Fluorescence-force spectroscopy at the single molecule level

single molecule detection;fluorescence microscopy;optical tweezers

Zhou, Ruobo

关键词: single molecule detection;    fluorescence microscopy;    optical tweezers;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/31921/Zhou_Ruobo.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

During the past decade, various powerful single-molecule techniques haveevolved and helped to address important questions in life sciences. As the singlemolecule techniques become mature, there is increasingly pressing need to maximize theinformation content of the analysis in order to be able to study more complex systemsthat better approximate in-vivo conditions. Here, we develop a fluorescence-forcespectroscopy method to combine single-molecule fluorescence spectroscopy with opticaltweezers. Optical tweezers are used to manipulate and observe mechanical properties onthe nanometer scale and piconewton force range. However, once the force range is in thelow piconewton range or less, the spatial resolution of optical tweezers decreasessignificantly. In combination with fluorescence spectroscopy, like single moleculeFörster (or fluorescence) resonance energy transfer (FRET) whose detectable distancerange is approximately 3-10 nm, we are able to observe nanometer fluctuations andinternal conformational changes in a low-force regime. The possibility to placefluorescent labels at nearly any desired position and a sophisticated design of theexperiment increases the amount of information that can be extracted in contrast to puremechanical or fluorescence experiments. We demonstrate the applications of this methodto various biological systems including: 1) to measure the effect of very low forces on thenanometer scale conformational transitions of the DNA four-way (Holliday) junction; 2)to dissect protein diffusion and dissociation mechanisms on single stranded DNA, 3) tocalibrate FRET-based in-vivo force sensors and 4) to study mechanical unfolding ofsingle proteins. The results could not have been obtained with fluorescence or forcemeasurement 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 detectsmall conformational dynamics corresponding to sub-nanometer distance changes ofsingle molecules in a FRET-insensitive short range (< 3 nm), extending the detectabledistance range of our fluorescence-force spectroscopy method.