Combined High-Resolution Optical Tweezers and Multicolor Single-Molecule Fluorescence with an Automated Single-Molecule Assembly Line

Abstract

We present an instrument that combines high-resolution optical tweezers and multicolor confocal fluorescence spectroscopy along with automated single-molecule assembly. The multicolor allows the simultaneous observation of multiple molecules or multiple degrees of freedom, which allows, for example, the observation of multiple proteins simultaneously within a complex. The instrument incorporates three fluorescence excitation lasers, with a reliable alignment scheme, which will allow three independent fluorescent probe or FRET measurements and also increases flexibility in the choice of fluorescent molecules. We demonstrate the ability to simultaneously measure angstrom-scale changes in tether extension and fluorescence signals. Simultaneous tweezers and fluorescence measurement are particularly challenging because of fluorophore photobleaching, even more so if multiple fluorophores are to be measured. Therefore, (1) fluorescence excitation and detection is interlaced with time-shared dual optical traps. (2) We investigated the photostability of common fluorophores. The mean number of photons emitted before bleaching was unaffected by the trap laser and decreased only slightly with increasing excitation laser intensity. Surprisingly, we found that Cy5 outperforms other commonly used fluorophores by more than fivefold. (3) We devised computer-controlled automation, which conserves fluorophore lifetime by quickly detecting fluorophore-labeled molecule binding, turning off lasers, and moving to add the next fluorophore-labeled component. The single-molecule assembly line enables the precise assembly of multimolecule complexes while preserving fluorophores.

DNA replication machinery: Insights from in vitro single-molecule approaches

“…Furthermore, to elucidate the complex dynamics and mechano-chemical processes that govern the operation of a multi-nucleoprotein complex such as the replisome, it will be necessary to interrogate different variables of the system simultaneously. In this regard, the recent development of hybrid methods that combine single-molecule manipulation with fluorescence microscopy [103] , [170] , [171] , [172] , [173] , [174] , [175] will enable to correlate the real-time kinetics of DNA replication to the structural organization and/or to inter- or intramolecular structural changes of the replisome components. Progress in this area will be conditioned by advances in chemical methods that allow efficient fluorescent labeling of proteins without affecting their function.…”

DNA replication: In vitro single-molecule manipulation data analysis and models

“…The analysis and modeling techniques described in this review have proven to be useful for the interpretation of the activities of replisome components when working in isolation (or in pairs) and importantly, have set the stage for the analysis of replication traces of fully reconstituted replisomes in the future. Analysis and modeling of single-molecule manipulation data will evolve hand by hand with the development of new methods that enable increased resolution, multiplexing or access to measure multiple variables of the system at the same time [40] , [20] , [2] . The methods described in this document have been used or could be adapted to study RNA polymerases [1] , [33] , [32] , [19] , [25] , [91] and other molecular motors [98] , [90] .…”

Optical tweezer and TIRF microscopy for single molecule manipulation of RNA/DNA nanostructures including their rubbery property and single molecule counting

“…simultaneously measured angstrom-scale changes in tether extension and fluorescence signals (Chuang et al . 2019 ) ( Fig. 16A , 16B ).…”