In:
Scientific Reports, Springer Science and Business Media LLC, Vol. 7, No. 1 ( 2017-05-08)
Abstract:
Analysis of RNA dynamics and localization at the single-molecule level in living cells has been predominantly achieved by engineering target RNAs with large insertions of tandem repeat sequences that are bound by protein-based or oligonucleotide-based fluorescent probes. Thus, individual RNAs are tagged by multiple fluorescent probes, making them detectable by fluorescence microscopy. Since large insertions may affect RNA processes including trafficking and localization, here we present a strategy to visualize single RNA transcripts in living cells using molecular beacons (MBs) - fluorogenic oligonucleotide probes - with minimal target engineering. The MBs are composed of 2′-O-methyl RNAs with a fully phosphorothioate-modified loop domain (2Me/PS LOOP MBs), an architecture that elicits marginal levels of nonspecific signals in cells. We showed that MBs can detect single transcripts containing as few as 8 target repeat sequences with ~90% accuracy. In both the nucleus and the cytoplasm, mRNAs harboring 8 repeats moved faster than those with 32 repeats, suggesting that intracellular activities are less impeded by smaller engineered insertions. We then report the first MB-based imaging of intracellular dynamics and localization of single long noncoding RNAs (lncRNAs). We envision the proposed minimally-engineered, MB-based technology for live-cell single-molecule RNA imaging could facilitate new discoveries in RNA research.
Type of Medium:
Online Resource
ISSN:
2045-2322
DOI:
10.1038/s41598-017-01740-1
Language:
English
Publisher:
Springer Science and Business Media LLC
Publication Date:
2017
detail.hit.zdb_id:
2615211-3
