Double-Stranded RNA-Specific Templated Reaction with Triplex Forming PNA

RNA, originally perceived as a simple information transfer biopolymer, is emerging as an important regulator in cellular processes. A number of non-coding RNAs are double-stranded and there is a need for technologies to reliably detect and image such RNAs for biological and biomedical research. Herein we report double-stranded RNA-specific templated reaction resulting from PNA-reagent conjugates that are brought within reactive distance through the formation of sequence-specific triplexes onto double-stranded RNA. The reaction makes use of a ruthenium-based photocatalyst that reduces a pyridinium-based immolative linker, unmasking a profluorophore. The reaction was shown to proceed with signal amplification and to be selective for double-stranded RNA over DNA as well as single-stranded RNA. The generality of the triplex formation was enabled by non-canonical nucleobases that extend the Hoogsteen base-pairing repertoire. The technology was applied to a templated reaction using pre-microRNA 31.