Delivering Molecular Beacons via an Electroporation-Based Approach Enables Live-Cell Imaging of Single RNA Transcripts and Genomic Loci
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Molecular beacons (MBs) are synthetic oligonucleotide probes that are designed to fluoresce upon hybridization to complementary nucleic acid targets. In contrast to genetically encoded probes that can be readily introduced into cells via standard transfection procedures, using MBs to obtain reliable intracellular measurements entails a reliable delivery method that maximizes MB entry while minimizing cell damage. One promising approach is microporation, a microliter volume electroporation-based method that exhibits reduced harmful events as compared with traditional electroporation methods. In this chapter, we describe in detail microporation steps for MB delivery that we have utilized over the past several years, followed by examples demonstrating successful MB-based imaging of specific RNA transcripts and genomic loci at the single-molecule level.
Key wordsMolecular beacons Microporation Tandem repeats Single-molecule
This project was supported by grants from the National Key R&D Program of China (No. 2016YFA0501603), the National Natural Science Foundation of China (No. 31771583), and the Beijing Municipal R&D Key Project (Grant No. Z151100003915081).
- 5.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
- 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