A versatile and high-throughput flow-cell system combined with fluorescence imaging for simultaneous single-molecule force measurement and visualization

Abstract

A flow-cell offers many advantages for single-molecule studies. But, its merit as a quantitative single-molecule tool has long been underestimated. In this work, we developed a gas-pumped fully calibrated flow-cell system combined with fluorescence imaging for simultaneous single-molecule force measurement and visualization. Such a flow-cell system has considered the hydrodynamic drags on biomolecules and hence can apply and measure force up to more than 100 pN in sub-pN precision with an ultra-high force stability (force drift <0.01 pN in 10 minutes) and tuning accuracy (∼0.04 pN). Meanwhile, it also allows acquiring force signals and fluorescence images at the same time, parallelly tracking hundreds of protein motors in real time as well as monitoring the conformational changes of biomolecules under a well-controlled force, as demonstrated by a series of single-molecule experiments in this work, including the studies of DNA overstretching dynamics, transcription under force and DNA folding/unfolding dynamics. Interesting findings, such as the very tight association of single-stranded binding (SSB) proteins with ssDNA and the reversed transcription, have also been made. These results together lay down an essential foundation for a flow-cell to be used as a versatile, quantitative and high-throughput tool for single-molecule manipulation and visualization.