title: Protein induced fluorescence enhancement as a single molecule assay with short distance sensitivity.
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.
authors.affiliation: Bioengineering Department, University of Illinois, Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: Helen
authors.initials: H
authors.lastname: Hwang
authors.forename: Hajin
authors.initials: H
authors.lastname: Kim
authors.forename: Sua
authors.initials: S
authors.lastname: Myong
mesh_terms: D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D015245:Deoxyribonuclease BamHI / Q000378:metabolism; D009841:Oligonucleotides / Q000235:genetics; D011485:Protein Binding; D012313:RNA / Q000378:metabolism*; D011938:Rec A Recombinases / Q000378:metabolism; D011971:Receptors, Immunologic; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D009841:Oligonucleotides; D011971:Receptors, Immunologic; D012313:RNA; D004247:DNA; D011938:Rec A Recombinases; D015245:Deoxyribonuclease BamHI; C452820:RIGI protein, human; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases