Single-molecule binding experiments on long time scales

Abstract

Single-molecule binding experiments on long time scales Elenko, Mark P.; Szostak, Jack W.; van Oijen, Antoine M. Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. We describe an approach for performing single-molecule binding experiments on time scales from hours to days, allowing for the observation of slower kinetics than have been previously investigated by single-molecule techniques. Total internal reflection fluorescence microscopy is used to image the binding of labeled ligand to molecules specifically coupled to the surface of an optically transparent flow cell. Long-duration experiments are enabled by ensuring sufficient positional, chemical, thermal, and image stability. Principal components of this experimental stability include illumination timing, solution replacement, and chemical treatment of solution to reduce photodamage and photobleaching; and autofocusing to correct for spatial drift. © 2010 American Institute of Physics. ͓doi:10.1063/1.3473936͔ I. SINGLE-MOLECULE MICROSCOPY FOR BINDINGBinding is arguably the most fundamental of biomolecular interactions, and as such has been investigated using a wide array of methods. Canonical solution-phase methods are often limited to nonequilibrium measurements, are restricted in the rate and K d ranges they can access, and provide only ensemble-averaged molecular information. As such, it can be challenging to capture the fine structure of molecular behavior with bulk-phase methods. We seek to take advantage of the blooming of single-molecule techniques that have already enabled the exploration of previously inaccessible biomolecular phenomena, including enzymatic memory, molecular motor motion, folding, conformational change, and catalysis. [1][2][3][4] The study of these phenomena at the ultimate level of sensitivity, that of a single molecule, allows for the observation of variations between and within single molecules under equilibrium conditions.Single-molecule experiments have typically explored molecular interactions taking place at short time scales up to seconds or a few minutes. These typical time scales place a severe limit on the kinetic space that can be explored. For a diffusion-limited ͑10 8 M −1 s −1 ͒ bimolecular interaction, a very tight K d of 10 nM translates to an off rate of 1 s −1 , resulting in a duration of the binding cycle that is readily captured in a short time frame. Many bimolecular interactions of interest, however, display nondiffusion limi...

Single-molecule approaches to characterizing kinetics of biomolecular interactions

“…From the fluorescence 'on' times, the dissociation rate constant could be determined, whereas the 'off' times provided information on the association rate constant. Correction of mechanical drift and reduction of photobleaching by a noncontinuous illumination allowed these experiments to take place over many days, so that the slow binding kinetics of the aptamers could be studied [35].…”

Studying small molecule–aptamer interactions using MicroScale Thermophoresis (MST)

“…Other methods are isocratic elution [9], equilibrium filtration [10,11] and in-line probing [12][13][14]. Gel-shift assays, stopped-flow fluorescence spectroscopy [15,16], Fluorescence anisotropy (FA) [17,18] and single molecule fluorescence imaging [19,20] are also widely used. The innovative MicroScale Thermophoresis technology (MST) combines various aspects of the above-mentioned methods and is a powerful novel tool for scientists to characterize small molecule-aptamer interactions.…”

Role of substrate unbinding in Michaelis–Menten enzymatic reactions

“…Single-molecule approaches to biomolecular interaction kinetics (24-26) allow for direct measurements of binding and unbinding rates (27)(28)(29). In addition, the combination of single-molecule florescence (16, 18) with single-molecule force spectroscopy (23, 30) marks itself as an especially promising tool in unbinding studies because the fluorescence readout for catalytic activity is expected to be correlated with selective, force-induced, changes of the activation barrier for unbinding (23,30).…”