Recent technical advances in the study of nucleic acid modifications

Abstract

Enzyme-mediated chemical modifications of nucleic acids are indispensable regulators of gene expression. Our understanding of the biochemistry and biological significance of these modifications has largely been driven by an ever-evolving landscape of technologies that enable accurate detection, mapping, and manipulation of these marks. Here we provide a summary of recent technical advances in the study of nucleic acid modifications with a focus on techniques that allow accurate detection and mapping of these modifications. For each modification discussed (N⁶-methyladenosine, 5-methylcytidine, inosine, pseudouridine, and N⁴-acetylcytidine), we begin by introducing the "gold standard" technique for its mapping and detection, followed by a discussion of techniques developed to address any shortcomings of the gold standard. By highlighting the commonalities and differences of these techniques, we hope to provide a perspective on the current state of the field and to lay out a guideline for development of future technologies.

Keywords: 5-methylcytidine; DNA modification; N(4)-acetylcytidine; N(6)-methyladenosine; RNA modification; inosine; pseudouridine.

Figure 1.. Biogenesis reactions for each modification discussed

(A) Generation of m⁶A and inosine from adenosine. (B) Generation of 5-methylcytidine from cytidine and oxidation reactions of 5-methylcytidine. (C) Generation of ψ from uridine. (D) Generation of ac⁴C from cytidine.

Figure 2.. Chemical and biochemical reactions for each technique discussed

(A) (Bio)chemical modifications used in the study of m⁶A: biochemical modification of m⁶A to hm⁶A by the enzyme FTO and subsequent chemical modification to d⁶A by DTT and biochemical modification of A to a⁶A by writer enzymes and subsequent chemical modification to Cyc-A by iodination. (B) (Bio)chemical modifications used in the study of 5mC/m⁵C: chemical deamination of C to dU by bisulfite, chemical oxidation of 5hmC to 5fC by KRuO₄ and biochemical modification of 5hmC to 5ghmc by the enzyme β-GT, biochemical deamination of 5mC to T by APOBEC, chemical modification of 5caC to dihydrouridine (DHU) by pyridine borane, and structure of 5-azacytidine. (C) Chemical modifications used in the study of inosine: chemical modification of inosine to ce¹I by acrylonitrile and chemical modification of guanosine with glyoxal and further modification with borate. (D) Chemical modifications used in the study of ψ: chemical modification of ψ with CMCT and chemical cleavage of uridine with hydrazine. (E) Chemical modification used in the study of ac⁴C: chemical reduction of ac⁴C by NaCNBH₃.

Figure 3.. Workflows/principles and drawbacks of the gold standard techniques for each modification

(A) Workflow of meRIP/m⁶A-IP-seq. (B) Principle of bisulfite conversion. (C) Principle of RNA/DNA differences. (D) Drawbacks of meRIP/m⁶A-IP-seq. (E) Drawbacks of bisulfite conversion. (F) Drawback of RNA/DNA differences.

Figure 4.. Underlying principles relevant for several techniques

(A) Enrichment of modified RNA by IP. (B) Use of reader proteins to target or enrich for modified RNA. Active moieties can be fused to the reader protein to act as an epitope tag for enrichment or to further modify the RNA. (C) Differential enzyme activity/chemical reactivity as a result of modification. (D) Induction of RT stops or mutations by modified bases. (E) Covalent trapping of RNA-modifying enzymes. (F) Further chemical modification of RNA to alter base-pairing, to alter susceptibility to further modification, or to protect the RNA from digestion.