Biology subjects

Ganser, L. R.

Publications and source records attributed to Ganser, L. R..

6 recordsLinked to original sources

bioRxiv2025

RNA G-Quadruplexes Function as a Tunable Switch of FUS Phase Separation

FUS undergoes liquid-liquid phase separation (LLPS) to support essential cellular functions, but aberrant phase transitions promote toxic aggregation in neurodegenerative disease. Short RNA oligonucleotides can reverse this behavior, yet the structural determinants that govern RNA activity remain poorly defined. Here, we identify RNA G-quadruplexes (rG4s) as tunable structural motifs that potently modulate FUS LLPS. rG4 activity depends on its concentration and is modulated by rG4 length and stability: increasing repeat number switches rG4s from inhibitor to nucleator of FUS assembly, whereas chemical modifications that stabilize rG4 enhance inhibitory function and render these activities resilient to ionic perturbation. Although short rG4s interact with both soluble and condensed FUS, they preferentially engage the soluble pool, likely shifting the equilibrium toward dispersion. Leveraging these mechanistic insights, we developed a bioinformatic pipeline that uncovered more rG4 inhibitors that robustly reverse FUS LLPS and aggregation. Our findings establish rG4s as chemically programmable regulators of protein phase behavior and provide a blueprint for engineering RNA-based therapeutics that dissolve pathogenic FUS assemblies. More broadly, this work directly links RNA secondary structure to distinct functional outcomes in phase behavior, establishing a structure-function paradigm for RNA control of condensates, demonstrating implications in both fundamental biology and therapeutic development. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/685846v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1db3ea8org.highwire.dtl.DTLVardef@eec825org.highwire.dtl.DTLVardef@96187eorg.highwire.dtl.DTLVardef@2204ce_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Switch-like Compaction of Poly(ADP-ribose) Upon Cation Binding

Poly(ADP-ribose) (PAR) is a homopolymer of adenosine diphosphate ribose that is added to proteins as a post-translational modification to regulate numerous cellular processes. PAR also serves as a scaffold for protein binding in macromolecular complexes, including biomolecular condensates. It remains unclear how PAR achieves specific molecular recognition. Here, we use single-molecule fluorescence resonance energy transfer (smFRET) to evaluate PAR flexibility under different cation conditions. We demonstrate that, compared to RNA and DNA, PAR has a longer persistence length and undergoes a sharper transition from extended to compact states in physiologically relevant concentrations of various cations (Na+, Mg2+, Ca2+, and spermine). We show that the degree of PAR compaction depends on the concentration and valency of cations. Furthermore, the intrinsically disordered protein FUS also served as a macromolecular cation to compact PAR. Taken together, our study reveals the inherent stiffness of PAR molecules, which undergo switch-like compaction in response to cation binding. This study indicates that a cationic environment may drive recognition specificity of PAR. SignificancePoly(ADP-ribose) (PAR) is an RNA-like homopolymer that regulates DNA repair, RNA metabolism, and biomolecular condensate formation. Dysregulation of PAR results in cancer and neurodegeneration. Although discovered in 1963, fundamental properties of this therapeutically important polymer remain largely unknown. Biophysical and structural analyses of PAR have been exceptionally challenging due to the dynamic and repetitive nature. Here, we present the first single-molecule biophysical characterization of PAR. We show that PAR is stiffer than DNA and RNA per unit length. Unlike DNA and RNA which undergoes gradual compaction, PAR exhibits an abrupt switch-like bending as a function of salt concentration and by protein binding. Our findings points to unique physical properties of PAR that may drive recognition specificity for its function.

biophysics↗

bioRxiv2022

RNA conformational propensities determine cellular activity

Cellular processes are the product of interactions between biomolecules, which associate to form biologically active complexes 1. These interactions are mediated by intermolecular contacts, which if disrupted, lead to alterations in cell physiology. Nevertheless, the formation of intermolecular contacts nearly universally requires changes in the conformations of the interacting biomolecules. As a result, binding affinity and cellular activity crucially depend not only on the strength of the contacts, but also on the inherent propensities to form binding-competent conformational states2,3. Thus, conformational penalties are ubiquitous in biology and must be known in order to quantitatively model binding energetics for protein and nucleic acid interactions4,5. However, conceptual and technological limitations have hindered our ability to dissect and quantitatively measure how conformational propensities impact cellular activity. Here, we systematically altered and determined the propensities for forming the protein-bound conformation of HIV-1 TAR RNA. These propensities quantitatively predicted the binding affinities of TAR to the RNA-binding region of the Tat protein and predicted the extent of HIV-1 Tat-dependent transactivation in cells. Our results establish the role of ensemble-based conformational propensities in cellular activity and reveal an example of a cellular process driven by an exceptionally rare and short-lived RNA conformational state.

biophysics↗

bioRxiv2020

Revealing the high propensity of RNAs to non-specifically bind drug-like small molecules

Identifying small molecules that selectively bind a single RNA target while discriminating against all other cellular RNAs is an important challenge in RNA-targeted drug discovery. Much effort has been directed toward identifying drug-like small molecules that minimize electrostatic and stacking interactions that lead to non-specific binding of aminoglycosides and intercalators to a variety of RNAs. Many such compounds have been reported to bind RNAs and inhibit their cellular activities, however the ability of such compounds to discriminate against RNA stem-loops commonly found in the transcriptome has not been thoroughly assessed in all cases. Here, we examined the propensities of three drug-like compounds, previously shown to bind and inhibit the cellular activity of three distinct RNAs, to non-specifically bind two HIV-1 stem-loop RNAs: the transactivation response element (TAR) and stem IIB in the rev response element (RREIIB). All three compounds bound to TAR and RREIIB in vitro, and two inhibited TAR-dependent transactivation and RRE-dependent viral export in cell-based assays while also exhibiting substantial off-target interactions consistent with non-specific cellular activity. A survey of X-ray and NMR structures of RNA-small molecule complexes revealed that drug-like molecules form hydrogen bonds with functional groups commonly accessible in canonical stem-loop RNA motifs, much like aminoglycosides, and in contrast to ligands that specifically bind riboswitches. Our results support extending the group of non-selective RNA-binders beyond aminoglycosides and intercalators to encompass drug-like compounds with capacity for non-specific hydrogen-bonding and reinforce the importance of assaying for off-target interactions and RNA selectivity in vitro and in cells when assessing novel RNA-binders.

biochemistry↗

bioRxiv2019

Stabilizing non-native excited states as a therapeutic strategy for targeting RNA

Many regulatory RNAs undergo changes in their structure from the dominant ground-state (GS) toward short-lived low-abundance excited-states (ES) that reorganize local elements of secondary structure. ESs are increasingly observed in vitro and implicated in the folding and biological activities of regulatory RNAs and as targets for developing therapeutics. However, whether these ESs also form with comparable abundance within the complex cellular environment remains unknown. Here, we developed an approach for assessing the relative stability and abundance of RNA ESs within the functional cellular context. The approach uses point substitution mutations to increase the population of an inactive ES relative to the active GS. The cellular activity of such ES-stabilizing mutants then provides an indirect measure of any residual population of the active GS within the functional cellular context. Compensatory rescue mutations that restore the GS are used to control for changes in cellular activity arising due to changes in sequence. The approach is applied to probe ESs in two highly conserved and functionally important regulatory RNAs from HIV-1: the transactivation response element (TAR) and the Rev response element (RRE). For both RNAs, ES-stabilizing mutations inhibited cellular activity to a degree that correlates with the extent to which they stabilize the ES relative to the GS in vitro. These results indicate that the non-native ESs of TAR and RRE likely form in cells with abundances comparable to those measured in vitro and their targeted stabilization provides a new avenue for developing anti-HIV therapeutics.

biochemistry↗

bioRxiv2017

High Performance Virtual Screening by Targeting a High-resolution RNA Dynamic Ensemble

Dynamic ensembles that capture the flexibility of RNA three-dimensional (3D) structures hold great promise in advancing RNA-targeted drug discovery. Here, we experimentally screened the transactivation response element (TAR) RNA from human immunodeficiency virus type-1 (HIV-1) against ~100,000 small molecules. We used this dataset, along with 240 known hit molecules, to evaluate virtual screening (VS) against a high-resolution TAR ensemble determined by combining NMR spectroscopy and molecular dynamics (MD) simulations. Ensemble-based VS (EBVS) scores molecules with an area under the receiver operator characteristic curve (ROC AUC) of 0.87 with ~50% of all hits falling within the top 2% of scored molecules, and also correctly predicts the different TAR inter-helical structures when bound to six molecules. The prediction accuracy decreased significantly with decreasing accuracy of the target ensemble or when docking against a single RNA structure. These results demonstrate that experime ...

biochemistry↗