DAMP-RNA: DNAzyme-Assisted Methylation Profiling of RNA

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 that employs RNA-cleaving deoxyribozymes (DNAzymes) to detect 2'-O-ribose methylations. The validity of this protocol was demonstrated by analysis of two well-characterized 2'-O-methylated adenines in human U1 and U2 snRNAs. By using siRNA to target NOP58 and antisense oligonucleotides to scaRNA7, we showcased the sensitivity and specificity of our approach in detecting variations in 2'-O-methylation levels. Combining the DNAzyme cleavage with RT-qPCR provides a robust and efficient way to investigate 2'-O-methylation in any cellular RNA regardless of its abundance, yielding valuable insights into the dynamics of RNA modifications. Despite certain limitations, this method stands out for its clarity and reproducibility, paving the way for further exploration in the field of RNA biology.