Repurposing an adenine riboswitch into a fluorogenic imaging and sensing tag. Academic Article uri icon

Overview

abstract

  • Fluorogenic RNA aptamers are used to genetically encode fluorescent RNA and to construct RNA-based metabolite sensors. Unlike naturally occurring aptamers that efficiently fold and undergo metabolite-induced conformational changes, fluorogenic aptamers can exhibit poor folding, which limits their cellular fluorescence. To overcome this, we evolved a naturally occurring well-folded adenine riboswitch into a fluorogenic aptamer. We generated a library of roughly 1015 adenine aptamer-like RNAs in which the adenine-binding pocket was randomized for both size and sequence, and selected Squash, which binds and activates the fluorescence of green fluorescent protein-like fluorophores. Squash exhibits markedly improved in-cell folding and highly efficient metabolite-dependent folding when fused to a S-adenosylmethionine (SAM)-binding aptamer. A Squash-based ratiometric sensor achieved quantitative SAM measurements, revealed cell-to-cell heterogeneity in SAM levels and revealed metabolic origins of SAM. These studies show that the efficient folding of naturally occurring aptamers can be exploited to engineer well-folded cell-compatible fluorogenic aptamers and devices.

publication date

  • December 22, 2021

Research

keywords

  • Aptamers, Nucleotide
  • Biosensing Techniques
  • Optical Imaging

Identity

PubMed Central ID

  • PMC8967656

Scopus Document Identifier

  • 85121545282

Digital Object Identifier (DOI)

  • 10.1016/j.chembiol.2021.04.006

PubMed ID

  • 34937909

Additional Document Info

volume

  • 18

issue

  • 2