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 Peking University researchers 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/PSLOOP MBs), an architecture that elicits marginal levels of nonspecific signals in cells. The researchers show 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. They then report the first MB-based imaging of intracellular dynamics and localization of single long noncoding RNAs (lncRNAs). They envision the proposed minimally-engineered, MB-based technology for live-cell single-molecule RNA imaging could facilitate new discoveries in RNA research.
MB-based imaging of single NEAT1 transcripts in the nucleus
Live-cell detection of NEAT1 colocalization with PSP1α proteins. 5 µM of anti-repeat MBs or control MBs were injected into HeLa-NEAT1-8x cells transfected with pEYFP-PSP1α constructs. Representative fluorescent images acquired within a few minutes post-injection are shown. (Scale bar, 10 µm).