Dual-TERM enables simultaneous profiling of 5' and 3' terminal modifications in small RNAs by integrating enzymatic...

Small RNAs carry diverse terminal modifications that reflect their biogenesis and affect their stability and function. However, simultaneously discriminating these modifications remains challenging with existing methods. Here, we present Dual TERminal Modification analysis (Dual-TERM), a gel-based strategy that integrates enzymatic pretreatment with phenylboronic acid polyacrylamide gel electrophoresis (PBA-PAGE) to resolve 5' and 3' terminal modifications in small RNAs. Using synthetic RNAs, we demonstrated that Dual-TERM reliably distinguished four major 3' terminal ribose modifications (3'-OH, 3'-Nm, 3'-P, and 3'-cP) based on large band shifts and detected 5' phosphorylation status based on slight mobility changes. We validated the method with endogenous small RNAs-including miRNAs, piRNAs, and 5' tRNA halves-from mammals and plants, confirming species-specific modification patterns. With its simplicity and sensitivity, Dual-TERM provides a versatile tool for characterizing terminal modifications in small RNAs and has the potential to be adapted for high-throughput profiling when coupled with next-generation sequencing.