Microfluidic mixer designed for performing single-molecule kinetics with confocal detection on timescales from milliseconds to minutes

Microfluidic mixing in combination with single-molecule spectroscopy allows the investigation of complex biomolecular processes under non-equilibrium conditions. Here we present a protocol for building, installing and operating microfluidic mixing devices optimized for this purpose. The mixer is fabricated by replica molding with polydimethylsiloxane (PDMS), which allows the production of large numbers of devices at a low cost using a single microfabricated silicon mold. The design is based on hydrodynamic focusing combined with diffusive mixing and allows single-molecule kinetics to be recorded over five orders of magnitude in time, from 1 ms to ∼100 s. Owing to microfabricated particle filters incorporated in the inlet channels, the devices provide stable flow for many hours to days without channel blockage, which allows reliable collection of high-quality data. Modular design enables rapid exchange of samples and mixing devices, which are mounted in a specifically designed holder for use with a confocal microscopy detection system. Integrated Peltier elements provide temperature control from 4 to 37 °C. The protocol includes the fabrication of a silicon master, production of the microfluidic devices, instrumentation setup and data acquisition. Once a silicon master is available, devices can be produced and experiments started within ∼1 d of preparation. We demonstrate the performance of the system with single-molecule Förster resonance energy transfer (FRET) measurements of kinetics of protein folding and conformational changes. The dead time of 1 ms, as predicted from finite element calculations, was confirmed by the measurements

Single-molecule FRET reveals the energy landscape of the full-length SAM-I riboswitch

S-adenosyl-L-methionine (SAM) ligand binding induces major structural changes in SAM-I riboswitches, through which gene expression is regulated via transcription termination. Little is known about the conformations and motions governing the function of the full-length Bacillus subtilis yitJ SAM-I riboswitch. Therefore, we have explored its conformational energy landscape as a function of Mg^2+ and SAM ligand concentrations using single-molecule Förster resonance energy transfer (smFRET) microscopy and hidden Markov modeling analysis. We resolved four conformational states both in the presence and the absence of SAM and determined their Mg^2+-dependent fractional populations and conformational dynamics, including state lifetimes, interconversion rate coefficients and equilibration timescales. Riboswitches with terminator and antiterminator folds coexist, and SAM binding only gradually shifts the populations toward terminator states. We observed a pronounced acceleration of conformational transitions upon SAM binding, which may be crucial for off-switching during the brief decision window before expression of the downstream gene

Single-molecule spectroscopy of protein conformational dynamics in live eukaryotic cells

Single-molecule methods have become widely used for quantifying the conformational heterogeneity and structural dynamics of biomolecules in vitro. Their application in vivo, however, has remained challenging owing to shortcomings in the design and reproducible delivery of labeled molecules, the range of applicable analysis methods, and suboptimal cell culture conditions. By addressing these limitations in an integrated approach, we demonstrate the feasibility of probing protein dynamics from milliseconds down to the nanosecond regime in live eukaryotic cells with confocal single-molecule Förster resonance energy transfer (FRET) spectroscopy. We illustrate the versatility of the approach by determining the dimensions and submicrosecond chain dynamics of an intrinsically disordered protein; by detecting even subtle changes in the temperature dependence of protein stability, including in-cell cold denaturation; and by quantifying the folding dynamics of a small protein. The methodology opens possibilities for assessing the effect of the cellular environment on biomolecular conformation, dynamics and function