Method for the site-specific enzymatic labelling of nucleic acids in vitro by incorporation of unnatural nucleotides

Patent metadata

Field	Value
Publication number	US-12281137-B2
Application number	US-202318112952-A
Country	US
Kind code	B2
Filing date	Feb 22, 2023
Priority date	Aug 8, 2013
Publication date	Apr 22, 2025
Grant date	Apr 22, 2025

Abstract

Provided herein are analogs of unnatural nucleotides bearing predominantly hydrophobic nucleobase analogs that form unnatural base pairs during DNA polymerase-mediated replication of DNA or RNA polymerase-mediated transcription of RNA. In this manner, the unnatural nucleobases can be introduced in a site-specific way into oligonucleotides (single or double stranded DNA or RNA), where they can provide for site-specific cleavage, or can provide a reactive linker than can undergo functionalization with a cargo-bearing reagent by means of reaction with a primary amino group or by means of click chemistry with an alkyne group of the unnatural nucleobase linker.

First claim

What is claimed is:

1. A nucleobase pair comprising a first nucleobase analog having the structure β9b, and a second nucleobase analog having any of the structures α15a or α15b:

wherein each X is independently carbon or nitrogen,
wherein each R 1 is independently hydrogen, alkyl group, a reactive linker comprising a reactive center a linker coupled to a cargo,
wherein each R 2 is optional and when present is independently hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, azide, nitro group, a reactive linker comprising a reactive center, or a linker coupled to a cargo,
wherein each Y is independently sulfur, oxygen, selenium, or a secondary amine,
wherein each E is independently oxygen, sulfur or selenium,
and the wavy line indicates a point of bonding to a ribosyl, deoxyribosyl, or dideoxyribosyl moiety or an analog thereof, wherein the ribosyl, deoxyribosyl, or dideoxyribosyl moiety, or analog thereof is in free form, is bound to a mono-phosphate, diphosphate, triphosphate, α-thiotriphosphate, β-thiophosphate, or γ-thiophosphate group, or is included in an RNA or a DNA or in an RNA analog or a DNA analog.

2. A double-stranded oligonucleotide comprising the nucleobase pair of claim 1 .

3. The nucleobase pair of claim 1 , wherein the ribosyl, deoxyribosyl, or dideoxyribosyl moiety or analog thereof is included in an RNA.

4. The nucleobase pair of claim 1 , wherein the ribosyl, deoxyribosyl, or dideoxyribosyl moiety or analog thereof is included in a DNA.

5. The nucleobase pair of claim 1 , wherein each X in structure β9b is carbon.

6. The nucleobase pair of claim 1 , wherein each R 2 in structure β9b is hydrogen.

7. The nucleobase pair of claim 1 , wherein Y is sulfur.

8. The nucleobase pair of claim 7 , wherein E in structure β9b is sulfur.

9. The nucleobase pair of claim 1 , wherein the first nucleobase analog is

10. The nucleobase pair of claim 1 , wherein the first nucleobase analog is

11. The nucleobase pair of claim 1 , wherein each X in structure α15b and α15a is carbon.

12. The nucleobase pair of claim 1 , wherein the second nucleobase analog has the structure α15b.

13. The nucleobase pair of claim 12 , wherein R 1 in structure α15b is methyl.

14. The nucleobase pair of claim 13 , wherein E in structure α15b is oxygen.

15. The nucleobase pair of claim 12 , wherein each R 2 in structure α15b is hydrogen.

16. The nucleobase pair of claim 12 , wherein the second nucleobase analog is

17. The nucleobase pair of claim 1 , wherein the second nucleobase analog has the structure α15a.

18. The nucleobase pair of claim 17 , wherein at least one R 2 is CN.

19. The nucleobase pair of claim 17 , wherein R 1 in structure α15a is methyl.

20. The nucleobase pair of claim 19 , wherein E in structure α15a is oxygen.

21. The nucleobase pair of claim 17 , wherein the second nucleobase analog is

22. The nucleobase pair of claim 1 , wherein the second nucleobase analog has any of the structures

wherein each R is independently hydrogen, or the reactive linker.

23. The nucleobase pair of claim 1 , wherein the first nucleobase analog is

24. The nucleobase pair of claim 23 , wherein the second nucleobase analog is

25. The nucleobase pair of claim 1 , wherein the first nucleobase analog is

and the second nucleobase analog is

26. The nucleobase pair of claim 1 , wherein the first nucleobase analog is

and the second nucleobase analog is

27. The nucleobase pair of claim 1 , wherein the first nucleobase analog is

and the second nucleobase analog is

28. The nucleobase pair of claim 1, 7, or 8 , wherein at least one R 2 in structure β9b is halogen.

29. The nucleobase pair of claim 8 , wherein each X in structure β9b is carbon.

30. The nucleobase pair of claim 1 , wherein the first nucleobase analog is

31. The nucleobase pair of claim 1 , wherein the first nucleobase analog is

and the second nucleobase analog is

32. A nucleobase pair comprising a first nucleobase analog having the structure

and a second nucleobase analog having the structure

wherein the wavy line indicates a point of bonding to a ribosyl, deoxyribosyl, or dideoxyribosyl moiety, wherein the ribosyl, deoxyribosyl, or dideoxyribosyl moiety (i) has its 5′-hydroxy group in free form or bound to a mono-phosphate, diphosphate, triphosphate, α-thiotriphosphate, β-thiophosphate, γ-thiophosphate group, or (ii) is included in an RNA or a DNA.

33. A nucleobase pair comprising a first nucleobase analog having the structure

and a second nucleobase analog having the structure

wherein the wavy line indicates a point of bonding to a ribosyl, deoxyribosyl, or dideoxyribosyl moiety, wherein the ribosyl, deoxyribosyl, or dideoxyribosyl moiety (i) has its 5′-hydroxy group in free form or bound to a mono-phosphate, diphosphate, triphosphate, α-thiotriphosphate, β-thiophosphate, or γ-thiophosphate group, or (ii) is included in an RNA or a DNA.

34. A double-stranded oligonucleotide comprising the nucleobase pair of claim

Assignees

• Scripps Research Inst

Inventors

• Romesberg Floyd E
• Malyshev Denis A
• Li Lingjun
• Lavergne Thomas
• Li Zhengtao

Classifications

• C12P19/34

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C12N15/1024

  In vivo mutagenesis using high mutation rate "mutator" host strains by inserting genetic material, e.g. encoding an error prone polymerase, disrupting a gene for mismatch repair · CPC title

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C07H21/04

  with deoxyribosyl as saccharide radical · CPC title

• C12Q1/6832

  Enhancement of hybridisation reaction · CPC title