Next-generation sequencing techniques have led to a new quantitative dimension in the biological sciences. In particular, integrating sequencing techniques with biophysical tools allows sequence-dependent mechanistic studies. Using the millions of DNA clusters that are generated during sequencing to perform high-throughput binding affinity and kinetics measurements enabled the construction of energy landscapes in sequence space, uncovering relationships between sequence, structure, and function. Here, we review the approaches to perform ensemble fluorescence experiments on next-generation sequencing chips for variations of DNA, RNA, and protein sequences. As the next step, we anticipate that these fluorescence experiments will be pushed to the single-molecule level, which can directly uncover kinetics and molecular heterogeneity in an unprecedented high-throughput fashion. Molecular biophysics in sequence space, both at the ensemble and single-molecule level, leads to new mechanistic insights. The wide spectrum of applications in biology and medicine ranges from the fundamental understanding of evolutionary pathways to the development of new therapeutics.

Single-molecule FRET is a versatile tool to study nucleic acids and proteins at the nanometer scale. However, currently, only a couple of FRET pairs can be reliably measured on a single object, which makes it difficult to apply single-molecule FRET for structural analysis of biomolecules. Here, we present an approach that allows for the determination of multiple distances between FRET pairs in a single object. We use programmable, transient binding between short DNA strands to resolve the FRET efficiency of multiple fluorophore pairs. By allowing only a single FRET pair to be formed at a time, we can determine the pair distance with subnanometer precision. The distance between other pairs are determined by sequentially exchanging DNA strands. We name this multiplexing approach FRET X for FRET via DNA eXchange. Our FRET X technology will be a tool for the high-resolution analysis of biomolecules and nanostructures.

Detection of specific nucleic acid sequences is invaluable in biological studies such as genetic disease diagnostics and genome profiling. Here, we developed a highly sensitive and specific detection method that combines an advanced oligonucleotide ligation assay with multicolor single-molecule fluorescence. We demonstrated that under our experimental conditions, 7-nucleotide long DNA barcodes have the optimal short length to ascertain specificity while being long enough for sufficient ligation. Using four spectrally separated fluorophores to label DNA barcodes, we simultaneously distinguished four DNA target sequences differing by only a single nucleotide. Our single-molecule approach will allow for accurate identification of low-abundance molecules without the need for target DNA preamplification.