Selective Chemical Functionalization at N6-Methyladenosine Residues in DNA Enabled by Visible-Light-Mediated Photoredox Catalysis

Abstract

Selective chemistry that modifies the structure of DNA and RNA is essential to understanding the role of epigenetic modifications. We report a visible-light-activated photocatalytic process that introduces a covalent modification at a C(sp³)-H bond in the methyl group of N6-methyl deoxyadenosine and N6-methyl adenosine, epigenetic modifications of emerging importance. A carefully orchestrated reaction combines reduction of a nitropyridine to form a nitrosopyridine spin-trapping reagent and an exquisitely selective tertiary amine-mediated hydrogen-atom abstraction at the N6-methyl group to form an α-amino radical. Cross-coupling of the putative α-amino radical with nitrosopyridine leads to a stable conjugate, installing a label at N6-methyl-adenosine. We show that N6-methyl deoxyadenosine-containing oligonucleotides can be enriched from complex mixtures, paving the way for applications to identify this modification in genomic DNA and RNA.

Figure 1

Evolution of a strategy for the chemical modification of N⁶mdA in DNA. (A) Canonical DNA bases and N⁶mdA. (B) Proposed mechanism for biochemical demethylation of N⁶mA via Fe-dependent enzyme-controlled hydrogen atom abstraction and oxygen-rebound. (C) Plan for covalent modification at N⁶mdA via trapping of an “on-DNA” α-amino radical intermediate.

Figure 2

Design plan for a N⁶mdA-selective functionalization process. (A) Nitrosoarene-derived STRs; can a stable precursor, such as a nitroarene, be used for in situ generation of reactive spin trapping reagents? (B) Selective HAA at N⁶mdA. (C) Design plan for photoredox-facilitated covalent modification of N⁶mdA based on the merger of selective HAA and spin trapping via in situ generation of nitrosoarenes.

Figure 3

Visible light-mediated photoredox strategy for covalent functionalization of N⁶mdA. (A) Oligonucleotide conjugation via HAA and spin trapping via an oxidative photocatalytic quenching cycle with [Ru(II)(phen)₃]Cl_2. (B) The use of modular nitropyridine probes in oligonucleotide functionalization and subsequent elaboration by Huisgen cycloaddition. (C) Selectivity parameters in the oligonucleotide functionalization are defined as “HAA selectivity” (reflecting the position of C–H bond cleavage) and “Probe selectivity” (reflecting the selectivity of reaction via nitrosopyridine vs nitropyridine). (D) Scope of N⁶mdA functionalization using standard conditions detailed in panels A and B. For the reaction of 5c, Ru(bpz)₃(PF₆)₂ was used as a catalyst.

Figure 4

Pull-down strategy for the enrichment of N⁶mdA-containing oligonucleotides. (A) A pull down procedure involving photoredox functionalization with an alkyne-derived nitropyridine, Huisgen cycloaddition with a biotin-derived azide, immobilization on streptavidin coated magnetic beads, oligonucleotide separation by sequential washing, and selective cleavage of N⁶mdA-derived oligonucleotides delivers an enrichment of >50:1. (B) Pull down experiments using 99nt ssDNA and 99bp dsDNA in the presence and absence of salmon sperm (SS) DNA demonstrate enrichment in complex mixtures of DNA sequences; the blue dot indicates the position of N⁶mdA. dsDNA displaying the methyl group (red) of the N⁶mdA nucleobase (blue) in the major groove.