The Development of Instrumentation to Increase the Accessibility and Throughput of Single-Molecule FRET

Current drug discovery approaches use high-throughput ensemble methods combined with
large libraries of small molecules to measure and quantify the drug-ligand interactions. These
methods currently suffer from ensemble averaging, meaning only the average conformational
state is measured. Diffusion-based single-molecule Förster resonance energy transfer (FRET)
can measure tightly bound drug candidates with high affinity and has the potential to overcome
the limitations of these methods by identifying multiple conformational states and, in the
process, minimises sample consumption. However, this technique suffers from high acquisition
times which limits the throughput of this method. Furthermore, high instrument costs and a
lack of open-source hardware have limited the use of single-molecule FRET to specialists. We
aim to extend this method by making it affordable, easy to use and applicable for high throughput drug discovery.

Here we present a commercially available, affordable, single-spot, single-molecule microscope
that allows flexibility for single-molecule researchers. In addition, an open-source, compact
and cost-effective excitation module for single-molecule imaging and spectroscopy is
presented. By comparing this module to commercially available lasers, this excitation module
reported a similar degree in accuracy of single-molecule FRET measurements for three well
studied oligonucleotides. By combining common confocal microscopy principles with single�molecule methods we also studied the performance of a multi-spot setup for high-throughput single-molecule FRET. Here we observed single-molecules at high frame rates (1000 frames
per second) and observed FRET in a multi-spot setup using a transmission electron microscopy
(TEM) grid.

We envisage that this research will make this powerful single-molecule technique more widely
available for users in academia and industry and more applicable to the drug discovery process.