期刊
BIOMATERIALS
卷 100, 期 -, 页码 172-183出版社
ELSEVIER SCI LTD
DOI: 10.1016/j.biomaterials.2016.05.022
关键词
Molecular beacons; 2 '-O-methyl RNA; Phosphorothioate; RNA imaging; Single-molecule detection; RNA dynamics
资金
- National Natural Science Foundation of China [81371613]
- Beijing Natural Science Foundation [7162114]
- China's 1000 Young Talent Award program
Molecular Beacons (MBs) composed of 2 '-O-methyl RNA (2Me) and phosphorothioate (PS) linkages throughout the backbone (2Me/PSFua MBs) have enabled long-term imaging of RNA in living cells, but excess PS modification can induce nonspecific binding, causing false-positive signals. In this study, we evaluate the intracellular stability of MBs composed of 2Me with various PS modifications, and found that false-positive signals could be reduced to marginal levels when the MBs possess a fully PS-modified loop domain and a phosphodiester stem (2Me/PSLOOP MB). Additionally, 2Me/PSLOOP MBs exhibited uncompromised hybridization kinetics, prolonged functionality and >88% detection accuracy for single RNA transcripts, and could do so without interfering with gene expression or cell growth. Finally, 2Me/PSLOOP MBs could image the dynamics of single mRNA transcripts in the nucleus and the cytoplasm simultaneously, regardless of whether the MBs targeted the 5'- or the 3'-UTR. Together, these findings demonstrate the effectiveness of loop-domain PS modification in reducing nonspecific signals and the potential for sensitive and accurate imaging of individual RNAs at the single-molecule level. With the growing interest in the role of RNA localization and dynamics in health and disease, 2Me/PSLOOP MBs could enable new discoveries in RNA research. (C) 2016 The Author(s). Published by Elsevier Ltd.
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