Time lapse sequencing: a convertible-nucleoside approach to enrichment-free analysis of RNA dynamics
The invention provides methods and compositions for generating mutations in new nucleic acid molecules through incorporation of a transformable nucleoside into the nucleic acid and subsequent transformation of the nucleoside through oxidative-nucleophilic-aromatic-substitution chemistry, referred to as TimeLapse chemistry. The invention further provides methods for detecting the mutations, referred to as TimeLapse-seq.
1. A method for identifying newly synthesized nucleic acid molecules comprising:
a) contacting a cell with a modified base, wherein the modified base is incorporated into at least one newly generated nucleic acid molecule;
b) isolating the nucleic acid molecules from the cell;
c) contacting the isolated nucleic acid molecules with an oxidant and a nucleophile, wherein the nucleophile is selected from the group consisting of 2,2,2-trifluoroethylamine (TFEA), benzylamine (C 6 H 5 CH 2 NH 2 ), aniline (C 6 H 5 NH 2 ), 1,1-dimethylethylenediamine ((CH 3 ) 2 NCH 2 CH 2 NH 2 ), ethanolamine (C 2 H 7 NO) and 4-(Triflouromethyl)benzylamine (CF 3 C 6 H 4 CH 2 NH 2 ),
and further wherein the modified base is converted to an analog of a different base;
d) detecting the presence of the analog of a different base or a. mutation that is the result of amplification thereof; and
e) identifying a nucleic acid molecule comprising the analog of a different base as being a newly generated nucleic acid molecule.
2. The method of claim 1 , wherein the oxidant is selected from the group consisting of hydrogen peroxide (H 2 O 2 ), sodium iodate (NaIO 3 ), potassium permagnate (KMnO 4 ), sodium periodate (NaIO 4 ), and meta-Chloroperoxybenzoic acid (mCPBA).
3. The method of claim 1 , wherein the oxidant is NaIO 4 and the nucleophile is TFEA.
4. The method of claim 1 , wherein the nucleic acid molecule is selected from the group consisting of: a ribonucleic acid molecule and a deoxyribonucleic acid molecule.
5. The method of claim 1 , wherein the modified base is selected from the group consisting of 4-thiouridine (s 4 U), 6-thioguanine (s 6 G), 6-thiodeoxyguanosine (s 6 dG) and 4-thiothymine (s 4 T).
6. The method of claim 1 , wherein the mutation is selected from the group consisting of: a U to C mutation and a G to A mutation.
7. The method of claim 1 , wherein the modified base is selected from the group consisting of s 4 T and s 4 U and the modified base is converted into an analog of cytidine.
8. The method of claim 1 , wherein the modified base is selected from the group consisting of s 6 G and s 6 dG, and wherein the modified base is converted into an analog of adenine.
9. The method of claim 1 , wherein the step of detecting comprises sequencing the nucleic acid molecules.