Foundation model for efficient biological discovery in single-molecule data.

Modern data-intensive techniques offer ever deeper insights into biology, but render the process of discovery increasingly complex. For example, exploiting the unique ability of single-molecule fluorescence microscopy (SMFM)1-5. to uncover rare but critical intermediates often demands manual inspection of time traces and iterative ad hoc approaches that are difficult to systematize. To facilitate systematic and efficient discovery from SMFM data, we introduce META-SiM, a transformer-based foundation model pre-trained on diverse SMFM analysis tasks. META-SiM achieves high performance-rivaling best-in-class algorithms-on a broad range of analysis tasks including trace selection, classification, segmentation, idealization, and stepwise photobleaching analysis. Additionally, the model produces high-dimensional embedding vectors that encapsulate detailed information about each trace, which the web-based META-SiM Projector (https://www.simol-projector.org) casts into lower-dimensional space for efficient whole-dataset visualization, labeling, comparison, and sharing. Combining this Projector with the objective metric of Local Shannon Entropy enables rapid identification of condition-specific behaviors, even if rare or subtle. As a result, by applying META-SiM to an existing single-molecule Förster resonance energy transfer (smFRET) dataset6, we discover a previously unobserved intermediate state in pre-mRNA splicing. META-SiM thus removes bottlenecks, improves objectivity, and both systematizes and accelerates biological discovery in complex single-molecule data.

BNP-Track: a framework for superresolved tracking.

Superresolution tools, such as PALM and STORM, provide nanoscale localization accuracy by relying on rare photophysical events, limiting these methods to static samples. By contrast, here, we extend superresolution to dynamics without relying on photodynamics by simultaneously determining emitter numbers and their tracks (localization and linking) with the same localization accuracy per frame as widefield superresolution on immobilized emitters under similar imaging conditions (≈50 nm). We demonstrate our Bayesian nonparametric track (BNP-Track) framework on both in cellulo and synthetic data. BNP-Track develops a joint (posterior) distribution that learns and quantifies uncertainty over emitter numbers and their associated tracks propagated from shot noise, camera artifacts, pixelation, background and out-of-focus motion. In doing so, we integrate spatiotemporal information into our distribution, which is otherwise compromised by modularly determining emitter numbers and localizing and linking emitter positions across frames. For this reason, BNP-Track remains accurate in crowding regimens beyond those accessible to other single-particle tracking tools.

Beyond ligand binding: Single molecule observation reveals how riboswitches integrate multiple signals to balance bacterial gene regulation.

Riboswitches are specialized RNA structures that orchestrate gene expression in response to sensing specific metabolite or ion ligands, mostly in bacteria. Upon ligand binding, these conformationally dynamic RNA motifs undergo structural changes that control critical gene expression processes such as transcription termination and translation initiation, thereby enabling cellular homeostasis and adaptation. Because RNA folds rapidly and co-transcriptionally, riboswitches make use of the low complexity of RNA sequences to adopt alternative, transient conformations on the heels of the transcribing RNA polymerase (RNAP), resulting in kinetic partitioning that defines the regulatory outcome. This review summarizes single molecule microscopy evidence that has begun to unveil a sophisticated network of dynamic, kinetically balanced interactions between riboswitch architecture and the gene expression machinery that, together, integrate diverse cellular signals.

A nascent riboswitch helix orchestrates robust transcriptional regulation through signal integration.

Widespread manganese-sensing transcriptional riboswitches effect the dependable gene regulation needed for bacterial manganese homeostasis in changing environments. Riboswitches - like most structured RNAs - are believed to fold co-transcriptionally, subject to both ligand binding and transcription events; yet how these processes are orchestrated for robust regulation is poorly understood. Through a combination of single-molecule and bulk approaches, we discover how a single Mn2+ ion and the transcribing RNA polymerase (RNAP), paused immediately downstream by a DNA template sequence, are coordinated by the bridging switch helix P1.1 in the representative Lactococcus lactis riboswitch. This coordination achieves a heretofore-overlooked semi-docked global conformation of the nascent RNA, P1.1 base pair stabilization, transcription factor NusA ejection, and RNAP pause extension, thereby enforcing transcription readthrough. Our work demonstrates how a central, adaptable RNA helix functions analogous to a molecular fulcrum of a first-class lever system to integrate disparate signals for finely balanced gene expression control.

Molecular basis of mRNA delivery to the bacterial ribosome.

Protein synthesis begins with the formation of a ribosome-mRNA complex. In bacteria, the 30S ribosomal subunit is recruited to many mRNAs through base pairing with the Shine Dalgarno (SD) sequence and RNA binding by ribosomal protein bS1. Translation can initiate on nascent mRNAs and RNA polymerase (RNAP) can promote recruitment of the pioneering 30S subunit. Here we examined ribosome recruitment to nascent mRNAs using cryo-EM, single-molecule fluorescence co-localization, and in-cell crosslinking mass spectrometry. We show that bS1 delivers the mRNA to the ribosome for SD duplex formation and 30S subunit activation. Additionally, bS1 mediates the stimulation of translation initiation by RNAP. Together, our work provides a mechanistic framework for how the SD duplex, ribosomal proteins and RNAP cooperate in 30S recruitment to mRNAs and establish transcription-translation coupling.

Ultrasensitive amplification-free quantification of a methyl CpG-rich cancer biomarker by single-molecule kinetic fingerprinting.

The most well-studied epigenetic marker in humans is the 5-methyl modification of cytosine in DNA, which has great potential as a disease biomarker in liquid biopsies of cell-free DNA. Currently, quantification of DNA methylation relies heavily on bisulfite conversion followed by PCR amplification and NGS or microarray analysis. PCR is subject to potential bias in differential amplification of bisulfite-converted methylated versus unmethylated sequences. Here, we combine bisulfite conversion with single-molecule kinetic fingerprinting to develop an amplification-free assay for DNA methylation at the branched-chain amino acid transaminase 1 (BCAT1) promoter. Our assay selectively responds to methylated sequences with a limit of detection below 1 fM and a specificity of 99.9999%. Evaluating complex genomic DNA matrices, we reliably distinguish 2-5% DNA methylation at the BCAT1 promoter in whole blood DNA from completely unmethylated whole-genome amplified DNA. Taken together, these results demonstrate the feasibility and sensitivity of our amplification-free, single-molecule quantification approach to improve the early detection of methylated cancer DNA biomarkers.

A nascent riboswitch helix orchestrates robust transcriptional regulation through signal integration.

Widespread manganese-sensing transcriptional riboswitches effect the dependable gene regulation needed for bacterial manganese homeostasis in changing environments. Riboswitches - like most structured RNAs - are believed to fold co-transcriptionally, subject to both ligand binding and transcription events; yet how these processes are orchestrated for robust regulation is poorly understood. Through a combination of single molecule and bulk approaches, we discovered how a single Mn 2+ ion and the transcribing RNA polymerase (RNAP), paused immediately downstream by a DNA template sequence, are coordinated by the bridging switch helix P1.1 in the paradigmatic Lactococcus lactis riboswitch. This coordination achieves a heretofore-overlooked semi-docked global conformation of the nascent RNA, P1.1 base pair stabilization, transcription factor NusA ejection, and RNAP pause extension, thereby enforcing transcription readthrough. Our work demonstrates how a central, adaptable RNA helix functions analogous to a molecular fulcrum of a first-class lever system to integrate disparate signals for finely balanced gene expression control.

Are non-protein coding RNAs junk or treasure?: An attempt to explain and reconcile opposing viewpoints of whether the human genome is mostly transcribed into non-functional or functional RNAs.

The human genome project's lasting legacies are the emerging insights into human physiology and disease, and the ascendance of biology as the dominant science of the 21st century. Sequencing revealed that >90% of the human genome is not coding for proteins, as originally thought, but rather is overwhelmingly transcribed into non-protein coding, or non-coding, RNAs (ncRNAs). This discovery initially led to the hypothesis that most genomic DNA is "junk", a term still championed by some geneticists and evolutionary biologists. In contrast, molecular biologists and biochemists studying the vast number of transcripts produced from most of this genome "junk" often surmise that these ncRNAs have biological significance. What gives? This essay contrasts the two opposing, extant viewpoints, aiming to explain their bases, which arise from distinct reference frames of the underlying scientific disciplines. Finally, it aims to reconcile these divergent mindsets in hopes of stimulating synergy between scientific fields.

Regulation of bacterial gene expression by non-coding RNA: It is all about time!

Commensal and pathogenic bacteria continuously evolve to survive in diverse ecological niches by efficiently coordinating gene expression levels in their ever-changing environments. Regulation through the RNA transcript itself offers a faster and more cost-effective way to adapt than protein-based mechanisms and can be leveraged for diagnostic or antimicrobial purposes. However, RNA can fold into numerous intricate, not always functional structures that both expand and obscure the plethora of roles that regulatory RNAs serve within the cell. Here, we review the current knowledge of bacterial non-coding RNAs in relation to their folding pathways and interactions. We posit that co-transcriptional folding of these transcripts ultimately dictates their downstream functions. Elucidating the spatiotemporal folding of non-coding RNAs during transcription therefore provides invaluable insights into bacterial pathogeneses and predictive disease diagnostics. Finally, we discuss the implications of co-transcriptional folding andapplications of RNAs for therapeutics and drug targets.

A rhythmically pulsing leaf-spring DNA-origami nanoengine that drives a passive follower.

Molecular engineering seeks to create functional entities for modular use in the bottom-up design of nanoassemblies that can perform complex tasks. Such systems require fuel-consuming nanomotors that can actively drive downstream passive followers. Most artificial molecular motors are driven by Brownian motion, in which, with few exceptions, the generated forces are non-directed and insufficient for efficient transfer to passive second-level components. Consequently, efficient chemical-fuel-driven nanoscale driver-follower systems have not yet been realized. Here we present a DNA nanomachine (70 nm × 70 nm × 12 nm) driven by the chemical energy of DNA-templated RNA-transcription-consuming nucleoside triphosphates as fuel to generate a rhythmic pulsating motion of two rigid DNA-origami arms. Furthermore, we demonstrate actuation control and the simple coupling of the active nanomachine with a passive follower, to which it then transmits its motion, forming a true driver-follower pair.

Single-molecule FRET observes opposing effects of urea and TMAO on structurally similar meso- and thermophilic riboswitch RNAs.

Bacteria live in a broad range of environmental temperatures that require adaptations of their RNA sequences to maintain function. Riboswitches are regulatory RNAs that change conformation upon typically binding metabolite ligands to control bacterial gene expression. The paradigmatic small class-I preQ1 riboswitches from the mesophile Bacillus subtilis (Bsu) and the thermophile Thermoanaerobacter tengcongensis (Tte) adopt similar pseudoknot structures when bound to preQ1. Here, we use UV-melting analysis combined with single-molecule detected chemical denaturation by urea to compare the thermodynamic and kinetic folding properties of the two riboswitches, and the urea-countering effects of trimethylamine N-oxide (TMAO). Our results show that, first, the Tte riboswitch is more thermotolerant than the Bsu riboswitch, despite only subtle sequence differences. Second, using single-molecule FRET, we find that urea destabilizes the folded pseudoknot structure of both riboswitches, yet has a lower impact on the unfolding kinetics of the thermodynamically less stable Bsu riboswitch. Third, our analysis shows that TMAO counteracts urea denaturation and promotes folding of both the riboswitches, albeit with a smaller effect on the more stable Tte riboswitch. Together, these findings elucidate how subtle sequence adaptations in a thermophilic bacterium can stabilize a common RNA structure when a new ecological niche is conquered.

Critical Assessment of Condensate Boundaries in Dual-Color Single Particle Tracking.

Biomolecular condensates are membraneless cellular compartments generated by phase separation that regulate a broad variety of cellular functions by enriching some biomolecules while excluding others. Live-cell single particle tracking of individual fluorophore-labeled condensate components has provided insights into a condensate's mesoscopic organization and biological functions, such as revealing the recruitment, translation, and decay of RNAs within ribonucleoprotein (RNP) granules. Specifically, during dual-color tracking, one imaging channel provides a time series of individual biomolecule locations, while the other channel monitors the location of the condensate relative to these molecules. Therefore, an accurate assessment of a condensate's boundary is critical for combined live-cell single particle-condensate tracking. Despite its importance, a quantitative benchmarking and objective comparison of the various available boundary detection methods is missing due to the lack of an absolute ground truth for condensate images. Here, we use synthetic data of defined ground truth to generate noise-overlaid images of condensates with realistic phase separation parameters to benchmark the most commonly used methods for condensate boundary detection, including an emerging machine-learning method. We find that it is critical to carefully choose an optimal boundary detection method for a given dataset to obtain accurate measurements of single particle-condensate interactions. The criteria proposed in this study to guide the selection of an optimal boundary detection method can be broadly applied to imaging-based studies of condensates.

Biomolecular condensates in kidney physiology and disease.

The regulation and preservation of distinct intracellular and extracellular solute microenvironments is crucial for the maintenance of cellular homeostasis. In mammals, the kidneys control bodily salt and water homeostasis. Specifically, the urine-concentrating mechanism within the renal medulla causes fluctuations in extracellular osmolarity, which enables cells of the kidney to either conserve or eliminate water and electrolytes, depending on the balance between intake and loss. However, relatively little is known about the subcellular and molecular changes caused by such osmotic stresses. Advances have shown that many cells, including those of the kidney, rapidly (within seconds) and reversibly (within minutes) assemble membraneless, nano-to-microscale subcellular assemblies termed biomolecular condensates via the biophysical process of hyperosmotic phase separation (HOPS). Mechanistically, osmotic cell compression mediates changes in intracellular hydration, concentration and molecular crowding, rendering HOPS one of many related phase-separation phenomena. Osmotic stress causes numerous homo-multimeric proteins to condense, thereby affecting gene expression and cell survival. HOPS rapidly regulates specific cellular biochemical processes before appropriate protective or corrective action by broader stress response mechanisms can be initiated. Here, we broadly survey emerging evidence for, and the impact of, biomolecular condensates in nephrology, where initial concentration buffering by HOPS and its subsequent cellular escalation mechanisms are expected to have important implications for kidney physiology and disease.

Spontaneous Confinement of mRNA Molecules at Biomolecular Condensate Boundaries.

Cellular biomolecular condensates, termed ribonucleoprotein (RNP) granules, are often enriched in messenger RNA (mRNA) molecules relative to the surrounding cytoplasm. Yet, the spatial localization and diffusion of mRNAs in close proximity to phase separated RNP granules are not well understood. In this study, we performed single-molecule fluorescence imaging experiments of mRNAs in live cells in the presence of two types of RNP granules, stress granules (SGs) and processing bodies (PBs), which are distinct in their molecular composition and function. We developed a photobleaching- and noise-corrected colocalization imaging algorithm that was employed to determine the accurate positions of individual mRNAs relative to the granule's boundaries. We found that mRNAs are often localized at granule boundaries, an observation consistent with recently published data. We suggest that mRNA molecules become spontaneously confined at the RNP granule boundary similar to the adsorption of polymer molecules at liquid-liquid interfaces, which is observed in various technological and biological processes. We also suggest that this confinement could be due to a combination of intermolecular interactions associated with, first, the screening of a portion of the RNP granule interface by the polymer and, second, electrostatic interactions due to a strong electric field induced by a Donnan potential generated across the thin interface.

Riboswitches as therapeutic targets: promise of a new era of antibiotics.

INTRODUCTION: The growth of antibiotic resistance among bacterial pathogens is an impending global threat that can only be averted through the development of novel antibacterial drugs. A promising answer could be the targeting of riboswitches, structured RNA elements found almost exclusively in bacteria. AREAS COVERED: This review examines the potential of riboswitches as novel antibacterial drug targets. The limited mechanisms of action of currently available antibiotics are summarized, followed by a delineation of the functional mechanisms of riboswitches. We then discuss the potential for developing novel approaches that target paradigmatic riboswitches in the context of their bacterial gene expression machinery. EXPERT OPINION: We highlight potential advantages of targeting riboswitches in their functional form, embedded within gene expression complexes critical for bacterial survival. We emphasize the benefits of this approach, including potentially higher species specificity and lower side effects.

Structural basis for control of bacterial RNA polymerase pausing by a riboswitch and its ligand.

Folding of nascent transcripts can be modulated by the RNA polymerase (RNAP) that carries out their transcription, and vice versa. A pause of RNAP during transcription of a preQ1 riboswitch (termed que-PEC) is stabilized by a previously characterized template consensus sequence and the ligand-free conformation of the nascent RNA. Ligand binding to the riboswitch induces RNAP pause release and downstream transcription termination; however, the mechanism by which riboswitch folding modulates pausing is unclear. Here, we report single-particle cryo-electron microscopy reconstructions of que-PEC in ligand-free and ligand-bound states. In the absence of preQ1, the RNA transcript is in an unexpected hyper-translocated state, preventing downstream nucleotide incorporation. Strikingly, on ligand binding, the riboswitch rotates around its helical axis, expanding the surrounding RNAP exit channel and repositioning the transcript for elongation. Our study reveals the tight coupling by which nascent RNA structures and their ligands can functionally regulate the macromolecular transcription machinery.

Using Single-Molecule FRET to Evaluate DNA Nanodevices at Work.

The observation of DNA nanodevices at a single molecule (i.e., device) level and in real time provides rich information that is typically masked in ensemble measurements. Single-molecule fluorescence resonance energy transfer (smFRET) offers a means to directly follow dynamic conformational or compositional changes that DNA nanodevices undergo while operating, thereby retrieving insights critical for refining them toward optimal function. To be successful, smFRET measurements require careful execution and meticulous data analysis for robust statistics. Here we outline the elemental steps for smFRET experiments on DNA nanodevices, starting from microscope slide preparation for single-molecule observation to data acquisition and analysis.

BNP-Track: A framework for superresolved tracking.

Assessing dynamic processes at single molecule scales is key toward capturing life at the level of its molecular actors. Widefield superresolution methods, such as STORM, PALM, and PAINT, provide nanoscale localization accuracy, even when distances between fluorescently labeled single molecules ("emitters") fall below light's diffraction limit. However, as these superresolution methods rely on rare photophysical events to distinguish emitters from both each other and background, they are largely limited to static samples. In contrast, here we leverage spatiotemporal correlations of dynamic widefield imaging data to extend superresolution to simultaneous multiple emitter tracking without relying on photodynamics even as emitter distances from one another fall below the diffraction limit. We simultaneously determine emitter numbers and their tracks (localization and linking) with the same localization accuracy per frame as widefield superresolution does for immobilized emitters under similar imaging conditions (≈50nm). We demonstrate our results for both in cellulo data and, for benchmarking purposes, on synthetic data. To this end, we avoid the existing tracking paradigm relying on completely or partially separating the tasks of emitter number determination, localization of each emitter, and linking emitter positions across frames. Instead, we develop a fully joint posterior distribution over the quantities of interest, including emitter tracks and their total, otherwise unknown, number within the Bayesian nonparametric paradigm. Our posterior quantifies the full uncertainty over emitter numbers and their associated tracks propagated from origins including shot noise and camera artefacts, pixelation, stochastic background, and out-of-focus motion. Finally, it remains accurate in more crowded regimes where alternative tracking tools cannot be applied.

Utilizing functional cell-free extracts to dissect ribonucleoprotein complex biology at single-molecule resolution.

Cellular machineries that drive and regulate gene expression often rely on the coordinated assembly and interaction of a multitude of proteins and RNA together called ribonucleoprotein complexes (RNPs). As such, it is challenging to fully reconstitute these cellular machines recombinantly and gain mechanistic understanding of how they operate and are regulated within the complex environment that is the cell. One strategy for overcoming this challenge is to perform single molecule fluorescence microscopy studies within crude or recombinantly supplemented cell extracts. This strategy enables elucidation of the interaction and kinetic behavior of specific fluorescently labeled biomolecules within RNPs under conditions that approximate native cellular environments. In this review, we describe single molecule fluorescence microcopy approaches that dissect RNP-driven processes within cellular extracts, highlighting general strategies used in these methods. We further survey biological advances in the areas of pre-mRNA splicing and transcription regulation that have been facilitated through this approach. Finally, we conclude with a summary of practical considerations for the implementation of the featured approaches to facilitate their broader future implementation in dissecting the mechanisms of RNP-driven cellular processes. This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems.

Probing Transient Riboswitch Structures via Single Molecule Accessibility Analysis.

Riboswitches are a class of RNA motifs in the untranslated regions of bacterial messenger RNAs (mRNAs) that can adopt different conformations to regulate gene expression. The binding of specific small molecule or ion ligands, or other RNAs, influences the conformation the riboswitch adopts. Single Molecule Kinetic Analysis of RNA Transient Structure (SiM-KARTS) offers an approach for probing this structural isomerization, or conformational switching, at the level of single mRNA molecules. SiM-KARTS utilizes fluorescently labeled, short, sequence-complementary DNA or RNA oligonucleotide probes that transiently access a specific RNA conformation over another. Binding and dissociation to a surface-immobilized target RNA of arbitrary length are monitored by Total Internal Reflection Fluorescence Microscopy (TIRFM) and quantitatively analyzed, via spike train and burst detection, to elucidate the rate constants of isomerization, revealing mechanistic insights into riboswitching.