Disentangling a Complex Biomolecular World with Single-Molecule Resolution

Biological systems rely on a complex and precisely controlled mix of biomolecules to sustain life as we know it. In addition to their compositional heterogeneity, individual biomolecules undergo dynamic rearrangements to fulfil their cellular function: they move, reversibly interact, and alternate between multiple conformations. Disentangling these compositional and dynamic complexities of biological systems poses a formidable challenge to established ensemble techniques. In this review, we discuss two single-molecule techniques - nanopore recordings and single-molecule Förster Resonance Energy Transfer (smFRET) measurements - and highlight their powerful abilities to unravel mixtures and resolve biomolecular dynamics with the ultimate resolution of single molecules. Applications range from identifying the vast sequence space populated by nucleic acids and stoichiometries observed in small messenger molecules, to detecting time-varying conformations and interactions of large multi-domain proteins. This non-exhaustive review aims to introduce non-expert readers to the unique benefits of single-molecule experiments, which can overcome ensemble and time averaging as well as dynamic range limitations, and therefore offer unique, quantitative descriptions of the intriguingly complex biomolecular mechanisms found within and around us.