Monitoring the Conformational Fluctuations of DNA Hairpins Using Single-Pair Fluorescence Resonance Energy Transfer
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- 作者：Jocelyn R. Grunwell ; Jennifer L. Glass ; Thilo D. Lacoste ; Ashok A. Deniz ; Daniel S. Chemla ; and Peter G. Schultz
- 刊名：Journal of the American Chemical Society
- 出版年：2001
- 出版时间：May 9, 2001
- 年：2001
- 卷：123
- 期：18
- 页码：4295 - 4303
- 全文大小：230K
- 年卷期：v.123,no.18(May 9, 2001)
- ISSN：1520-5126
文摘
We present single-pair fluorescence resonance energy transfer (spFRET) observations of individualopening and closing events of surface-immobilized DNA hairpins. Two glass-surface immobilization strategiesemploying the biotin-streptavidin interaction and a third covalent immobilization strategy involving formationof a disulfide bond to a thiol-derivatized glass surface are described and evaluated. Results from image andtime-trace data from surface-immobilized molecules are compared with those from freely diffusing molecules,which are unperturbed by surface interactions. Using a simple two-state model to analyze the open and closedtime distributions for immobilized hairpins, we calculate the lifetimes of the two states. For hairpins with aloop size of 40 adenosines and a stem size of either seven or nine bases, the respective closed-state lifetimesare 45 ± 2.4 and 103 ± 6.0 ms, while the respective open-state lifetimes are 133 ± 5.5 and 142 ± 22 ms.These results show that the open state of the hairpin is favored over the closed state of the hairpin under theseconditions, consistent with previous diffusion fluorescence correlation spectroscopy (FCS) experiments onpoly(A)-loop hairpins. The measured open-state lifetime is about 30 times longer than the calculated 3 msopen-state lifetime for both hairpins based on a closing rate scaling factor derived from a previous FCS studyfor hairpins in diffusion with 12-30 thymidines in their loops. As predicted, the closed-state lifetime is dependenton the stem length and is independent of the loop characteristics. Our findings indicate that current modelsshould consider sequence dependence in calculating ssDNA thermostability. The surface immobilizationchemistries and other experimental techniques described here should prove useful for studies of single-moleculepopulations and dynamics.