Format

Send to

Choose Destination
Nucleic Acids Res. 2017 Aug 21;45(14):e130. doi: 10.1093/nar/gkx504.

FASTmiR: an RNA-based sensor for in vitro quantification and live-cell localization of small RNAs.

Author information

1
Bio-Imaging Center, Delaware Biotechnology Institute, University of Delaware, Newark, DE 19711, USA.
2
Department of Plant and Soil Sciences, University of Delaware, Newark, DE 19716, USA.
3
Nanobioscience Constellation, State University of New York- Polytechnic Institute, College of Nanoscale Science and Engineering, Albany, NY 12203, USA.
4
Center for Bioinformatics and Computational Biology, University of Delaware, Newark, DE 19711, USA.
5
Donald Danforth Plant Science Center, 975 North Warson Road, St Louis, MO 63132, USA.
6
University of Missouri-Columbia, Division of Plant Sciences, 52 Agriculture Lab, Columbia, MO 65211, USA.

Abstract

Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), play a variety of important regulatory roles in many eukaryotes. Their small size has made it challenging to study them directly in live cells. Here we describe an RNA-based fluorescent sensor for small RNA detection both in vitro and in vivo, adaptable for any small RNA. It utilizes an sxRNA switch for detection of miRNA-mRNA interactions combined with a fluorophore-binding sequence 'Spinach', a GFP-like RNA aptamer for which the RNA-fluorophore complex exhibits strong and consistent fluorescence under an excitation wavelength. Two example sensors, FASTmiR171 and FASTmiR122, can rapidly detect and quantify the levels of miR171 and miR122 in vitro. The sensors can determine relative levels of miRNAs in total RNA extracts with sensitivity similar to small RNA sequencing and northern blots. FASTmiR sensors were also used to estimate the copy number range of miRNAs in total RNA extracts. To localize and analyze the spatial distribution of small RNAs in live, single cells, tandem copies of FASTmiR122 were expressed in different cell lines. FASTmiR122 was able to quantitatively detect the differences in miR122 levels in Huh7 and HEK293T cells demonstrating its potential for tracking miRNA expression and localization in vivo.

PMID:
28586459
PMCID:
PMC5737440
DOI:
10.1093/nar/gkx504
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Silverchair Information Systems Icon for PubMed Central
Loading ...
Support Center