RNA‐Cleaving Deoxyribozymes Differentiate Methylated Cytidine Isomers in RNA

Abstract

Deoxyribozymes are emerging as modification‐specific endonucleases for the analysis of epigenetic RNA modifications. Here, we report RNA‐cleaving deoxyribozymes that differentially respond to the presence of natural methylated cytidines, 3‐methylcytidine (m3C), N4‐methylcytidine (m4C), and 5‐methylcytidine (m5C), respectively. Using in vitro selection, we found several DNA catalysts, which are selectively activated by only one of the three cytidine isomers, and display 10‐ to 30‐fold accelerated cleavage of their target m3C‐, m4C‐ or m5C‐modified RNA. An additional deoxyribozyme is strongly inhibited by any of the three methylcytidines, but effectively cleaves unmodified RNA. The mXC‐detecting deoxyribozymes are programmable for the interrogation of natural RNAs of interest, as demonstrated for human mitochondrial tRNAs containing known m3C and m5C sites. The results underline the potential of synthetic functional DNA to shape highly selective active sites.

Synthesis of N4-acetylated 3-methylcytidine phosphoramidites for RNA solid-phase synthesis

“…Additionally, new RNA sequencing methods, AlkAniline-Seq [ 18 ] and HAC-seq [ 19 ], have been developed to detect m 3 C modifications in transcriptome-wide manner. Furthermore, deoxyribozyme tools to detect m 3 C, m 4 C and m 5 C have been selected in vitro and are able to distinguish the methylation position based on distinct kinetic signatures of their RNA catalyzed backbone cleavage reaction at the site of modification [ 20 ].…”

High-Throughput Activity Profiling of RNA-Cleaving DNA Catalysts by Deoxyribozyme Sequencing (DZ-seq)

“…In our efforts to develop tools for site-specific detection of epigenetic modifications in RNA, we performed in vitro selections to identify RNA-cleaving deoxyribozymes that differentiate between modified and unmodified RNA. [23][24][25] Recently, we reported DNAzymes that distinguish between unmodified cytosine (C), 3-methylcytidine (m 3 C), N 4 methylcytidine (m 4 C) and 5-methylcytidine (m 5 C). 25 Using Sanger sequencing and analysis of sequence enrichment in the NGS data with subsequent characterization of candidate catalysts by gel shift-based kinetic assays, we found few DNA enzymes with the desired properties.…”

“…[23][24][25] Recently, we reported DNAzymes that distinguish between unmodified cytosine (C), 3-methylcytidine (m 3 C), N 4 methylcytidine (m 4 C) and 5-methylcytidine (m 5 C). 25 Using Sanger sequencing and analysis of sequence enrichment in the NGS data with subsequent characterization of candidate catalysts by gel shift-based kinetic assays, we found few DNA enzymes with the desired properties. Screening of further deoxyribozymes was time-consuming and laborious as every candidate sequence had to be individually assayed for its cleavage activity.…”

“…We applied DZ-seq to analyze endpoint activity (10 mM Mg 2+ , 6 h, 37 °C) of in-vitro selection pools AK, AL, AM and AN which have been trained previously to cleave NC-, Nm 3 C-, Nm 4 C-and Nm 5 C-dinucleotide containing all-RNA substrates, respectively (N = A, C, G, U). 25 We prepared in total 16 sequencing libraries by combining eight deoxyribozyme libraries from rounds 7 and 18 of each in-vitro selection with four RNA substrates (R1−R4, Table S2) in various combinations. Eight sequencing libraries contained DNA pools ligated to the unmodified NC-substrate R1.…”

“…However, most of the data points in the DZ-seq dataset agreed well with the in-trans selectivity profiles of the previously characterized deoxyribozymes (exemplary data points are highlighted in Figure 2B for deoxyribozymes AL112, AM101 and AK104). 25 Hence, DZ-seq can be used for identification of deoxyribozymes that differentiate between modified and unmodified RNA substrates in intermolecular cleavage reactions. To this end, we picked candidate sequences AM328-AM332 (D14−D18) which were predicted by DZ-seq to preferentially cleave Gm 3 C-, Am 3 C-, Cm 3 C-, Gm 4 C-and Am 5 Ccontaining RNAs, respectively (Figure 2C).…”