Tandem Spinach Array for mRNA Imaging in Living Bacterial Cells

Sci Rep. 2015 Nov 27;5:17295. doi: 10.1038/srep17295.

Abstract

Live cell RNA imaging using genetically encoded fluorescent labels is an important tool for monitoring RNA activities. A recently reported RNA aptamer-fluorogen system, the Spinach, in which an RNA aptamer binds and induces the fluorescence of a GFP-like 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) ligand, can be readily tagged to the RNA of interest. Although the aptamer-fluorogen system is sufficient for imaging highly abundant non-coding RNAs (tRNAs, rRNAs, etc.), it performs poorly for mRNA imaging due to low brightness. In addition, whether the aptamer-fluorogen system may perturb the native RNA characteristics has not been systematically characterized at the levels of RNA transcription, translation and degradation. To increase the brightness of these aptamer-fluorogen systems, we constructed and tested tandem arrays containing multiple Spinach aptamers (8-64 aptamer repeats). Such arrays enhanced the brightness of the tagged mRNA molecules by up to ~17 fold in living cells. Strong laser excitation with pulsed illumination further increased the imaging sensitivity of Spinach array-tagged RNAs. Moreover, transcriptional fusion to the Spinach array did not affect mRNA transcription, translation or degradation, indicating that aptamer arrays might be a generalizable labeling method for high-performance and low-perturbation live cell RNA imaging.

MeSH terms

  • Aptamers, Nucleotide / chemical synthesis
  • Aptamers, Nucleotide / chemistry*
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / metabolism
  • Escherichia coli / genetics*
  • Escherichia coli / metabolism
  • Escherichia coli / ultrastructure
  • Lac Operon
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Molecular Imaging / methods*
  • Oligonucleotide Array Sequence Analysis / methods
  • Protein Biosynthesis
  • RNA, Bacterial / genetics
  • RNA, Bacterial / metabolism
  • RNA, Bacterial / ultrastructure*
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • RNA, Messenger / ultrastructure*
  • Spinacia oleracea / chemistry*
  • Spinacia oleracea / metabolism
  • Tandem Repeat Sequences
  • Transcription, Genetic

Substances

  • Aptamers, Nucleotide
  • Luminescent Proteins
  • RNA, Bacterial
  • RNA, Messenger
  • red fluorescent protein
  • bacteriophage T7 induced DNA polymerase
  • DNA-Directed DNA Polymerase