Multiplexed Photoactivation of mRNA with Single-Cell Resolution

ACS Chem Biol. 2020 Jul 17;15(7):1773-1779. doi: 10.1021/acschembio.0c00205. Epub 2020 Jun 12.

Abstract

We demonstrate sequential optical activation of two types of mRNAs in the same mammalian cell through the sequential photocleavage of small molecule caging groups ("photocages") tethered to the 5'-untranslated region (5'-UTR) of mRNAs. Synthetic photocages were conjugated onto target mRNA using RNA-TAG, an enzymatic site-specific RNA modification technique. Translation of mRNA was severely reduced upon conjugation of the photocages onto the 5'-UTR. However, subsequent photorelease of the cages from the mRNA transcript triggered activation of translation with single-cell spatiotemporal resolution. To achieve sequential photoactivation of two mRNAs in the same cell, we synthesized a pair of photocages that can be selectively cleaved from mRNA upon photoirradiation with different wavelengths of light. Sequential photoactivation of two mRNAs enabled precise optical control of translation of two unique transcripts. We believe that this modular approach to precisely and rapidly control gene expression will serve as a powerful tool in future biological studies that require controlling translation of multiple transcripts with high spatiotemporal resolution.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • 5' Untranslated Regions
  • Coumarins / chemical synthesis
  • Coumarins / chemistry*
  • Coumarins / radiation effects
  • HEK293 Cells
  • Humans
  • Light
  • Protein Biosynthesis / drug effects
  • Protein Biosynthesis / radiation effects*
  • RNA, Messenger / chemistry
  • RNA, Messenger / genetics*
  • RNA, Messenger / radiation effects

Substances

  • 5' Untranslated Regions
  • Coumarins
  • RNA, Messenger