Sungchul Hohng

A Practical Guide to Single Molecule FRET

Single-molecule fluorescence resonance energy transfer (smFRET) is one of the most general and adaptable single-molecule techniques. Despite the explosive growth in the application of smFRET to answer biological questions in the last decade, the technique has been practiced mostly by biophysicists. We provide a practical guide to using smFRET, focusing on the study of immobilized molecules that allow measurements of single-molecule reaction trajectories from 1 ms to many minutes. We discuss issues a biologist must consider to conduct successful smFRET experiments, including experimental design, sample preparation, single-molecule detection and data analysis. We also describe how a smFRET-capable instrument can be built at a reasonable cost with off-the-shelf components and operated reliably using well-established protocols and freely available software.

Single‐Molecule Four‐Color FRET

We developed a single-molecule four-color FRET technique both in confocal and in total-internal-reflection fluorescence microscopies. Real-time determination of six inter-fluorophore FRET efficiencies allowed us to probe the correlated motion of four arms of the Holliday junction. The technique was also applied to assess the correlation of RecA-mediated strand exchange events at both ends of a synaptic complex.

Observation of internal cleavage and ligation reactions of a ribozyme

We have used single-molecule spectroscopy to untangle conformational dynamics and internal chemistry in the hairpin ribozyme. The active site of the ribozyme is stably formed by docking two internal loops, but upon cleavage undocking is accelerated by two orders of magnitude. The markedly different kinetic properties allow us to differentiate cleaved and ligated forms, and thereby observe multiple cycles of internal cleavage and ligation of a ribozyme in a uniquely direct way. The position of the internal equilibrium is biased toward ligation, but the cleaved ribozyme undergoes several undocking events before ligation, during which products may dissociate. Formation of the stably docked active site, rapid undocking after cleavage, and a strong bias toward ligation should combine to generate a stable circular template for the synthesis of the viral (+) strand and thus ensure a productive replication cycle.

Spectroscopic observation of RNA chaperone activities of Hfq in post-transcriptional regulation by a small non-coding RNA

Hfq protein is vital for the function of many non-coding small (s)RNAs in bacteria but the mechanism by which Hfq facilitates the function of sRNA is still debated. We developed a fluorescence resonance energy transfer assay to probe how Hfq modulates the interaction between a sRNA, DsrA, and its regulatory target mRNA, rpoS. The relevant RNA fragments were labelled so that changes in intra- and intermolecular RNA structures can be monitored in real time. Our data show that Hfq promotes the strand exchange reaction in which the internal structure of rpoS is replaced by pairing with DsrA such that the Shine-Dalgarno sequence of the mRNA becomes exposed. Hfq appears to carry out strand exchange by inducing rapid association of DsrA and a premelted rpoS and by aiding in the slow disruption of the rpoS secondary structure. Unexpectedly, Hfq also disrupts a preformed complex between rpoS and DsrA. While it may not be a frequent event in vivo, this melting activity may have implications in the reversal of sRNA-based regulation. Overall, our data suggests that Hfq not only promotes strand exchange by binding rapidly to both DsrA and rpoS but also possesses RNA chaperoning properties that facilitates dynamic RNA–RNA interactions.

Kinetic mechanism for viral dsRNA length discrimination by MDA5 filaments

The viral sensor MDA5 distinguishes between cellular and viral dsRNAs by length-dependent recognition in the range of ∼0.5–7 kb. The ability to discriminate dsRNA length at this scale sets MDA5 apart from other dsRNA receptors of the immune system. We have shown previously that MDA5 forms filaments along dsRNA that disassemble upon ATP hydrolysis. Here, we demonstrate that filament formation alone is insufficient to explain its length specificity, because the intrinsic affinity of MDA5 for dsRNA depends only moderately on dsRNA length. Instead, MDA5 uses a combination of end disassembly and slow nucleation kinetics to “discard” short dsRNA rapidly and to suppress rebinding. In contrast, filaments on long dsRNA cycle between partial end disassembly and elongation, bypassing nucleation steps. MDA5 further uses this repetitive cycle of assembly and disassembly processes to repair filament discontinuities, which often are present because of multiple, internal nucleation events, and to generate longer, continuous filaments that more accurately reflect the length of the underlying dsRNA scaffold. Because the length of the continuous filament determines the stability of the MDA5–dsRNA interaction, the mechanism proposed here provides an explanation for how MDA5 uses filament assembly and disassembly dynamics to discriminate between self vs. nonself dsRNA.

Single-molecule three-color FRET with both negligible spectral overlap and long observation time.

Full understanding of complex biological interactions frequently requires multi-color detection capability in doing single-molecule fluorescence resonance energy transfer (FRET) experiments. Existing single-molecule three-color FRET techniques, however, suffer from severe photobleaching of Alexa 488, or its alternative dyes, and have been limitedly used for kinetics studies. In this work, we developed a single-molecule three-color FRET technique based on the Cy3-Cy5-Cy7 dye trio, thus providing enhanced observation time and improved data quality. Because the absorption spectra of three fluorophores are well separated, real-time monitoring of three FRET efficiencies was possible by incorporating the alternating laser excitation (ALEX) technique both in confocal microscopy and in total-internal-reflection fluorescence (TIRF) microscopy.

Single-molecule approach to immunoprecipitated protein complexes: insights into miRNA uridylation

Single‐molecule techniques have been used for only a subset of biological problems because of difficulties in studying proteins that require cofactors or post‐translational modifications. Here, we present a new method integrating single‐molecule fluorescence microscopy and immunopurification to study protein complexes. We used this method to investigate Lin28‐mediated microRNA uridylation by TUT4 (terminal uridylyl transferase 4, polyU polymerase), which regulates let‐7 microRNA biogenesis. Our real‐time analysis of the uridylation by the TUT4 immunoprecipitates suggests that Lin28 functions as a processivity factor of TUT4. Our new technique, SIMPlex (single‐molecule approach to immunoprecipitated protein complexes), provides a universal tool to analyse complex proteins at the single‐molecule level.

Intrinsic Z-DNA Is Stabilized by the Conformational Selection Mechanism of Z-DNA-Binding Proteins

Z-DNA, a left-handed isoform of Watson and Crick’s B-DNA, is rarely formed without the help of high salt concentrations or negative supercoiling. However, Z-DNA-binding proteins can efficiently convert specific sequences of the B conformation into the Z conformation in relaxed DNA under physiological salt conditions. As in the case of many other specific interactions coupled with structural rearrangements in biology, it has been an intriguing question whether the proteins actively induce Z-DNAs or passively trap transiently preformed Z-DNAs. In this study, we used single-molecule fluorescence assays to observe intrinsic B-to-Z transitions, protein association/dissociation events, and accompanying B-to-Z transitions. The results reveal that intrinsic Z-DNAs are dynamically formed and effectively stabilized by Z-DNA-binding proteins through efficient trapping of the Z conformation rather than being actively induced by them. Our study provides, for the first time, detailed pictures of the intrinsic B-to-Z transition dynamics and protein-induced B-to-Z conversion mechanism at the single-molecule level.

Light emission from the shadows: Surface plasmon nano-optics at near and far fields

When light illuminates a thick metal film perforated with small holes, shadows appear. At the nanoscopic level, however, light can be emitted predominantly from the metal surfaces between the holes—shadows can be indeed brighter than the lighted holes. The symmetry of the near-field emission pattern is determined by the symmetry of the surface plasmon waves. Surprisingly, these nanoscopic emission patterns from the metal can be preserved to the far-field region, where the pattern becomes sinusoidal. This unusual behavior of light emission from the shadows is explained by efficient wave vector selection.

Human Argonaute 2 Has Diverse Reaction Pathways on Target RNAs

Argonaute is a key enzyme of various RNA silencing pathways. We use single-molecule fluorescence measurements to characterize the reaction mechanisms of the core-RISC (RNA-induced silencing complex) composed of human Argonaute 2 and a small RNA. We found that target binding of core-RISC starts at the seed region, resulting in four distinct reaction pathways: target cleavage, transient binding, stable binding, and Argonaute unloading. The target cleavage requires extensive sequence complementarity and dramatically accelerates core-RISC recycling. The stable binding of core-RISC is efficiently established with the seed match only, providing a potential explanation for the seed-match rule of miRNA (microRNA) target selection. Target cleavage on perfect-match targets sensitively depends on RNA sequences, providing an insight into designing more efficient siRNAs (small interfering RNAs).