Light-controlled twister ribozyme with single-molecule detection resolves RNA function in time and space

Arthur Korman, Huabing Sun, Boyang Hua, Haozhe Yang, Joseph N. Capilato, Rakesh Paul, Subrata Panja, Taekjip Ha, Marc M. Greenberg, Sarah A. Woodson

Research output: Contribution to journalArticle

Abstract

Small ribozymes such as Oryza sativa twister spontaneously cleave their own RNA when the ribozyme folds into its active conformation. The coupling between twister folding and self-cleavage has been difficult to study, however, because the active ribozyme rapidly converts to product. Here, we describe the synthesis of a photocaged nucleotide that releases guanosine within microseconds upon photosolvolysis with blue light. Application of this tool to O. sativa twister achieved the spatial (75 μm) and temporal (≤30 ms) control required to resolve folding and self-cleavage events when combined with single-molecule fluorescence detection of the ribozyme folding pathway. Real-time observation of single ribozymes after photo-deprotection showed that the precleaved folded state is unstable and quickly unfolds if the RNA does not react. Kinetic analysis showed that Mg2+ and Mn2+ ions increase ribozyme efficiency by making transitions to the high energy active conformation more probable, rather than by stabilizing the folded ground state or the cleaved product. This tool for light-controlled single RNA folding should offer precise and rapid control of other nucleic acid systems.

Original languageEnglish (US)
Article numberpnas2003425117
JournalProceedings of the National Academy of Sciences of the United States of America
Volume117
Issue number22
DOIs
StatePublished - Jun 2 2020

Keywords

  • Nucleic acid chemistry
  • Photocaged nucleotide
  • RNA folding
  • Ribozyme catalysis
  • Single-molecule FRET

ASJC Scopus subject areas

  • General

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