Journal
ANALYTICAL BIOCHEMISTRY
Volume 429, Issue 2, Pages 92-98Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2012.07.010
Keywords
Molecular beacon; mRNA; Quantitative analysis; Probing
Funding
- Basic Science Program through National Research Foundation of Korea (KRF)
- Ministry of Education, Science, and Technology (MEST) [2011-0021956, 2012-001680]
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Quantitation of poly(A)-RNA, time-dependent visualization of intracellular poly(A)(+)-RNA localization in living mammalian cells, and time-resolved intracellular binding dynamics of molecular beacons at the single-molecule level using a fluorescence resonance energy transfer (FRET)-based molecular beacon are described. FRET-based molecular beacons were designed as poly(A)-targeting probes to be oligonucleotides that contained Cy5 and Cy3 fluorescent dyes at the strand ends and a poly(A)-targeting sequence inside the strand. Our ratiometric analysis using poly(A)-targeting probes allowed for highly specific and wide-ranging detection (from 1.25 nM to 0.5 mu M) of poly(A)-RNA, as well as for determination of K-d values, and revealed a distribution of the probe itself and localization of the target RNA sequence in cells. Furthermore, time-dependent FRET-mediated fluorescence changes at the single-molecule level caused by the folding-induced gradual conformation changes in live cells were observed. (C) 2012 Elsevier Inc. All rights reserved.
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