Incorporation of unnatural nucleotides and methods of use in vivo thereof

Patent metadata

Field	Value
Publication number	US-12497642-B2
Application number	US-201816629211-A
Country	US
Kind code	B2
Filing date	Jul 10, 2018
Priority date	Jul 11, 2017
Publication date	Dec 16, 2025
Grant date	Dec 16, 2025

Abstract

Disclosed herein are in vivo methods, compositions, and kits for producing nucleic acids which comprises at least one unnatural base. Disclosed herein are in vivo methods of producing a nucleic acid with an expanded genetic alphabet, the method comprising incorporating at least one unnatural base in the nucleic acid. Disclosed herein are semi-synthetic organisms comprising an expanded genetic alphabet, wherein the genetic alphabet comprises at least one unnatural base. Disclosed herein are compositions that include a heterologous or recombinant polymerase and methods of use thereof. Further, disclosed herein are kits that are useful for stably incorporating an unnatural nucleic acid into a nucleic acid molecule, e.g., using the methods provided by the present invention in in vitro condition or under a cell free condition.

First claim

What is claimed is:

1 . A method of producing DNA in an Escherichia coli , the method comprising incorporating into DNA present in an Escherichia coli at least a first unnatural base and the first unnatural base comprises:

and wherein the first unnatural base pairs with a second unnatural base to form an unnatural base pair (UBP), wherein the second unnatural base comprises,

and wherein the Escherichia coli expresses a nucleotide triphosphate transporter from Phaeodactylum tricornutum.

2 . The method of claim 1 , wherein the first unnatural base is bonded to an unnatural sugar moiety.

3 . The method of claim 2 , wherein the unnatural sugar moiety comprises a modification selected from:
(i) a modification at the 2′ position comprising:
(a) F; SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 F; O-alkyl, S-alkyl, N-alkyl; O-alkenyl, S-alkenyl, N-alkenyl; O-alkynyl, S-alkynyl, N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl is substituted or unsubstituted C 1 -C 10 alkyl, C 2 -C 10 alkenyl, and C 2 -C 10 alkynyl, respectively;
(b) —O[(CH 2 ) n O] m CH 3 , —O(CH 2 ) n OCH 3 , —O(CH 2 ) n NH 2 , —O(CH 2 ) n CH 3 , —O(CH 2 ) n —ONH 2 , or —O(CH 2 ) n ON[(CH 2 ) n CH 3 )] 2 , where n and m are each independently from 1 to 10; or (c) C 1 to C 10 alkyl, substituted C 1 to C 10 alkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , substituted silyl, a reporter group, an intercalator, or a group for improving the pharmacokinetic properties of an oligonucleotide;
(ii) a modification at the 5′ position comprising: 5′-vinyl or 5′-methyl; (iii) a modification at the 4′ position comprising: 4′-S; and (iv) any combination thereof.

4 . An Escherichia coli expressing a nucleotide triphosphate transporter from Phaeodactylum tricornutum and comprising DNA that comprises at least a first unnatural base, wherein the first unnatural base comprises:

and wherein the first unnatural base pairs with a second unnatural base to form an unnatural base pair (UBP), wherein the second unnatural base comprises

5 . The Escherichia coli of claim 4 , wherein the first unnatural base is bonded to an unnatural sugar moiety.

6 . The Escherichia coli of claim 5 , wherein the unnatural sugar moiety comprises a modification selected from the group:
(i) a modification at the 2′ position comprising: (a) F; SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 F; O-alkyl, S-alkyl, N-alkyl; O-alkenyl, S-alkenyl, N-alkenyl; O-alkynyl, S-alkynyl, N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl is substituted or unsubstituted C 1 -C 10 alkyl, C 2 -C 10 alkenyl, and C 2 -C 10 alkynyl, respectively; (b) —O[(CH 2 ) n O] m CH 3 , —O(CH 2 ) n OCH 3 , —O(CH 2 ) n NH 2 , —O(CH 2 ) n CH 3 , —O(CH 2 ) n —ONH 2 , or —O(CH 2 ) n ON[(CH 2 ) n CH 3 )] 2 , where n and m are each independently from 1 to 10; or (c) C 1 to C 10 alkyl, substituted C 1 to C 10 alkyl SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , substituted silyl, a reporter group, an intercalator, or a group for improving the pharmacokinetic properties of an oligonucleotide; (ii) a modification at the 5′ position comprising: 5′-vinyl or 5′-methyl; (iii) a modification at the 4′ position comprising: 4′-S, and (iv) any combination thereof.

7 . A method of producing DNA in an Escherichia coli , the method comprising incorporating at least a first unnatural base into DNA present in an Escherichia coli cell expressing a nucleotide triphosphate transporter from Phaeodactylum tricornutum , wherein the first unnatural base comprises:

wherein the first unnatural base pairs with a second unnatural base to form an unnatural base pair (UBP) in the DNA, and wherein the second unnatural base comprises:

8 . An Escherichia coli expressing a nucleotide triphosphate transporter from Phaeodactylum tricornutum and comprising DNA that comprises at least a first unnatural base, wherein the first unnatural base comprises:

and wherein the first unnatural base pairs with a second unnatural base to form an unnatural base pair (UBP) in the DNA, and wherein the second unnatural base comprises:

Assignees

• Scripps Research Inst

Inventors

• Romesberg Floyd E
• Zhang Yorke
• Feldman Aaron W
• Dien Vivian T

Classifications

• C12N1/20

  Bacteria; Culture media therefor · CPC title

• 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

• Y02A50/30

  Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change · CPC title

• C12Y207/07007

  DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase · CPC title

• A61K31/712

  Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose · CPC title