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

Abstract

N 6 -methyladenosine (m 6 A) is the most prevalent modified base in eukaryotic mRNA and long noncoding RNA (lncRNA). Although candidate sites for the m 6 A modification are identified at the transcriptomic level, methods for site-specific quantification of absolute m 6 A modification levels are still limited. Herein, we present a facile method implementing a deoxyribozyme, VMC10, which preferentially cleaves the unmodified RNA. We leveraged reverse transcription and real-time quantitative PCR along with key control experiments to quantify the methylation fraction of specific m 6 A sites. We validated the accuracy of this method with synthetic RNA in which methylation fractions ranged from 0% to 100% and applied our method to several endogenous sites that were previously identified in sequencing-based studies. This method provides a time-and cost-effective approach for absolute quantification of the m 6 A fraction at specific loci, with the potential for multiplexed quantifications, expanding the current toolkit for studying RNA modifications. AUTHOR CONTRIBUTIONSM.B. J.Z. and J.F. designed the experiments. M.B., J.Z., and E.H. performed the experiments. M.B. analyzed the data. Q.D. synthesized the 35-40 mer synthetic oligonucleotides. M.B., J.Z. and J.F. wrote the manuscript.

N6‐Isopentenyladenosine in RNA Determines the Cleavage Site of Endonuclease Deoxyribozymes

“…[21] In the future,i tw ill be interesting to investigate how other tRNAm odifications, including hydroxylated or thiomethylated i 6 Aa nalogues are recognized and how they influence the catalytic activities of AB08, AC17 and future evolved variants for the study of native tRNAs. [22] Thep resented data demonstrate the surprising plasticity of DNAsc atalytic ability.T he reported modes of action are distinct from previously observed responses of DNAenzymes to m 6 Ai nR NA,a nd no other DNAc atalysts sensitive to nucleobase modifications have been reported. Thed irect comparison of how i 6 Aa nd m 6 Aa ffect the catalytic activity Figure 4.…”

“…[22] Thep resented data demonstrate the surprising plasticity of DNAsc atalytic ability.T he reported modes of action are distinct from previously observed responses of DNAenzymes to m 6 Ai nR NA,a nd no other DNAc atalysts sensitive to nucleobase modifications have been reported. Thed irect comparison of how i 6 Aa nd m 6 Aa ffect the catalytic activity Figure 4. a) Predicted secondary structure of AC17 shown to cut unmodified RNA at G16 and i 6 A-RNA at G15.…”

“…[5] Them 6 A-sensitive DNAzymes were generally applicable to analyse the presence of m 6 Ai nD GACH sequence motifs,a ss hown for lncRNAs and as et of C/D box snoRNAs, [3] and these analyses were recently extended to other RNAs. [6] Thedetailed mechanism, how DNAe nzymes recognize m 6 Aa nd modulate the cleavage response is currently unknown. Best discrimination was observed by the VMC10 DNAe nzyme,w hich was inhibited by m 6 Aand cleaved unmodified RNAupto150-fold faster.…”

Fundamental studies of functional nucleic acids: aptamers, riboswitches, ribozymes and DNAzymes

“…Such enzymes are useful for the enrichment of sparsely methylated RNA, and can be used to validate predicted m 6 A sites in isolated cellular RNA. 145,249 Besides the development of tools for epitranscriptomic research, it is of fundamental interest to understand if and how DNA enzymes distinguish different methylation/ modification states of the target RNA. In the absence of any crystal structure, the responsible supramolecular contacts remain speculative.…”

N6‐Isopentenyladenosine in RNA Determines the Cleavage Site of Endonuclease Deoxyribozymes

“…Inhibition of this mechanisms by 2′‐ O ‐methylated nucleotides was used to analyse ribose methylation in rRNA . The m 6 A‐sensitive DNAzymes were generally applicable to analyse the presence of m 6 A in DGACH sequence motifs, as shown for lncRNAs and a set of C/D box snoRNAs, and these analyses were recently extended to other RNAs . The detailed mechanism, how DNA enzymes recognize m 6 A and modulate the cleavage response is currently unknown.…”