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).
T7 RNA polymerase is the most widely used enzyme in RNA synthesis, and it is also used for RNA labeling in position-selective labeling of RNA (PLOR). PLOR is a liquid–solid hybrid phase method that has been developed to introduce labels to specific positions of RNA. Here, we applied PLOR as a single-round transcription method to quantify the terminated and read-through products in transcription for the first time. Various factors, including pausing strategies, Mg2+, ligand and the NTP concentration at the transcriptional termination of adenine riboswitch RNA have been characterized. This helps to understand transcription termination, which is one of the least understood processes in transcription. Additionally, our strategy can potentially be used to study the co-transcription behavior of general RNA, especially when continuous transcription is not desired.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.