Quantitative detection of 2′-O-methylated residues in non-coding RNAs using DNAzymes and quantitative RT-PCR

Abstract

In recent decades, post-transcriptional RNA modifications have emerged as a critical regulatory network in cellular processes. The emergence of advanced technologies, including next-generation sequencing (e.g., Illumina) and third-generation sequencing (e.g., Nanopore and PacBio), has significantly improved the detection of RNA modifications in both coding (mRNAs) and non-coding RNAs (ncRNAs). However, these powerful and transcriptome-wide approaches often generate a significant number of false positive hits, highlighting the need for complementary methods allowing validation of the RNA modification profile as well as more precise quantification. In this study, we present further optimization of a straightforward and sensitive protocol using RNA-cleaving deoxyribozymes (DNAzymes) to validate 2′-O-methylations. The validity of this protocol was demonstrated by the analysis of two well-characterized 2′-O-methylated adenines in human U1 and U2 small nuclear RNAs (snRNAs). Using siRNA targeting NOP58 and antisense oligonucleotides targeting scaRNA7, we showcased the sensitivity and specificity of our approach in detecting variations in 2′-O-methylation levels. The DAMP-RNA (DNAzyme-Assisted Methylation Profiling of RNA) method, combining the DNAzyme cleavage with RT-qPCR provides a robust and efficient way to investigate known 2′-O-methylation in any cellular RNA regardless of its abundance, yielding valuable insights into the dynamics of RNA modifications. Despite its limitations, this method represents a significant advancement, enhancing clarity and reproducibility in the epitranscriptomics field, paving the way for further exploration in the RNA biology.