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

Helen Hwang, Hajin Kim, Sua Myong

Research output: Contribution to journalArticlepeer-review

172 Scopus citations

Abstract

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.

Original languageEnglish
Pages (from-to)7414-7418
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume108
Issue number18
DOIs
StatePublished - May 3 2011

Keywords

  • Cis-trans isomerization
  • DNA-protein interaction
  • Label free protein
  • RNA-protein interaction

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