Biology subjects

Smitskamp, Q.

Publications and source records attributed to Smitskamp, Q..

6 recordsLinked to original sources

bioRxiv2026

A multimodal, correlative magnetic tweezers-TIRF platform for high-throughput single-molecule interrogations

Single-molecule techniques can resolve biological reactions at unmatched detail, but their low throughput and single-modality readouts have kept them out of data-intensive pipelines such as omics and drug discovery, and beyond reach of low-yield biological systems. Here we introduce a multimodal platform integrating high-throughput magnetic tweezers with ultra-wide-and flat-field objective-based total internal reflection fluorescence, enabling simultaneous force, torque, multicolor fluorescence, and temperature-dependent measurements on up to thousands of individual molecules in parallel and in real time. We demonstrate accurate single-molecule Forster resonance energy transfer (smFRET) for prism-based spectral imaging, capture temperature-dependent hairpin folding dynamics at high temporal resolution with smFRET and use correlative torque-fluorescence measurements to unravel the open-complex formation dynamics during bacterial transcription initiation. By unifying high resolution, throughput, and multimodal readout, this platform enables multidimensional dissection of complex biomolecular reactions with high statistical confidence, unlocking single-molecule biophysics for integration with drug discovery, omics, and cryo-EM workflows.

biophysics↗

bioRxiv2026

The nucleoside analog CMX521 inhibits coronavirus RNA-dependent RNA polymerase via a two-pronged mechanism

The SARS-CoV-2 pandemic has underscored the urgent need for broad-spectrum antivirals in pandemic preparedness efforts. Nucleoside analogs targeting viral polymerases are often considered in this context. Here, we employ ensemble biochemical assays and single-molecule magnetic tweezers to characterize the detailed mechanism of action of the adenosine analog CMX521 (developed through Phase 1 clinical studies), a broad-spectrum antiviral against caliciviruses and coronaviruses, against SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). The triphosphate form of CMX521 is efficiently incorporated by RdRp, even against saturating ATP concentrations. Analog incorporation induces only a brief pause in nascent RNA synthesis. When embedded in the template strand, CMX521 causes the polymerase to stall [~]9 s on average due to impaired uridine opposite incorporation. Multiple CMX521 residues in the template strand completely inhibit polymerase elongation. When the coronavirus polymerase is associated with the viral helicase, CMX521 strongly promotes copy-back RNA synthesis suggesting a second inhibitory mechanism for CMX521. Collectively, our findings establish a two-pronged mode of coronavirus polymerase inhibition by CMX521.

microbiology↗

bioRxiv2026

Accurate single-bead force calibration in high-throughput magnetic tweezers reveals the mechanism of directional transcription termination by MTERF1

High-throughput force spectroscopy assays, such as with magnetic tweezers, enable reconstruction of biomolecular reaction energy landscapes and provide access to rare events with deep statistics. Precise force calibration is essential for accurately describing complex reactions, which can be hindered by sample heterogeneity, such as bead-to-bead difference in magnetic content. Here, we describe an in-situ force calibration methodology for high-throughput magnetic tweezers that enables the calibration for each individual bead with an accuracy of up to 3%, limited only by the statistical resolution. We apply this approach to characterize the directional transcription termination molecular mechanism by the polar roadblock mitochondrial transcription termination factor 1 (MTERF1). Establishing a SpyTag-SpyCatcher surface-attachment strategy, we performed force-jump experiments on the same tethers for up to 11 hours. We showed that directional DNA unwinding is sufficient to explain the polar roadblock activity of MTERF1. Accurate force spectroscopy further reveals that the unlocking transition is rate-limited by a single kinetic barrier, with a transition-state distance consistent with structural interpretations. Together, these results provide a mechanistic and broadly applicable model for the asymmetric stability of MTERF1 and other nucleic acid polar roadblocks and establish a robust force spectroscopy framework for high-throughput magnetic tweezers experiments.

biophysics↗

bioRxiv2025

Template switching by coronavirus polymerase requires helicase activity and is stimulated by remdesivir and molnupiravir

Polymerase template switching is an essential mechanism in coronaviruses (CoVs) that enables both sub-genomic (sg) RNA synthesis and increases genomic diversity via RNA recombination. Despite its importance, the molecular mechanism of CoV polymerase template switching remains unclear. Using magnetic tweezers, we show that the CoV non-structural protein (nsp) 13-helicase drives polymerase template switching, followed by copy-back RNA synthesis. This activity requires nsp13-helicase ATPase activity and a duplex RNA downstream of the CoV polymerase. This novel function of nsp13-helicase is targeted by the nucleotide analogs remdesivir and molnupiravir, whose incorporation in the nascent strand increases CoV polymerase template switching probability, leading to defective RNA production. We propose a novel mechanism of action where incorporation of these analogs dramatically reduces full length genome copy number by stimulating polymerase template switching. Our study further demonstrates nsp13-helicases central role in CoV replication and how this enzyme function can be indirectly targeted by analogs.

microbiology↗

bioRxiv2024

Single-molecule magnetic tweezers reveals that TAV2b-derived peptides underwind and stabilize double-stranded RNA

Double-stranded RNA (dsRNA) has evolved into a key tool in understanding and regulating biological processes, with promising implications in therapeutics. However, its efficacy is often limited due to instability in biological settings. Recently, the development of peptidic dsRNA binders derived from naturally occurring RNA-binding proteins has emerged as a favorable starting point to address this limitation. Nevertheless, it remains unclear how these high affinity dsRNA binders alter the structure and flexibility of dsRNA. To this end, we employed single-molecule magnetic tweezers experiments to investigate the effects of TAV2b-derived peptidic dsRNA binders on the mechanical properties of dsRNA. Torque spectroscopy assays demonstrated that these peptides underwind dsRNA, while also stabilizing the duplex. Additionally, force spectroscopy experiments demonstrate that a wild type TAV2b peptide derivative extends the contour length and lowers the bending rigidity of dsRNA, while a homodimeric version triggers the formation of higher order complexes at forces below 1 pN. Our study presents a quantitative approach to investigate how these peptides alter the structure of dsRNA, and whether peptide structural design alters the affinity to dsRNA and its stability. This approach could inform the design of more potent and effective dsRNA binders in the efforts to advance RNA therapeutics.

biophysics↗

bioRxiv2024

An alternative mechanism for activation of innate immune signaling by MDA5

Long double-stranded (ds) RNA in the cytosol acts as a potent inflammatory molecule recognized by the receptor MDA5, triggering the innate immune response. Mutations affecting MDA5 ATPase activity lead to severe pathological conditions. MDA5 nucleoprotein filament assembly-disassembly dynamic is proposed to regulate dsRNA recognition, though the exact mechanism remains unclear. Here, we employed magnetic tweezers to monitor the assembly and manipulate MDA5 filaments at the single dsRNA level. Following a slow nucleation event, MDA5 assembles cooperatively and directionally into (partial) filaments and utilizes ATP hydrolysis to compact dsRNA via single-stranded (ss) RNA loop extrusion, even against a significant opposing force. This compacted state is further stabilized by oligomerization of MDA5s caspase recruitment domain (CARD) and requires high force to be disrupted. ssRNA gaps impaired compaction, suggesting a new mechanism for dsRNA recognition. We propose that MDA5 mediated dsRNA compaction captures viral dsRNA, preventing further usage for viral replication.

biophysics↗