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

Patent metadata

Field	Value
Publication number	US-11634451-B2
Application number	US-201916518715-A
Country	US
Kind code	B2
Filing date	Jul 22, 2019
Priority date	Aug 8, 2013
Publication date	Apr 25, 2023
Grant date	Apr 25, 2023

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 compound comprising a nucleobase analog of the following formula

wherein each X is independently carbon or nitrogen;
wherein R 1 is hydrogen, an alkyl group, a reactive linker comprising a reactive center adapted to bond to a cargo reagent comprising a cargo and a group of reactivity complementary to the reactive center, or a coupled linker to which a cargo is bonded;
wherein when an X is nitrogen, the corresponding R 2 is absent, and when an X is carbon, the corresponding R 2 is present and is independently hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, an azide group, a reactive linker comprising a reactive center adapted to bond to a cargo reagent comprising a cargo and a group of reactivity complementary to the reactive center, or a coupled linker to which a cargo is bonded;
wherein E is oxygen, sulfur or selenium;
wherein the wavy line indicates a bond to a ribosyl moiety, deoxyribosyl moiety, or dideoxyribosyl moiety, wherein the ribosyl moiety, deoxyribosyl moiety, or dideoxyribosyl moiety is in free form, bound to a monophosphate, diphosphate, triphosphate, α-thiotriphosphate, β-thiotriphosphate, or γ-thiotriphosphate group, or is included in an RNA or a DNA;

and wherein:
(i) the ribosyl moiety is incorporated into an RNA oligonucleotide chain;
(ii) the ribosyl moiety bears a triphosphate or an α-thiotriphosphate group bonded to a 5′-hydroxyl thereof;
(iii) the deoxyribosyl moiety bears an α-thiotriphosphate group bonded to a 5′-hydroxyl thereof;
(iv) the nucleobase analog has a structure selected from:

(v) the wavy line indicates a bond to the ribosyl moiety bound to a monophosphate or triphosphate group;
(vi) at least one R 2 is alkenyl, alkynyl, methanethiol, methaneseleno, a reactive linker comprising a reactive center adapted to bond a cargo reagent comprising a cargo and a group of reactivity complementary to the reactive center, or a coupled linker to which a cargo is bonded; or
(vii) E is not oxygen.

2. The compound of claim 1 , wherein the reactive center comprises an amino group, an acetylenic group, a thiol group, an aldehyde group, or an azide group.

3. The compound of claim 1 , wherein the ribosyl moiety bears a triphosphate or an α-thiotriphosphate group bonded to a 5′-hydroxyl thereof, or the deoxyribosyl moiety bears an α-thiotriphosphate group bonded to a 5′-hydroxyl thereof.

4. The compound of claim 1 , wherein the ribosyl or deoxyribosyl moiety is incorporated into an RNA or DNA oligonucleotide chain.

5. The compound of claim 1 , wherein at least one R 2 independently comprises a group selected from: a —C≡C—CH 2 NHR 3 group, wherein R 3 is hydrogen or is an amino-protecting group; —C≡C—(CH 2 ) n —C≡CH, wherein n is 1, 2, 3, 4, 5, or 6; and —C≡C—(CH 2 ) n1 —O—(CH 2 ) n2 —C≡CH, wherein n1 and n2 are each independently 1, 2, or 3.

6. The compound of claim 1 , wherein the nucleobase analog has a structure selected from:

7. The compound of claim 1 , wherein E is sulfur.

8. The compound of claim 1 , wherein each X is independently carbon.

9. The compound of claim 1 , wherein the wavy line indicates a bond to the ribosyl moiety.

10. The compound of claim 1 , wherein the wavy line indicates a bond to the deoxyribosyl moiety.

11. The compound of claim 1 , wherein the wavy line indicates a bond to the ribosyl or the deoxyribosyl moiety, and the ribosyl or the deoxyribosyl moiety is in free form.

12. The compound of claim 1 , wherein the wavy line indicates a bond to the ribosyl or the deoxyribosyl moiety, and the ribosyl or the deoxyribosyl moiety is bound to the monophosphate group.

13. The compound of claim 1 , wherein the wavy line indicates a bond to the ribosyl moiety, and the ribosyl moiety is bound to the triphosphate group.

14. The compound of claim 1 , wherein the wavy line indicates a bond to the deoxyribosyl moiety, and the deoxyribosyl moiety is bound to the triphosphate group.

15. The compound of claim 2 , wherein the reactive center comprises an azide group.

16. The compound of claim 2 , wherein the reactive center comprises an acetylenic group.

17. The compound of claim 1 , wherein E is oxygen.

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

19. The compound of claim 18 , wherein the nucleobase analog has the structure

20. The compound of claim 19 , wherein the ribosyl moiety or dideoxyribosyl moiety is in free form.

21. The compound of claim 19 , wherein the ribosyl moiety is incorporated into an RNA oligonucleotide chain.

22. The compound of claim 19 , wherein the ribosyl moiety bears a triphosphate bonded to a 5′-hydroxyl thereof, or the deoxyribosyl moiety bears and α-thiotriphosphate group bonded to a 5′-hydroxyl thereof.

23. The compound of claim 1 , wherein the nucleobase analog has a structure selected from:

24. The compound of claim 23 , wherein the ribosyl moiety, deoxyribosyl moiety, or dideoxyribosyl moiety is in free form.

25. The compound of claim 23 , wherein the ribosyl moiety is incorporated into an RNA oligonucleotide chain.

26. The compound of claim 23 , wherein the ribosyl moiety bears a triphosphate or an α-thiotriphosphate group bonded to a 5′-hydroxyl thereof, or the deoxyribosyl moiety bears an α-thiotriphosphate group bonded to a 5′-hydroxyl thereof.

27. The compound of claim 23 , wherein the nucleobase analog has the structure

28. The compound of claim 23 , wherein the nucleobase analog has the structure

29. The compound of claim 23 , wherein the nucleobase analog has the structure

30. The compound of claim 23 , wherein the nucleobase analog has the structure

31. The compound of claim 23 , wherein the nucleobase analog has the structure

32. The compound of claim 23 , wherein the nucleobase analog has the structure

33. The compound of claim 23 , wherein the nucleobase analog has the structure

34. The compou

Assignees

• Scripps Research Inst

Inventors

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

Classifications

• C07H21/00

  Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids · 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

• C07H19/00 Primary

  Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides ; Anhydro-derivatives thereof · CPC title

• C12P19/34

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

• C12Q1/6832

  Enhancement of hybridisation reaction · CPC title