Live-Cell Imaging of Long Noncoding RNAs Using Molecular Beacons
Long noncoding RNAs (lncRNAs) are a family of non-protein-coding RNA transcripts greater than 200 nucleotides in length that have been regarded as crucial modulators of gene expression in various biological and disease contexts, but mechanisms underlying such regulation still remains largely elusive. In addition to cell lysate-based approaches that have proven invaluable for studies of lncRNAs, live-imaging methods can add value by providing more in-depth information on lncRNA dynamics and localizations at the single-molecule level. Recently, we have developed a versatile imaging approach based on molecular beacons (MBs), which are a class of fluorogenic oligonucleotide-based probes with the capacity to convert RNA target hybridization into a measurable fluorescence signal. In this chapter, we describe the detailed protocol of using MBs to illuminate lncRNA transcripts at the single-molecule level in living cells.
Key wordsMolecular beacons lncRNA Tandem repeats Single-molecule
This project was supported by grants from the National Key R&D Program of China (Nos. 2016YFA0501603 and 2016YFA0100702), the National Natural Science Foundation of China (Nos. 31771583 and 81371613), and the Beijing Natural Science Foundation (7162114).
- 6.Chen M, Ma Z, Wu X, Mao S, Yang Y, Tan J, Krueger CJ, Chen AK (2017) A molecular beacon-based approach for live-cell imaging of RNA transcripts with minimal target engineering at the single-molecule level. Sci Rep 7(1):1550. https://doi.org/10.1038/s41598-017-01740-1CrossRefPubMedPubMedCentralGoogle Scholar
- 7.Yoon JH, Abdelmohsen K, Kim J, Yang X, Martindale JL, Tominaga-Yamanaka K, White EJ, Orjalo AV, Rinn JL, Kreft SG, Wilson GM, Gorospe M (2013) Scaffold function of long non-coding RNA HOTAIR in protein ubiquitination. Nat Commun 4:2939. https://doi.org/10.1038/ncomms3939CrossRefPubMedPubMedCentralGoogle Scholar
- 11.Zhao D, Yang Y, Qu N, Chen M, Ma Z, Krueger CJ, Behlke MA, Chen AK (2016) Single-molecule detection and tracking of RNA transcripts in living cells using phosphorothioate-optimized 2'-O-methyl RNA molecular beacons. Biomaterials 100:172–183. https://doi.org/10.1016/j.biomaterials.2016.05.022CrossRefPubMedGoogle Scholar