High-throughput smFRET analysis of freely diffusing nucleic acid molecules and associated proteins

Single-molecule Förster resonance energy transfer (smFRET) is a powerful technique for nanometer-scale studies of single molecules. Solution-based smFRET, in particular, can be used to study equilibrium intra- and intermolecular conformations, binding/unbinding events and conformational changes under biologically relevant conditions without ensemble averaging. However, single-spot smFRET measurements in solution are slow. Here, we detail a high-throughput smFRET approach that extends the traditional single-spot confocal geometry to a multispot one. The excitation spots are optically conjugated to two custom silicon single photon avalanche diode (SPAD) arrays. Two-color excitation is implemented using a periodic acceptor excitation (PAX), allowing distinguishing between singly- and doubly-labeled molecules. We demonstrate the ability of this setup to rapidly and accurately determine FRET efficiencies and population stoichiometries by pooling the data collected independently from the multiple spots. We also show how the high throughput of this approach can be used o increase the temporal resolution of single-molecule FRET population characterization from minutes to seconds. Combined with microfluidics, this high-throughput approach will enable simple real-time kinetic studies as well as powerful molecular screening applications

High-throughput smFRET analysis of freely diffusing nucleic acid molecules and associated proteins

Single-molecule Förster resonance energy transfer (smFRET) is a powerful technique for nanometer-scale studies of single molecules. Solution-based smFRET, in particular, can be used to study equilibrium intra- and intermolecular conformations, binding/unbinding events and conformational changes under biologically relevant conditions without ensemble averaging. However, single-spot smFRET measurements in solution are slow. Here, we detail a high-throughput smFRET approach that extends the traditional single-spot confocal geometry to a multispot one. The excitation spots are optically conjugated to two custom silicon single photon avalanche diode (SPAD) arrays. Two-color excitation is implemented using a periodic acceptor excitation (PAX), allowing distinguishing between singly- and doubly-labeled molecules. We demonstrate the ability of this setup to rapidly and accurately determine FRET efficiencies and population stoichiometries by pooling the data collected independently from the multiple spots. We also show how the high throughput of this approach can be used o increase the temporal resolution of single-molecule FRET population characterization from minutes to seconds. Combined with microfluidics, this high-throughput approach will enable simple real-time kinetic studies as well as powerful molecular screening applications

48-spot single-molecule FRET setup with periodic acceptor excitation

Single-molecule Förster resonance energy transfer (smFRET) allows measuring distances between donor and acceptor fluorophores on the 3–10 nm range. Solution-based smFRET allows measurement of binding-unbinding events or conformational changes of dye-labeled biomolecules without ensemble averaging and free from surface perturbations. When employing dual (or multi) laser excitation, smFRET allows resolving the number of fluorescent labels on each molecule, greatly enhancing the ability to study heterogeneous samples. A major drawback to solution-based smFRET is the low throughput, which renders repetitive measurements expensive and hinders the ability to study kinetic phenomena in real-time. Here we demonstrate a high-throughput smFRET system that multiplexes acquisition by using 48 excitation spots and two 48-pixel single-photon avalanche diode array detectors. The system employs two excitation lasers allowing separation of species with one or two active fluorophores. The performance of the system is demonstrated on a set of doubly labeled double-stranded DNA oligonucleotides with different distances between donor and acceptor dyes along the DNA duplex. We show that the acquisition time for accurate subpopulation identification is reduced from several minutes to seconds, opening the way to high-throughput screening applications and real-time kinetics studies of enzymatic reactions such as DNA transcription by bacterial RNA polymerase

Membrane insertion of—and membrane potential sensing by—semiconductor voltage nanosensors: Feasibility demonstration

We developed membrane voltage nanosensors that are based on inorganic semiconductor nanoparticles. We provide here a feasibility study for their utilization. We use a rationally designed peptide to functionalize the nanosensors, imparting them with the ability to self-insert into a lipid membrane with a desired orientation. Once inserted, these nanosensors could sense membrane potential via the quantum confined Stark effect, with a single-particle sensitivity. With further improvements, these nanosensors could potentially be used for simultaneous recording of action potentials from multiple neurons in a large field of view over a long duration and for recording electrical signals on the nanoscale, such as across one synapse