Bisulfite-free, base-resolution identification of cytosine modifications
View Patent ↗This disclosure provides methods for bisulfite-free identification in a nucleic acid sequence of the locations of 5-methylcytosine, 5-hydroxymethylcytosine, 5-carboxylcytosine and 5-formylcytosine.
1. A method for converting 5-carboxylcytosine (5caC) and/or 5-formylcytosine (5fC) to dihydrouracil (DHU) comprising contacting a nucleic acid sample comprising 5caC and/or 5fC with a borane reducing agent.
2. The method of claim 1 , wherein the borane reducing agent comprises an agent selected from the group consisting of 2-picoline borane (pic-BH3), borane, sodium borohydride, sodium cyanoborohydride, and sodium triacetoxyborohydride.
3. The method of claim 1 , wherein the borane reducing agent comprises 2-picoline borane.
4. The method of claim 1 comprising contacting the nucleic acid sample with an oxidizing agent prior to contacting with the borane reducing agent or contacting a target nucleic acid with an oxidizing agent to prepare the nucleic acid sample prior to contacting with the borane reducing agent.
5. The method of claim 4 , wherein the oxidizing agent is a ten-eleven translocation (TET) enzyme.
6. The method of claim 5 , wherein the TET enzyme comprises human TET1, human TET2, human TET3, murine TET1, murine TET2, murine TET3, Naegleria TET (NgTET), Coprinopsis cinerea (CcTET), or derivatives or analogues thereof.
7. The method of claim 4 , wherein the oxidizing agent comprises a chemical oxidizing agent.
8. The method of claim 7 , wherein the oxidizing agent is selected from the group consisting of potassium perruthenate (KRuO 4 ), Cu(II)/2,2,6,6-tetramethylpiperidine--oxyl (TEMPO), tetrapropylammonium perruthenate (TPAP), tetrabutylammonium perruthenate (TBAP), polymer-supported perruthenate (PSP), tetraphenylphosphoniurn ruthenate, copper salts or complexes of 3-carbamoyl-2, 2,5,5-tetramethyl-3-pyrrolin-1-yloxy (3-Carbamoyl-PROXYL), copper salts or complexes of 2-azaadamantane-N-oxyl (AZADO) , and copper salts or complexes of 9-azabicyclo [3.3.1] nonane N′-oxyl (ABNO).
9. The method of claim 1 , further comprising adding a blocking group to one or more modified cytosines in the nucleic acid sample.
10. The method of claim 9 , wherein the one or more modified cytosines comprises 5hmC.
11. The method of claim 10 , wherein the blocking group comprises a sugar or a uridine diphosphate (UDP)-linked sugar.
12. The method of claim 9 , wherein the blocking group is added prior to contacting with the borane reducing agent.
13. The method of claim 9 , wherein the one or more modified cytosines comprises 5caC or 5fC.
14. The method of claim 13 , wherein the blocking group comprises an aldehyde reactive compound.
15. The method of claim 14 , wherein the aldehyde reactive compound comprises a hydroxylamine derivative, a hydrazine derivative, or a hydrazide derivative.
16. The method of claim 15 , wherein adding the blocking group comprises contacting the nucleic acid sample with (i) a coupling agent and (ii) an amine, hydrazine, or hydroxylamine compound.
17. The method of claim 1 , further comprising sequencing the nucleic acid sample after contacting with the borane reducing agent to identify converted cytosine bases.