Protein induced fluorescence enhancement as a single molecule assay with short distance sensitivity.

Citation data:

Proceedings of the National Academy of Sciences of the United States of America, ISSN: 1091-6490, Vol: 108, Issue: 18, Page: 7414-8

Publication Year:
2011
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Citations 100
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Repository URL:
http://scholarworks.unist.ac.kr/handle/201301/7083
PMID:
21502529
DOI:
10.1073/pnas.1017672108; 10.1073/pnas.1017672108/
PMCID:
PMC3088603
Author(s):
Hwang, Helen; Kim, Hajin; Myong, Sua
Publisher(s):
Proceedings of the National Academy of Sciences; NATL ACAD SCIENCES
Tags:
Multidisciplinary; Cis-trans isomerization; DNA-protein interaction; Label free protein; RNA-protein interaction
article description
Single-molecule FRET has been widely used for monitoring protein-nucleic acids interactions. Direct visualization of the interactions, however, often requires a site-specific labeling of the protein, which can be circuitous and inefficient. In addition, FRET is insensitive to distance changes in the 0-3-nm range. Here, we report a systematic calibration of a single molecule fluorescence assay termed protein induced fluorescence enhancement. This method circumvents protein labeling and displays a marked distance dependence below the 4-nm distance range. The enhancement of fluorescence is based on the photophysical phenomenon whereby the intensity of a fluorophore increases upon proximal binding of a protein. Our data reveals that the method can resolve as small as a single base pair distance at the extreme vicinity of the fluorophore, where the enhancement is maximized. We demonstrate the general applicability and distance sensitivity using (a) a finely spaced DNA ladder carrying a restriction site for BamHI, (b) RNA translocation by DExH enzyme RIG-I, and (c) filament dynamics of RecA on single-stranded DNA. The high spatio-temporal resolution data and sensitivity to short distances combined with the ability to bypass protein labeling makes this assay an effective alternative or a complement to FRET.