METHOD FOR THE SITE-SPECIFIC ENZYMATIC LABELLING OF NUCLEIC ACIDS IN VITRO BY INCORPORATION OF UNNATURAL NUCLEOTIDES
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.
1 . A double-stranded oligonucleotide comprising a first strand and a second strand, wherein:
(a) the first strand comprises a first compound of the formula
wherein R 2 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, and azido; and
(b) the second strand comprises a second compound selected from the group consisting of
and wherein in the case of each of (a) and (b), the wavy line indicates a bond to a 2′-deoxyribosyl moiety.
2 . The double-stranded oligonucleotide of claim 1 , wherein the first compound and the second compound form a base pair in the double-stranded oligonucleotide.
3 . The double-stranded oligonucleotide of claim 2 , wherein the R 2 is hydrogen or halogen.
4 . The double-stranded oligonucleotide of claim 3 , wherein the R 2 is hydrogen.
5 . The double-stranded oligonucleotide of claim 3 , wherein the R 2 is halogen.
6 . The double-stranded oligonucleotide of claim 5 , wherein the halogen is a fluoro.
7 . The double-stranded oligonucleotide of claim 4 , wherein the second compound is selected from the group consisting of
8 . The double-stranded oligonucleotide of claim 7 , wherein the second compound is selected from the group consisting of
9 . The double-stranded oligonucleotide of claim 8 , wherein the second compound is selected from the group consisting of
10 . The double-stranded oligonucleotide of claim 9 , wherein the second compound is selected from the group consisting of
11 . The double-stranded oligonucleotide of claim 10 , wherein the second compound is
12 . The double-stranded oligonucleotide of claim 10 , wherein the second compound is
13 . A composition comprising:
(a) a 2′-deoxyribonucleic acid (DNA) molecule comprising a first compound of the formula
wherein R 2 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, and azido, and wherein the wavy line indicates a bond to a 2′-deoxyribosyl moiety; and
(b) a ribonucleic acid (RNA) molecule comprising a second compound selected from the group consisting of
wherein the wavy line indicates a bond to a ribosyl moiety.
14 . The composition of claim 13 , wherein the first compound and the second compound form a base pair.
15 . The composition of claim 14 , wherein the R 2 is hydrogen or halogen.
16 . The composition of claim 15 , wherein the R 2 is hydrogen.
17 . The composition of claim 16 , wherein the second compound is selected from the group consisting of
18 . The composition of claim 17 , wherein the second compound is selected from the group consisting of
19 . The composition of claim 18 , wherein the second compound is selected from the group consisting of
20 . The composition of claim 19 , wherein the second compound is selected from the group consisting of
21 . The composition of claim 20 , wherein the second compound is
22 . The composition of claim 21 , wherein the second compound is
23 . A composition comprising:
(a) a first ribonucleic acid (RNA) molecule comprising a first compound of the formula
wherein R 2 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, and azido; and
(b) a second ribonucleic acid (RNA) molecule comprising a second compound selected from the group consisting of
and wherein in the case of each of (a) and (b), the wavy line indicates a bond to a ribosyl moiety.
24 . The composition of claim 23 , wherein the first compound and the second compound form a base pair.
25 . The composition of claim 24 , wherein the R 2 is hydrogen.
26 . The composition of claim 25 , wherein the second compound is selected from the group consisting of
27 . The composition of claim 26 , wherein the second compound is selected from the group consisting of
28 . The composition of claim 27 , wherein the second compound is selected from the group consisting of
29 . The composition of claim 28 , wherein the second compound is
30 . The composition of claim 28 , wherein the second compound is