Short Oligonucleotides Facilitate Co-transcriptional Labeling of RNA at Specific Positions

Labeling RNA molecules at specific positions is critical for RNA research and applications. Such methods are in high demand but still a challenge, especially those that enable native co-synthesis rather than post-synthesis labeling of long RNAs. The method we developed in this work meets these requirements, in which a leader RNA is extended on the hybrid solid-liquid phase by an engineered transcriptional complex following the pause-restart mode. A custom-designed short oligonucleotide is used to functionalize the engineered complex. This remarkable co-transcriptional labeling method incorporates labels into RNAs in high yields with great flexibility. We demonstrate the method by successfully introducing natural modifications, a fluorescent nucleotide analogue and a donor-acceptor fluorophore pair to specific sites located at an internal loop, a pseudoknot, a junction, a helix, and the middle of consecutive identical nucleotides of various RNAs. This newly developed method overcomes efficiency and position-choosing constraints that have hampered routine strategies to label RNAs beyond 200 nucleotides (nt).