smFRET Assays Research Articles

An automated single-molecule FRET platform for high-content, multiwell plate screening of biomolecular conformations and dynamics

Single-molecule FRET (smFRET) has become a versatile tool for probing the structure and functional dynamics of biomolecular systems, and is extensively used to address questions ranging from biomolecular folding to drug discovery. Confocal smFRET measurements are amongst the widely used smFRET assays and are typically performed in a single-well format. Thus, sampling of many experimental parameters is laborious and time consuming. To address this challenge, we extend here the capabilities of confocal smFRET beyond single-well measurements by integrating a multiwell plate functionality to allow for continuous and automated smFRET measurements. We demonstrate the broad applicability of the multiwell plate assay towards DNA hairpin dynamics, protein folding, competitive and cooperative protein–DNA interactions, and drug-discovery, revealing insights that would be very difficult to achieve with conventional single-well format measurements. For the adaptation into existing instrumentations, we provide a detailed guide and open-source acquisition and analysis software.

Translational readthrough-inducing drug (TRID) effects on eukaryotic termination investigated at the single-molecule level.

Many genetic disorders are caused by premature stop codon (PSC) mutations, but only one TRID (Translational readthrough-inducing drug), ataluren, has been approved for clinical use. Recently, we used single-molecule TIRF with a cell-free in vitro assay and found that ataluren competitively inhibits productive release factor complex (RFC, eRF1.eRF3.GTP) binding to the pre-termination complex. We found that such inhibition occurs before or at the peptidyl-tRNA hydrolysis step (Huang et al., Nat. Comm., 2022, 13: 2413). Here we report new results using an sm-FRET assay with a Cy3-labeled peptidyl-tRNA bound in the ribosomal P-site adjacent to a UGA stop codon in the A-site and Cy5-labeled human eRF1. Upon RFC binding, we observe transient FRET efficiency, E = ∼0.25, between Cy5-eRF1 and Cy3-tRNA, consistent with the successful accommodation of eRF1 within the A-site. Following peptidyl-tRNA hydrolysis, we find a strong correlation between eRF1 and tRNA dissociation times, unlike the weak correlation between peptide release and tRNA dissociation times that we observed previously (Huang et al., ibid.). Additionally, we found that ataluren and added near-cognate suppressor tRNA each had a significant inhibitory effect on the arrival time of RFCs at the ribosome, and that ataluren in combination with the aminoglycoside G418 had an even stronger inhibitory effect compared with ataluren or G418 alone. These results support our earlier suggestion (Ng et al., PNAS, 2021, 118: e2020599118) that ataluren or ataluren-like TRIDs could potentiate the therapeutic effects of aminoglycosides on PSC diseases. We expect that our ongoing studies will aid in elucidating the readthrough mechanisms of other TRIDs leading to enhanced and safer treatments of PSC diseases.

Cotranscriptional Moonlighting of RsmC as an RNA Chaperone Protein

Ribosome is the ribonucleoprotein cellular machine that carries out protein biosynthesis in all kingdoms of life. During ribosome biogenesis, accurate coordination of rRNA transcription, folding, modification, processing, and assembly of r-proteins is essential to form a functional ribosome. The involvement of assembly factors is critical to synchronize various processes that co-occur with ribosome assembly. Here we report moonlighting of RsmC (ribosomal RNA small subunit methyltransferase C) as an RNA chaperone and RNA strand annealer during ribosome synthesis in cells. RsmC methylates an exocyclic amine of guanine (G) located at 1207 of helix 34 (h34) of 16S rRNA (E. coli standard nucleotide numbering). A 25-fold increase in the rate of annealing of h34 was observed in our FRET-based stopped-flow kinetic measurements. Furthermore, smFRET assays illustrated the ability of RsmC to denature the predicted hairpin structure of its substrate strand. Chaperone activity of RmsC may be critical for the formation of the 30S head domain.

Translocation kinetics and structural dynamics of ribosomes are modulated by the conformational plasticity of downstream pseudoknots.

Downstream stable mRNA secondary structures can stall elongating ribosomes by impeding the concerted movements of tRNAs and mRNA on the ribosome during translocation. The addition of a downstream mRNA structure, such as a stem-loop or a pseudoknot, is essential to induce -1 programmed ribosomal frameshifting (-1 PRF). Interestingly, previous studies revealed that -1 PRF efficiencies correlate with conformational plasticity of pseudoknots, defined as their propensity to form incompletely folded structures, rather than with the mechanical properties of pseudoknots. To elucidate the detailed molecular mechanisms of translocation and -1 PRF, we applied several smFRET assays to systematically examine how translocation rates and conformational dynamics of ribosomes were affected by different pseudoknots. Our results show that initial pseudoknot-unwinding significantly inhibits late-stage translocation and modulates conformational dynamics of ribosomal post-translocation complexes. The effects of pseudoknots on the structural dynamics of ribosomes strongly correlate with their abilities to induce -1 PRF. Our results lead us to propose a kinetic scheme for translocation which includes an initial power-stroke step and a following thermal-ratcheting step. This scheme provides mechanistic insights on how selective modulation of late-stage translocation by pseudoknots affects -1 PRF. Overall our findings advance current understanding of translocation and ribosome-induced mRNA structure unwinding.

Single-molecule FRET reveals proofreading complexes in the large fragment of Bacillus stearothermophilus DNA polymerase I.

There is increasing interest in the use of DNA polymerases (DNA pols) in next-generation sequencing strategies. These methodologies typically rely on members of the A and B family of DNA polymerases that are classified as high-fidelity DNA polymerases. These enzymes possess the ability to selectively incorporate the correct nucleotide opposite a templating base with an error frequency of only 1 in 106 insertion events. How they achieve this remarkable fidelity has been the subject of numerous investigations, yet the mechanism by which these enzymes achieve this level of accuracy remains elusive. Several smFRET assays were designed to monitor the conformational changes associated with the nucleotide selection mechanism(s) employed by DNA pols. smFRET has also been used to monitor the movement of DNA pols along a DNA substrate as well as to observe the formation of proof-reading complexes. One member among this class of enzymes, the large fragment of Bacillus stearothermophilus DNA polymerase I (Bst pol I LF), contains both 5′→3′ polymerase and 3′→5′ exonuclease domains, but reportedly lacks exonuclease activity. We have designed a smFRET assay showing that Bst pol I LF forms proofreading complexes. The formation of proofreading complexes at the single molecule level is strongly influenced by the presence of the 3′ hydroxyl at the primer-terminus of the DNA substrate. Our assays also identify an additional state, observed in the presence of a mismatched primer-template terminus, that may be involved in the transfer of the primer-terminus from the polymerase to the exonuclease active site.

Multispot single-molecule FRET: High-throughput analysis of freely diffusing molecules.

We describe an 8-spot confocal setup for high-throughput smFRET assays and illustrate its performance with two characteristic experiments. First, measurements on a series of freely diffusing doubly-labeled dsDNA samples allow us to demonstrate that data acquired in multiple spots in parallel can be properly corrected and result in measured sample characteristics consistent with those obtained with a standard single-spot setup. We then take advantage of the higher throughput provided by parallel acquisition to address an outstanding question about the kinetics of the initial steps of bacterial RNA transcription. Our real-time kinetic analysis of promoter escape by bacterial RNA polymerase confirms results obtained by a more indirect route, shedding additional light on the initial steps of transcription. Finally, we discuss the advantages of our multispot setup, while pointing potential limitations of the current single laser excitation design, as well as analysis challenges and their solutions.

A practical guide to studying G-quadruplex structures using single-molecule FRET.

In this article, we summarize the knowledge and best practices learned from bulk and single-molecule measurements to address some of the frequently experienced difficulties in single-molecule Förster resonance energy transfer (smFRET) measurements on G-quadruplex (GQ) structures. The number of studies that use smFRET to investigate the structure, function, dynamics, and interactions of GQ structures has grown significantly in the last few years, with new applications already in sight. However, a number of challenges need to be overcome before reliable and reproducible smFRET data can be obtained in measurements that include GQ. The annealing and storage conditions, the location of fluorophores on the DNA construct, and the ionic conditions of the experiment are some of the factors that are of critical importance for the outcome of measurements, and many of these manifest themselves in unique ways in smFRET assays. By reviewing these aspects and providing a summary of best practices, we aim to provide a practical guide that will help in successfully designing and performing smFRET studies on GQ structures.

Probing the Conformational Landscape of DNA Polymerases Using Diffusion-Based Single-Molecule FRET.

Monitoring conformational changes in DNA polymerases using single-molecule Förster resonance energy transfer (smFRET) has provided new tools for studying fidelity-related mechanisms that promote the rejection of incorrect nucleotides before DNA synthesis. In addition to the previously known open and closed conformations of DNA polymerases, our smFRET assays utilizing doubly labeled variants of Escherichia coli DNA polymerase I were pivotal in identifying and characterizing a partially closed conformation as a primary checkpoint for nucleotide selection. Here, we provide a comprehensive overview of the methods we used for the conformational analysis of wild-type DNA polymerase and some of its low-fidelity derivatives; these methods include strategies for protein labeling and our procedures for solution-based single-molecule fluorescence data acquisition and data analysis. We also discuss alternative single-molecule fluorescence strategies for analyzing the conformations of DNA polymerases in vitro and in vivo.

BLM unfolds G-quadruplexes in different structural environments through different mechanisms.

Mutations in the RecQ DNA helicase gene BLM give rise to Bloom's syndrome, which is a rare autosomal recessive disorder characterized by genetic instability and cancer predisposition. BLM helicase is highly active in binding and unwinding G-quadruplexes (G4s), which are physiological targets for BLM, as revealed by genome-wide characterizations of gene expression of cells from BS patients. With smFRET assays, we studied the molecular mechanism of BLM-catalyzed G4 unfolding and showed that ATP is required for G4 unfolding. Surprisingly, depending on the molecular environments of G4, BLM unfolds G4 through different mechanisms: unfolding G4 harboring a 3′-ssDNA tail in three discrete steps with unidirectional translocation, and unfolding G4 connected to dsDNA by ssDNA in a repetitive manner in which BLM remains anchored at the ss/dsDNA junction, and G4 was unfolded by reeling in ssDNA. This indicates that one BLM molecule may unfold G4s in different molecular environments through different mechanisms.

Elucidating the Transition Dynamics of HIV-1 Reverse Transcriptase using Single Molecule FRET

The reverse transcriptase (RT) of HIV has been a focus of research owing to its central role during the infection of a host cell. Although, currently there are thirteen antiretroviral drugs targeting RT in clinical therapy of HIV AIDS, HIV has developed resistance to these drugs. Understanding the mechanism of inhibition of RT's function and its interaction with nucleic acids will aid in discovering improved viral therapies. Previously, we developed an smFRET assay to study RT-nucleic acid interactions and showed that RT dynamically explores multiple conformations to find a nucleic acid target. The assay is extended to study the transition mechanism by which RT flips and shuttles on nucleic acids. Distinct effects of increased macromolecular crowding and increased ionic concentrations were exploited to conclude that RT uses tumbling like mechanism to flip ∼1800 on target site whereas RT uses hopping like mechanism to shuttle during target searching process. We propose that these two distinct mechanisms of RT mobility can be explored for developing new drug targets.

Development of an smFRET Assay to Monitor Spliceosome Activation in Budding Yeast

The addition of the U4/U6-U5 tri-snRNP to the spliceosome instigates many changes to create a catalytically active spliceosome. Specifically the U6 internal stem-loop (ISL), structurally reminiscent of catalytic domain V of self-splicing group II introns, must form. U6 arrives at pre-mRNA stably base-paired with U4 in the tri-snRNP. The U4/U6 interaction sequesters the U6 ISL, blocking premature activity. Unwinding, regulated by proteins Brr2 and Prp8, allows for mutually exclusive U2/U6 interactions required for catalysis to form. This ensures assembly is highly coordinated while allowing for the careful regulation required for fidelity. Because most splicing studies use crude extracts, wherein multiple reversible steps in spliceosome assembly proceed asynchronously, fundamental details of the kinetics and conformational rearrangements undergone by U6 during activation are unknown.Single-molecule FRET provides a method to monitor dynamics and conformation without isolating or synchronizing reaction intermediates. I am currently developing an smFRET assay to monitor U6 ISL formation as a readout for U4/U6 unwinding in spliceosome activation. I have created a dual-fluorophore labeled U6 RNA construct that enables me to discern whether the U6 ISL is formed or sequestered by U4. This construct is incorporated into U6 snRNPs and is functional in in vitro splicing assays. To facilitate imaging, I have optimized a protocol to isolate tri-snRNPs. Once optimization of this assay is complete, I will determine the kinetics and molecular mechanism of how Brr2 unwinds U4/U6.

Single-Molecule Studies of the Effects of Small Compounds on the Activity of Translation Initiation Factor eIF4A

The translation initiation factor eIF4A is the prototypical DEAD-box RNA helicase as a subunit of eIF4F. It facilitates the binding and scanning of the ribosome via unwinding secondary structures in the 5’ UTR of mRNAs during translation initiation. Here single-molecule Fluorescence Resonance Energy Transfer (sm-FRET) assay for both structured and unstructured substrates is performed and it shows that eIF4A unwinds the substrates in a discrete step manner although it is a highly nonprocessive motor. It also shows that eIF4A is a bidirectional helicase and the step size is about 6 base pair. Pateamine A and silvestrol are small-molecule modulators of eIF4A activity and they are identified as potent inhibitors of translation. It was demonstrated that Pateamine A and silvestrol act as a chemical inducer of dimerization and promote the interaction between eIF4A and RNA, however the molecular mechanisms by which they regulate eIF4A activities still remain elusive. Our sm-FRET assay shows that both pateamine A and silvestrol stimulate the helicase activity of eIF4A. Understanding how the processivity of eIF4A is influenced by these molecules will help regulating the translation at the initiation step.

Real-Time Initial Transcription by a Multisubunit RNA Polymerase

Abortive initiation, the repetitive synthesis of short RNA transcripts before promoter escape and elongation, is a highly dynamic process that can constitute the rate-limiting step for many gene promoters. Previous studies using single-molecule Förster Resonance Energy Transfer (smFRET) demonstrated that abortive initiation occurs via a DNA-scrunching mechanism mediated by RNA polymerase. While these studies, along with biochemical work, have yielded insight into abortive initiation, no single-molecule method has been able to directly capture RNA synthesis/release, or the associated conformational changes of the transcription complex. Here, we descibe a highly sensitive smFRET assay that, for the first time, monitors transcription bubble expansion and compaction during abortive RNA synthesis and release in real-time; we also performed similar analysis using an assay that reports on the movement of downstream DNA toward the RNA polymerase main channel. Using single-molecule time trajectories, we have observed multiple cycles (up to 50) of RNA synthesis by single RNA polymerase molecules and we used them to obtain the rates of RNA synthesis and release, as well as to study the dependence of the rates on nucleotide concentration and promoter sequence. Our initial results revealed that, surprisingly, the rate of nucleotide addition in initial transcription is 5-10 fold faster than elongation; moreover, we observed large heterogeneity between different RNA polymerase molecules with regards to specific steps in initial transcription; such heterogeneity may contribute to the noise associated with gene expression.

Single-molecule analysis reveals the kinetics and physiological relevance of MutL-ssDNA binding.

DNA binding by MutL homologs (MLH/PMS) during mismatch repair (MMR) has been considered based on biochemical and genetic studies. Bulk studies with MutL and its yeast homologs Mlh1-Pms1 have suggested an integral role for a single-stranded DNA (ssDNA) binding activity during MMR. We have developed single-molecule Förster resonance energy transfer (smFRET) and a single-molecule DNA flow-extension assays to examine MutL interaction with ssDNA in real time. The smFRET assay allowed us to observe MutL-ssDNA association and dissociation. We determined that MutL-ssDNA binding required ATP and was the greatest at ionic strength below 25 mM (KD = 29 nM) while it dramatically decreases above 100 mM (KD>2 µM). Single-molecule DNA flow-extension analysis suggests that multiple MutL proteins may bind ssDNA at low ionic strength but this activity does not enhance stability at elevated ionic strengths. These studies are consistent with the conclusion that a stable MutL-ssDNA interaction is unlikely to occur at physiological salt eliminating a number of MMR models. However, the activity may infer some related dynamic DNA transaction process during MMR.