6 -A-(CH 2 ) n —R 7 , R 6 —(CH 2 ) n —B—R 7 ,
R 6 —(CH═CH—) n —R 7 , R 6 -(A-CH═CH—) n —R 7 , R 6 —(CH═CH—B—) n —R 7 ,
R 6 -A-CH═CH—(CH 2 —) n —R 7 , R 6 —(—CH═CH—CH 2 ) n —B—R 7 ,
R 6 —(C≡C—) n —R 7 , R 6 -(A-C≡C—) n —R 7 , R 6 —(C≡C—B—) n —R 7 ,
R 6 -A-C≡C—(CH 2 —) n —R 7 , R 6 —(—C≡C—CH 2 ) n —B—R 7, and R 6 —(—C≡C—C 2 —CH 2 —) n —B—R 7 ; and
R 6 represents the attachment point at the C-5 position of a pyrimidine nucleobase, and
R 7 is the remaining Linker connected to the Polynucleotide 1 moiety, and A and B are selected from the group consisting of —NH—, —O—, —S—, —SS—, —CO—NH—,
—NH—CO—, —CO—O—, —O—CO—, —CO—S—, —S—CO—, —P(O) 2 ,—Si(CH 3 ) 2 — and —(CH 2 ) n —, wherein n ranges from 1 to 5, and
said Polymer 1 is a polyethylene glycol(PEG), and
coupling unit T connects the Polymer 1 and Polynucleotide 1 and consists of R3, which is a linkage selected from the group consisting of —O—, —NH—, —CONH—, —NHCO—, and —S—, and
R2 is selected independently from the group consisting of H, OH, halogen, —O—PO 3 H, SH, N 3 , NH 2 , O—CH 3 , O—CH 2 —O—CH 3 and O—CH 2 —CH═CH 2 .
40. A nucleotide conjugate (nuc-macromolecule) having the structure:
wherein:
Nuc-component consists a 2′-deoxyuridine-5′-triphosphate substituent attached at its 5 -position to the coupling unit; or a pharmaceutically acceptable salt thereof, and
Linker connects the nucleobase of the nuc-component and a Polynucleotide 1, and
Linker consisting of Coupling unit L, Polymer 1 and Coupling unit T, and
the length of the Linker is in the range of from 10 to 100 chain atoms, and said Linker is attached covalently to the nucleobase at the C-5 position of the uracil-1-yl nucleobase, and is attached covalently to said Polynucleotide 1, and
Polynucleotide 1 is a nucleic acid chain with the length ranging from 10 to 100 nucleotides, and
Coupling unit L is selected from the group consisting of 3-amino-allyl (R1) and 3-amino-propargyl (R1), wherein remaining linker is bound to amino-group of the said linkage moiety (R1), and
said Polymer 1 is a polyethylene glycol(PEG), and
Coupling unit T connects the linker to the polynucleotide 1 having the structure —CONH—, and
R2 is selected independently from the group consisting of H, OH, halogen, —O—PO 3 H, SH, N 3 , NH 2 , O—CH 3 , O—CH 2 —O—CH 3 and O—CH 2 —CH═CH 2 .
41. A nucleotide conjugate (nuc-macromolecule) having the structure:
wherein:
Nuc-component consists of a 2′-deoxyuridine-5′-triphosphate substituent attached at its 5 -position to the coupling unit; or a pharmaceutically acceptable salt thereof, and
Linker connects the nucleobase of the nuc-component and a Polynucleotide 1, and
Linker consists of Coupling unit L, Polymer 1 and Coupling unit T, and
the length of the Linker is in the range of from 10 to 100 chain atoms, and said Linker is attached covalently to the nucleobase at the C-5 position of the uracil-1-yl nucleobase, and is attached covalently to said Polynucleotide 1, and
Polynucleotide 1 is a nucleic acid chain with the length ranging from 10 to 100 nucleotides, and
Coupling unit L consists of R1 which is selected from 3 aminoallyl and 3-aminopropargyl, wherein the remaining linker is bound to amino group of the linkage moiety (R1), and
said Polymer 1 is a polyethylene glycol(PEG), and
Coupling unit T connects the linker to the polynucleotide 1 having the structure —CONH—.
42. A nucleotide conjugate (nuc-macromolecule) having the structure:
wherein:
Nuc-component consists of a 2′-deoxyuridine-5′-triphosphate substituent attached at its 5-position to the coupling unit; or a pharmaceutically acceptable salt thereof, and
Linker connects the nucleobase of the Nuc-component and a Polynucleotide 1, and
Linker consists of Coupling unit L, Polymer 1 and Coupling unit T, and
the length of the Linker is in the range of from 10 to 100 chain atoms, and said Linker is attached covalently to the nucleobase at the C-5 position of the uracil-1-yl nucleobase, and is attached covalently to said Polynucleotide 1, and
Polynucleotide 1 is a nucleic acid chain with the length ranging from 10 to 100 nucleotides, and
Coupling unit L consists of 3-amino-propargyl, wherein the remaining linker is bound to the amino group of the 3aminopropargyl moiety and
said Polymer 1 is a polyethylene glycol(PEG), and
Coupling unit T connects the linker to the polynucleotide 1 having the structure —CONH—.
43. A nucleotide conjugate (nuc-macromolecule) having the structure
wherein:
Nuc-component consists of a 2′-deoxyuridine-5′-triphosphate substituent attached at its 5 -position to the Coupling unit; or a pharmaceutically acceptable salt thereof, and
Linker is a cleavable linker component, which connects the nucleobase of the nuc-component and a Polynucleotide 1 of the Marker,
wherein the length of the Linker is in the range of from 20 to 1000 chain atoms, and said Linker is attached covalently to the nucleobase at the C-5 position of the uracil-1-yl nucleobase, and is attached covalently to said Polynucleotide 1 of the Marker at the 3′-end of said Polynucleotide 1, and
Polynucleotide 1 of is an oligonucleotide consisting of 10 to 100 nucleotide monophosphate moieties, and
Detectable Marker Unit is Cy3-fluorescent dye attached to the 5′-end of said oligonucleotide.