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  • Accepted Manuscript: Light-up and FRET aptamer reporters; evaluating their applications for imaging transcription in eukaryotic cells

Title: Light-up and FRET aptamer reporters; evaluating their applications for imaging transcription in eukaryotic cells

The regulation of RNA transcription is central to cellular function. Changes in gene expression drive differentiation and cellular responses to events such as injury. RNA trafficking can also have a large impact on protein expression and its localization. Thus, the ability to image RNA transcription and trafficking in real time and in living cells is a worthwhile goal that has been difficult to achieve. The availability of “light-up” aptamers that cause an increase in fluorescence of their ligands when bound by the aptamer have shown promise for reporting on RNA production and localization in vivo. Here we have investigated two light-up aptamers (the malachite green aptamer and the Spinach aptamers) for their suitabilities as reporters of RNA expression in vivo using two eukaryotic cell types, yeast and mammalian. Our analysis focused on the aptamer ligands, their contributions to background noise, and the impact of tandem aptamer strings on signal strength and ligand affinity. Whereas the background fluorescence is very low in vitro, this is not always true for cell imaging. Our results suggest the need for caution in using light-up aptamers as reporters for imaging RNA. In particular, images should be collected and analyzed by operators blinded to the samplemore » identities. The appropriate control condition of ligand with the cells in the absence of aptamer expression must be included in each experiment. This control condition establishes that the specific interaction of ligand with aptamer, rather than nonspecific interactions with unknown cell elements, is responsible for the observed fluorescent signals. As a result, high background signals due to nonspecific interactions of aptamer ligands with cell components can be minimized by using IMAGEtags (Intracellular Multiaptamer GEnetic tags), which signal by FRET and are promising RNA reporters for imaging transcription.« less
Authors:
 [1] ;  [2] ;  [1] ;  [3] ;  [1] ;  [4] ;  [4]
+ Show Author Affiliations
  1. Iowa State Univ., Ames, IA (United States)
  2. Iowa State Univ., Ames, IA (United States); Cornell Univ., Ithaca, NY (United States)
  3. Ames Lab., Ames, IA (United States); Univ. of Michigan Medical School, Ann Arbor, MI (United States)
  4. Ames Lab. and Iowa State Univ., Ames, IA (United States)
Publication Date:
Report Number(s):
IS-J-8995
Journal ID: ISSN 1046-2023; PII: S104620231530178X
Grant/Contract Number:
AC02-07CH11358
Type:
Accepted Manuscript
Journal Name:
Methods
Additional Journal Information:
Journal Volume: 98; Journal Issue: C; Journal ID: ISSN 1046-2023
Publisher:
Elsevier
Research Org:
Ames Laboratory (AMES), Ames, IA (United States)
Sponsoring Org:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; RNA imaging; aptamer; DFHBI; Malachite green; fluorescence; FRET
OSTI Identifier:
1254308
Alternate Identifier(s):
OSTI ID: 1251776
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  • Cited by: 9works
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