Probing single-molecule structure and dynamics with an anti-Brownian trap

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

We have made significant progress on the following two areas during the past year. Two manuscripts are being prepared (one for each area) with a goal of submission by the end of 2025 a) ABEL-FRET spectroscopy for precise molecular nanometry. We previously published our “ABEL-FRET” platform (Nat. Meth. 18, 816), which demonstrated the highest resolution of smFRET achieved. We follow up to answer a more fundamental question in the single-molecule FRET field: what is the ultimate distance resolution of smFRET as a structural biology tool? Critical to answering this question is a mastery of the fundamental precision limits ultimately set by photon shot noise. However, common experimental realizations of smFRET are usually subject to additional sources of uncertainty—originating from the detection instrumentation, dye photophysics, or excess heterogeneity introduced by surface immobilization—that manifest as extra broadening of the peaks in FRET efficiency (E) histograms. In experiments on DNA standards, ABEL-FRET shows near shot-noise limited performance, even on pooled measurements over many molecules, leading to remarkably narrow E distributions with σE < 0.01 and facilitating straightforward single base-pair resolution. These results suggest that surface immobilization could be a major source for extra broadening and motivate a renewed investigation into its influence on smFRET experiments. To facilitate direct comparisons with ABEL-FRET, we have constructed an instrument for performing conventional surface-immobilized measurements in both photon-counting confocal and EMCCD prism-based total internal reflection fluorescence (TIRF) modalities. This instrumentation suite allows the assessment of many experimental factors—like immobilization strategy, dye pair, and detector noise—for impacting FRET precision, and will provide insight into optimal experimental design for biological applications. b) RNA structure and dynamics during Cas9 assembly. We are applying ABEL-FRET to study the structure and dynamics of guide RNA, during Cas9 assembly. Compared to conventional smFRET, ABEL-FRET has key advantages (high resolution, tether-free dynamics and binding state determination) which makes it particularly suitable for RNA structure and dynamics. CRISPR-Cas9 uses a programmable guide RNA (gRNA) as a template to cleave DNA at specific sites and has become an indispensable gene editing tool. The structure-function of the holoenzyme (gRNA-Cas9 complex) has been extensively studied using traditional biochemistry, cryoEM and crystallography12, but the RNA-RNA and RNA-protein assembly processes that lead to functional ribonucleoprotein formation is less understood, due to likely structural heterogeneity and plasticity of RNA molecules. Here, we aim to track the conformation of the sequence-encoding segment on the gRNA throughout the individual steps of gRNA maturation, gRNA-Cas9 binding and substrate DNA binding, using ABEL-FRET. The first question we asked is how secondary structure formation on the crispr RNA (crRNA) affects the assembly process and/or Cas9 function. Due to the programmable nature of the crRNA, certain sequences are expected to form secondary structures with itself or other segments of the gRNA molecule. We first designed two crRNAs with different second structure forming propensities and probed with ABEL-FRET. Single-molecule FRET traces confirms the presence of secondary structure in both crRNAs as mean FRET efficiency values are higher than those of a random coil control strand (polyU). The secondary structure formation in both strands is transient and depend on Mg2+ concentration. In particular Mg2+ slows down the kinetics but does not significantly alter the equilibrium of secondary structure formation. From there, we continued to learn the incredibly rich structural remodeling and dynamics during Cas9 assembly, which will be summarized in an impending publication. This line of research in the lab has demonstrated that ABEL-FRET is an excellent tool to study RNA and will continue to yield biophysical insight on the structure and dynamics of gRNA before and after Cas9 assembly. We will likely expand this research to include other non-coding small RNA, ribozymes and RNPs.