mrRNA-directed DNA methylation enables selective mutant oncogene silencing without exogenous proteins

The outcome of recent demethylation therapy is limited by the unspecific hypomethylation of oncogenes. The mechanism underlying differential DNA methylation distribution in the same topological domain remains unclear. Here, we established an efficient framework based on heterogeneity across pan-cancers to systemically characterize DNA methylation regulatory boxes (mrBoxes). Through plasmid-based functional motif interactome analysis assay, the role of mrBoxes on DNA methylation was validated. Moreover, the U1 small nuclear ribonucleoprotein A (SNPRA) was found to regulate the chromatin localization of DNMT1 to mrBox. In this process, long noncoding RNAs were identified to act as a bridge, connecting the targeted mrBox element and SNRPA/DNMT1 complex. Based on this regulatory model, we synthesized exogenous methylation-regulating RNA (mrRNA), which contains only the mrBox-recognizing and SNRPA/DNMT1 complex-binding motif. Furthermore, we developed a methylation-regulating therapy called “OncoMutation Silence”, which regulates the DNA methylation of mutated oncogenes through mrRNA. The efficacy and specificity of this therapy have been verified in tumors with KrasG12D mutation. Collectively, this study reveals mrBox-related regulatory model of DNA methylation, and provides mrRNA-based tumor therapy for silencing specific mutated oncogenes.