N⁶‐Methyladenosine‐Sensitive RNA‐Cleaving Deoxyribozymes

Abstract

Deoxyribozymes are synthetic enzymes made of DNA that can catalyze the cleavage or formation of phosphodiester bonds and are useful tools for RNA biochemistry. Herein, we report new RNA‐cleaving deoxyribozymes to interrogate the methylation status of target RNAs, thereby providing an alternative method for the biochemical validation of RNA methylation sites containing N6‐methyladenosine, which is the most wide‐spread and extensively investigated natural RNA modification. The developed deoxyribozymes are sensitive to the presence of N6‐methyladenosine in RNA near the cleavage site. One class of these DNA enzymes shows faster cleavage of methylated RNA, while others are strongly inhibited by the modified nucleotide. The general applicability of the new deoxyribozymes is demonstrated for several examples of natural RNA sequences, including a lncRNA and a set of C/D box snoRNAs, which have been suggested to contain m6A as a regulatory element that influences RNA folding and protein binding.

Deoxyribozyme-based method for absolute quantification of N6-methyladenosine fractions at specific sites of RNA

“…In summary, here we present a method for quantifying the absolute methylation fraction of potential m 6 A sites using a previously developed VMC10 DR (27), expanding the toolkit for site-specific quantification of m 6 A. In addition, the method can be adjusted for high throughput quantification of m 6 A sites.…”

“…While this method is easy to implement, there are several limitations that need to be considered. Firstly, the assay utilizes VMC10 DR, which has high cleavage efficiencies only on DGACH sequences, limiting its application on a subset of m 6 A sites with the DAACH sequences (27). Secondly, DR digestion efficiency varies among different sequences.…”

“…To address these challenges, we present an easy-to-implement method for quantifying m 6 A fraction at specific loci from extracted total RNA. The method utilizes a recently reported deoxyribozyme (DR) by Sednev et al, VMC10, to discriminate between A and m 6 A containing RNA (27) (Fig. 1A).…”

“…We chose VMC10 DR because it cleaves unmethylated A with reasonably high and robust efficiency while its cleavage of m 6 A remains low even after a long incubation time (27). Sednev et al demonstrated that the remainder of intact RNA after VMC10 DR treatment is correlated with the known methylation level of specific sites of abundant endogenous RNAs (27). However, quantification of the absolute m 6 A fraction requires additional characterization and correction of potential false positive and false negative due to various factors.…”

DNAzyme-dependent Analysis of rRNA 2’-O-Methylation

“…We used the DNAzyme approach to investigate 2'-O-methylation of rRNA. One can also use this technique to analyze other RNA modifications, such as N 6 -methyladenosine 19 . Ribosomal RNA, due to its abundance, can be analyzed by electrophoresis and the cleavage products can be visualized under the UV light.…”