Method for labeling nucleic acid
The invention provides a method for labeling a nucleic acid comprising N6-methyl adenine. The method comprises forming an alpha-amino radical on the N6-methyl group of N 6 mAde, and capturing the alpha-amino radical with a radical acceptor comprising a nitrosopyridyl group. The presence of N 6 mAde in a nucleic acid may then be established by detection of the labeled nucleic acid, or the labeled nucleic acid may be extracted or further modified using the label. Also provided is a method for mapping the position of N 6 mAde within a target nucleic acid, and probe molecules and kits for use in the method.
1 . A method for labeling a nucleic acid comprising N6-methyl adenine (N 6 mAde), the method comprising:
i) forming an alpha-amino radical on the N6-methyl group of N 6 mAde; and
ii) capturing the alpha-amino radical with a radical acceptor comprising a nitrosopyridyl group (O═N-Py-).
2 . The method of claim 1 , wherein the alpha-amino radical is formed by contacting the nucleic acid comprising N 6 mAde with an amine-centered radical cation to abstract a hydrogen atom from the N6-methyl group of N 6 mAde.
3 . The method of claim 2 , comprising oxidizing an amine to generate the amine-centered radical cation.
4 . The method of claim 3 , wherein the amine is a tertiary amine having the formula:
where R is selected from a hydrogen atom, a hydroxyl group, a C 1-6 alkyl group, a C 1-6 alkoxy group, a C 1-6 acyloxy group, a C 1-6 reverse ester group or a group —C(OH)R 1 R 2 , wherein R 1 and R 2 are each selected from C 1-6 alkyl.
5 . The method of claim 3 , wherein a photocatalyst is used to oxidize the amine, optionally wherein the photocatalyst, in either the excited state or reduced form, has a reduction potential of at least +1.10 volts (V) vs saturated calomel electrode (SCE) to at most +1.45 V vs SCE.
6 . The method of claim 5 , wherein the photocatalyst is a transition metal photocatalyst.
7 . The method of claim 1 , wherein the radical acceptor is a probe having the formula (I):
where -Py- is a pyridinediyl group, -L- is a linker and —X is a label.
8 . The method of claim 7 , wherein -L- is:
where:
-L 1 - is selected from a covalent bond or a C 6-10 arylene group;
-L 2 - is selected from an amide linkage, an ester linkage, a carbonyl linkage, an amine linkage, or an ether linkage; and
-L 3 - is selected from C 1-10 alkylene and C 1-10 heteroalkylene.
9 . The method of claim 7 , wherein —X is a click reaction partner (—C 1 ) selected from the group consisting of C 2-20 alkynyl, C 2-20 alkenyl, isocyanide, azido, nitrone, nitrile oxide and tetrazine.
10 . The method of claim 8 , wherein the probe is formed insitu from a precursor having the formula (II):
where -Py- is a pyridinediyl group, -L- is a linker and —X is a label.
11 . The method of claim 10 , wherein the precursor is selected from compounds of formula P1 to P7:
12 . The method of claim 10 , wherein the probe is formed by reducing the precursor of formula (II).
13 . The method of claim 12 , wherein a photocatalyst is used to reduce the precursor of formula (II).
14 . The method of claim 7 , wherein the method further comprises:
iii) contacting the nucleic acid with a bifunctional probe having the formula:
where —C 2 is a complementary click reaction partner, -L 4 - is a linker and —X 2 is a label, wherein the bifunctional probe covalently binds to the nucleic acid.
15 . The method of claim 14 , wherein:
(i) —C 2 is an azido group (—N 3 ); or
(ii) the linker -L 4 - is:
where:
-L 4A - is C 1-4 alkylene;
-L 4B - is C 1-4 heteroalkylene; and
n is 0 to 8.
16 . The method of claim 14 , wherein —X 2 is an isolation label (—X Iso ) that binds to a binding agent, wherein the isolation label (—X Iso ) is biotin.