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Widom, J. R.

Publications and source records attributed to Widom, J. R..

8 recordsLinked to original sources
bioRxiv2026

Pyrrolocytosine as a Fluorescent Sensor of RNA-Small Molecule Interactions

Fluorescent base analogues have proven to be valuable probes of RNA structure. Pyrrolocytosine (pC), an analogue of cytosine, exhibits highly environment-sensitive fluorescence within RNA and is strongly quenched in hydrogen-bonded contexts. In this work, we incorporated pC into the aptamer domain of the B. subtilis guanine riboswitch to determine if this quenching mechanism could allow for sensing of RNA-small molecule interactions. We utilized complementary fluorescence spectroscopy methods to under-stand the functional impacts and ligand-sensitivity of pC substitutions distal to and within the binding pocket. We found that pC substitution within a static structural region exhibits the expected ligand-independent fluorescence properties. When placed in the ligand binding pocket, pC exhibits a moderately decreased affinity for the ligands guanine (G) and hypoxanthine (Hx) while maintaining high specificity for G. We found that pC behaves as a turn-off sensor, with distinct changes in fluorescence upon G or Hx binding. These differences are dependent on the number of hydrogen bonds formed with pC and are resolvable in both time-dependent and steady-state fluorescence measurements. Our results show that pC can be used to study RNA-small molecule interactions and can discriminate between similar ligands even when they have identical binding modes.

biophysics↗
bioRxiv2025

Alternative probe chemistries for single-molecule analysis of long non-coding RNA

Single-molecule microscopy has been widely used to study the structure and dynamics of RNA, but extension to larger systems such as long non-coding RNA (lncRNA) has proven challenging. Methods such as single-molecule kinetic analysis of RNA transient structure (SiM-KARTS), where the binding of a short, complementary oligonucleotide probe is used to determine accessibility of a specific region of the RNA, are promising. However, adapting SiM-KARTS to systems as complex as lncRNA requires careful optimization of experimental variables that have not been thoroughly explored. In this work, SiM-KARTS, thermal denaturation experiments, and circular dichroism spectroscopy were used to analyze the binding behaviors of probes with alternative backbone chemistries, specifically DNA with locked nucleic acid (LNA) residues incorporated and morpholinos. A segment of lncRNA that enabled control over the accessibility of the target sequence was used as a model. We show that optimizing probe backbone chemistry can allow for a more precise distinction between different structures of the target RNA, and for fine-tuning of probe binding stability without the structural impacts that other variables such as ionic concentration may have. Specifically, we demonstrate that LNA probes exhibit a high degree of structural sensitivity in both their binding and unbinding kinetics. We further show that when binding and unbinding rates are considered holistically, LNA probes allow traces arising from different target RNA structures to be individually classified with a high degree of accuracy. These results provide design principles for the application of SiM-KARTS to target RNAs of increased complexity such as lncRNA.

biophysics↗
bioRxiv2025

A pseudouridine synthase shapes tRNA structural dynamics through both catalysis and remodeling

Transfer RNA has long served as an exemplar of a thermodynamically stable, structured RNA. Yet it undergoes significant structural changes upon binding and catalysis by diverse modification enzymes. We leveraged optical binding assays and single-molecule FRET to observe the structural dynamics of two yeast tRNAs, in isolation, and upon interaction with the conserved pseudouridine synthase Pus4/TruB. We show that unmodified and pseudouridylated tRNAeMet(CAU) and tRNAThr(AGU) all sample open, compact and intermediate conformations, though tRNAThr(AGU) exhibits faster dynamics. Consistent with its role in modifying RNAs with different structural properties, Pus4 binds robustly to unmodified tRNA, tRNA that was pseudouridylated prior to engaging Pus4 ("pre-modified"), and even an unrelated riboswitch RNA. Pus4 binding to tRNAeMet(CAU) leads to additional tRNA conformational states. The ensemble of conformations explored by Pus4-bound pre-modified tRNA resolved within minutes back into open, compact and intermediate states. tRNAeMet(CAU) that is initially unmodified more gradually approached the ensemble of structures that were attained rapidly by pre-modified tRNA. Thus, Pus4 both catalyzes a lasting chemical change on tRNA and remodels it over time after catalysis, perhaps to promote subsequent steps of tRNA maturation.

biophysics↗
bioRxiv2024

Impacts of sequence and structure on pyrrolocytosine fluorescence in RNA

Fluorescence spectroscopy encompasses many useful methods for studying the structures and dynamics of biopolymers. Applications to nucleic acids require the use of extrinsic fluorophores such as fluorescent base analogues (FBAs), which mimic the native bases but have enhanced fluorescence quantum yields. In this work, we use multiple complementary methods to systematically investigate the sequence- and structure-dependence of the fluorescence of the FBA pyrrolocytosine (pC) within RNA. We demonstrate that pC is typically brightest in conformations in which it is base-stacked but not base-paired, properties that distinguish it from more widely used FBAs. This effect is strongly sequence-dependent, with adjacent adenosine and cytidine residues conferring the greatest contrast between stacked and unstacked structures. Structural heterogeneity was resolved in single-stranded RNA and fully complementary and mismatched double-stranded RNA using time-resolved fluorescence measurements and fluorescence-detected circular dichroism spectroscopy. Double-stranded contexts are distinguished from single-stranded contexts by the presence of inter-strand energy transfer from opposing bases, while base-paired pC is distinguished by its short excited state lifetime. This work will enhance the value of pC as a structural probe for biologically and medicinally significant RNAs by guiding the selection of labeling sites and interpretation of the resulting data. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/618031v2_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@17fe2c1org.highwire.dtl.DTLVardef@1001feforg.highwire.dtl.DTLVardef@130b5a9org.highwire.dtl.DTLVardef@e692dd_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGRAPHICAL ABSTRACTC_FLOATNO C_FIG

biophysics↗
bioRxiv2022

Disentangling contact and ensemble epistasis in a riboswitch

Mutations introduced into macromolecules often exhibit epistasis, where the effect of one mutation alters the effect of another. Knowledge of the mechanisms that lead to epistasis is important for understanding how macromolecules work and evolve, as well as for effective macromolecular engineering. Here we investigate the interplay between "contact epistasis" (epistasis arising from physical interactions between mutated residues) and "ensemble epistasis" (epistasis that occurs when a mutation redistributes the conformational ensemble of a macromolecule, thus changing the effect of the second mutation). We argue that the two mechanisms can be distinguished in allosteric macromolecules by measuring epistasis at differing allosteric effector concentrations. Contacts give rise to epistasis in the microscopic equilibrium constants describing the conformational ensemble. Ensemble epistasis manifests in thermodynamic observables, such as the energy of ligand binding or enzyme activation, that depend on the concentration of allosteric effector. Using this framework, we experimentally investigated the origins of epistasis in three mutant cycles introduced into the adenine riboswitch aptamer domain. We found evidence for both contact and ensemble epistasis in all cycles. Further, we found that the two mechanisms of epistasis can interact with each other. For example, in one mutant cycle we observe contact epistasis of 6 kcal/mol attenuated by the ensemble to only 1.5 kcal/mol in the final thermodynamic observable. Finally, our work yields simple heuristics for identifying contact and ensemble epistasis using limited experimental measurements. Statement of significanceMutations to protein or RNA molecules often have different effects when introduced individually versus together. To understand and engineer biological macromolecules, we must identify the mechanistic origins of this phenomenon. Here, we measured the interplay between direct, physical interactions between mutations ("contact epistasis") and indirect interactions mediated by conformational ensembles ("ensemble epistasis"). We introduced pairs of mutations into an RNA molecule that transitions between several different conformations. We found epistasis arising from both contacts and the ensemble, and that the two mechanisms could synergize with one another. Our work reveals that one must consider the effects of mutations on multiple conformations to understand epistasis and suggests a few rules-of-thumb for disentangling contact and ensemble epistasis in other macromolecules.

biophysics↗
bioRxiv2021

Impacts of Fluorescent Base Analogue Substitution on the Folding of a Riboswitch

Riboswitches are mRNA segments that regulate gene expression in response to ligand binding. The Class I preQ1 riboswitch consists of a stem-loop and an adenine-rich single-stranded tail ("L3"), which adopt a pseudoknot structure upon binding of the ligand preQ1. We inserted 2-aminopurine (2-AP), a fluorescent analogue of adenine (A), into the riboswitch at six different positions within L3. Here, 2-AP functions both as a spectroscopic probe and as a "mutation" that reveals how alteration of specific A residues impacts the riboswitch. Using fluorescence and circular dichroism spectroscopy, we found that 2-AP decreases the affinity of the riboswitch for preQ1 at all labeling positions tested, though modified and unmodified variants undergo the same global conformational changes at sufficiently high preQ1 concentration. 2-AP substitution is most detrimental to ligand binding at sites proximal to the ligand-binding pocket, while distal labeling sites exhibit the largest impacts on the stability of the L3 domain in the absence of ligand. Insertion of multiple 2-AP residues does not induce significant additional disruptions. Our results show that interactions involving the A residues in L3 play a critical role in ligand recognition by the preQ1 riboswitch, and that 2-AP substitution exerts complex and varied impacts on this riboswitch.

biochemistry↗
bioRxiv2018

Ligand Modulates Cross-Coupling between Riboswitch Folding and Transcriptional Pausing

HighlightsO_LIsmFRET, biochemistry and simulations probe co-transcriptional riboswitch folding\nC_LIO_LINascent RNA folding is disfavored by the DNA template and aided by RNAP\nC_LIO_LITranscriptional pausing is stabilized by RNA pseudoknot and destabilized by ligand\nC_LI\n\nSUMMARYNumerous classes of riboswitches have been found to regulate bacterial gene expression in response to physiological cues, offering new paths to anti-bacterial drugs. As common studies of isolated riboswitches lack the functional context of the transcription machinery, we here combine single-molecule, biochemical and simulation approaches to investigate the coupling between co-transcriptional folding of the pseudoknot-structured preQ1 riboswitch and RNA polymerase (RNAP) pausing. We show that pausing at a site immediately downstream of the riboswitch requires a ligand-free pseudoknot in the nascent RNA, a precisely spaced sequence resembling the pause consensus, and electrostatic and steric interactions with the RNAP exit channel. While interactions with RNAP stabilize the native fold of the riboswitch, binding of the ligand signals RNAP release from the pause. Our results demonstrate that the nascent riboswitch and its ligand actively modulate the function of RNAP and vice versa, a paradigm likely to apply to other cellular RNA transcripts.

biophysics↗