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Ha, Taekjip Aksimentiev, Aleksei Ha, Taekjip Selvin, Paul R. Stack, John D. Zhou, Ruobo 2012-06-27T21:19:26Z 2014-06-28T10:00:18Z 2012-05 2012-06-27T21:19:26Z 2012-05 During the past decade, various powerful single-molecule techniques have evolved and helped to address important questions in life sciences. As the single molecule techniques become mature, there is increasingly pressing need to maximize the information content of the analysis in order to be able to study more complex systems that better approximate in-vivo conditions. Here, we develop a fluorescence-force spectroscopy method to combine single-molecule fluorescence spectroscopy with optical tweezers. Optical tweezers are used to manipulate and observe mechanical properties on the nanometer scale and piconewton force range. However, once the force range is in the low piconewton range or less, the spatial resolution of optical tweezers decreases significantly. In combination with fluorescence spectroscopy, like single molecule Förster (or fluorescence) resonance energy transfer (FRET) whose detectable distance range is approximately 3-10 nm, we are able to observe nanometer fluctuations and internal conformational changes in a low-force regime. The possibility to place fluorescent labels at nearly any desired position and a sophisticated design of the experiment increases the amount of information that can be extracted in contrast to pure mechanical or fluorescence experiments. We demonstrate the applications of this method to various biological systems including: 1) to measure the effect of very low forces on the nanometer scale conformational transitions of the DNA four-way (Holliday) junction; 2) to dissect protein diffusion and dissociation mechanisms on single stranded DNA, 3) to calibrate FRET-based in-vivo force sensors and 4) to study mechanical unfolding of single proteins. The results could not have been obtained with fluorescence or force measurement alone, and clearly demonstrates the power and generality of our approach. Finally, we show that self-quenching of two identical fluorophores can be used to detect small conformational dynamics corresponding to sub-nanometer distance changes of single molecules in a FRET-insensitive short range (< 3 nm), extending the detectable distance range of our fluorescence-force spectroscopy method. Item withdrawn by Mark Zulauf (zulauf@illinois.edu) on 2012-04-10T18:54:33Z Item was in collections: University of Illinois Theses & Dissertations (ID: 1) No. of bitstreams: 2 Zhou_Ruobo-thesis.doc: 19659776 bytes, checksum: 02eb6a0652de1c68c6cc634e1c902dc0 (MD5) Zhou_Ruobo.pdf: 17863661 bytes, checksum: fc8d8105800a84c4e2ac31912223cfda (MD5) Made available in DSpace on 2012-06-27T21:19:26Z (GMT). No. of bitstreams: 4 Zhou_Ruobo.pdf: 17863617 bytes, checksum: ef8402cee19879b8a5c54bd7528a26ba (MD5) license.txt: 4058 bytes, checksum: 6d006621667539391f60ab5a7387c7a1 (MD5) Zhou_Ruobo-thesis.doc: 19660800 bytes, checksum: f163b31d0d97fd7aa4931d079fa9720b (MD5) 1_Zhou_Ruobo-thesis.doc: 19660800 bytes, checksum: f163b31d0d97fd7aa4931d079fa9720b (MD5) Item marked as restricted to the 'UIUC Users [automated]' Group (id=2) by William Ingram (wingram2@illinois.edu) on 2012-06-27T21:24:31Z Item is restricted until 2014-06-27T21:24:27Z Item reinstated by Sarah Shreeves (sshreeve@illinois.edu) on 2014-06-28T10:00:18Z Item was in collections: Dissertations and Theses - Physics (ID: 445) Graduate Theses and Dissertations at Illinois (ID: 204) No. of bitstreams: 4 Zhou_Ruobo.pdf: 17863617 bytes, checksum: ef8402cee19879b8a5c54bd7528a26ba (MD5) license.txt: 4058 bytes, checksum: 6d006621667539391f60ab5a7387c7a1 (MD5) Zhou_Ruobo-thesis.doc: 19660800 bytes, checksum: f163b31d0d97fd7aa4931d079fa9720b (MD5) 1_Zhou_Ruobo-thesis.doc: 19660800 bytes, checksum: f163b31d0d97fd7aa4931d079fa9720b (MD5) Item released from any restrictions by Sarah Shreeves (sshreeve@illinois.edu) on 2014-06-28T10:00:18Z http://hdl.handle.net/2142/31921 en Copyright 2012 Ruobo Zhou single molecule detection fluorescence microscopy optical tweezers Fluorescence-force spectroscopy at the single molecule level text Physics 1244 Physics 0240 University of Illinois at Urbana-Champaign Dissertation Ph.D. PHD:Physics -UIUC 10KS0240PHD