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Frontiers in Optics

Application of Single Molecule FRET Photon-correlation Spectroscopy to Studying DNA-Protein Interactions

Samantha Fore, Thomas R. Huser, Rod Balhorn, Monique Cosman, Yin Yeh

Published Jan 1, 2005• 0 citations
Publisher

Abstract

Photon antibunching correlation analysis yields an absolute measure of N from a small number of fluorescent molecules. By combining this technique with FRET, we show how it can obtain information on the numbers of proteins in DNA/protein complexes.
DOI
10.1364/fio.2005.fwk6

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The formation of protein complexes with other proteins and nucleic acids is critical to biological function. Although it is relatively easy to identify the components present in these complexes, it is often difficult to determine their exact stoichiometry and obtain information about the homogeneity of the sample from bulk measurements. We demonstrate the use of single molecule photon-pair correlation spectroscopy to distinguish between discrete numbers of molecules in biological complexes. Fluorescence photon antibunching is observed from a single molecule by employing time-correlated single photon counting in combination with a Hanbury-Brown and Twiss coincidence setup. In addition, pulsed laser excitation and time-tagged time-resolved data collection allow for the measurement of photon arrival times with nanosecond time resolution. The interphoton time distribution between consecutively arriving photons can be calculated and provides a measure of the second-order temporal correlation function. Analysis of this function yields an absolute measure of the number of molecules, N, present in a given complex. It is this ability to measure N that renders this technique powerful for determining stoichiometries in complex biological systems at the single molecule level. We investigate the counting efficiency and statistics of photon antibunching of specifically designed biological samples labeled with multiple copies of the same fluorescent dye and derive conclusions about its use in the analytical evaluation of complex biological samples.
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In optical microscopy fluorescent molecules are used to label target structures like proteins or DNA.With confocal microscopy, a complex of multiple fluorescent molecules is detected as a point spread function due to the diffraction limit. A particular challenge is to determine the number of molecules hiding behind the point spread function. In this work an extended method for determining the number of fluorescent molecules is presented. The method is based on photon antibunching, which is the phenomenon that a single fluorescent molecule can emit only one photon at a time. A statistical analysis of coincidently detected photons can be used to determine the number of photon emitters. In previous works the maximal number of molecules that can be distinguished was about 3. This has now been extended by doubling the number of detectors from 2 to 4, so that up to 4 simultaneously emitted photons can be detected. A new data analysis procedure was established according to the changes of the scheme. Simulations have shown that in theory up to 50 molecules can be resolved under realistic conditions. These predictions were experimentally validated with immobilized dsDNA labeled with 5 fluorophores. The consideration of photobleaching in the data analysis and the use of a photo-stabilizer enable up to 15 molecules to be determined. Thus, this method provides a promising tool for determining the stoichiometry of various biomolecular complex, which can not be achieved by normal microscopic methods.
We report the use of a Hanbury-Brown Twiss apparatus to measure auto- and cross-correlations from pairs of dye molecules tethered to single strands of DNA and coupled by Fluorescence Resonance Energy Transfer (FRET). We observe non-classical photon anti-bunching, and we note that certain features of the observed correlations most likely arise from conformational fluctuations in the biomolecular substrate. These techniques should provide experimental access to molecular dynamics at nanosecond (and longer) timescales.
We report high time-resolution measurements of photon statistics from pairs of dye molecules coupled by fluorescence resonance energy transfer (FRET). In addition to quantum-optical photon antibunching, we observe photon bunching on a time scale of several nanoseconds. We show by numerical simulation that configuration fluctuations in the coupled fluorophore system could account for minor deviations of our data from predictions of basic Förster theory. With further characterization we believe that FRET photon statistics could provide a unique tool for studying DNA mechanics on time scales from 10(-9)-10(-3) s.
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