Biology subjects

Stannard, A.

Publications and source records attributed to Stannard, A..

3 recordsLinked to original sources

bioRxiv2026

Recognition in Confinement: The Dynamics of Homologous Gene Pairing

How can protein-independent, side-by-side alignment of homologous double-stranded DNA (ds-DNA) arise as an early step in genome repair and genetic exchange, and what determines their kinetics under confinement? Although recombination proteins are known to facilitate this process, experiments indicate that an initial side-by-side arrangement of homologs can occur even in their absence. Helical Coherence Theory (HCT) proposes that sequence-dependent distortions of the double helix lead to commensurate charge patterns between interacting dsDNAs, favouring homologous over non-homologous alignment. However, HCT has largely been developed for straight, rigid dsDNA rods, an approximation limited to roughly one persistence length ({ell}B {approx} 50 nm), leaving the question open as to how recognition manifests in longer, fluctuating chains. Moreover, the dynamics and timescales of homolog searching under confinement, and the internal dynamics of the paired state, such as transient formation of local unpaired regions ("bubbles") and end fraying, remain unexplored. Here we extend HCT to dsDNA chains of multiple persistence lengths under confinement using coarse-grained simulations with a custom HCT-based force field, in order to come closer to understanding how homologous genes pair in vivo. We simulate two homologous chains within spherical cavities of radii 0.3-1 times their free-space gyration radius under two limiting ionic conditions. We found that confinement warrants homologous pairing; it occurs on microsecond timescales and depends non-monotonically on confinement size, with an optimal confinement that accelerates pairing while avoiding tangled states and metastable trapping in an ion-dependent manner. To resolve pairing dynamics, we develop a kinetic theory and fit it to simulation trajectories, distinguishing rates of independent pairing modes underlying bubble formation and fraying. Together, these results support a physical mechanism for homologous pairing beyond the rigid-rod limit and suggest how confinement can promote both encounter and stabilisation within the HCT framework.

biophysics↗

bioRxiv2024

Reduced non-specific binding of super-resolution DNA-PAINT markers by Shielded DNA-PAINT labeling protocols

The DNA-based single molecule super-resolution imaging approach, DNA-PAINT, can achieve nanometer resolution of single targets. However, the approach can suffer from significant non-specific background signals originating from non-specifically bound DNA-conjugated DNA-PAINT secondary antibodies as shown here. Using dye-modified oligonucleotides the location of DNA-PAINT secondary antibody probes can easily be observed with widefield imaging prior to beginning a super-resolution measurement. This reveals that a substantial proportion of DNA probes can accumulate, non-specifically, within the nucleus, as well as across the cytoplasm, of cells. Here, Shielded DNA-PAINT labeling is introduced, a method using partially or fully double-stranded docking strand sequences, prior to labeling, in buffers with increased ionic strength to greatly reduce non-specific interactions in the nucleus as well as the cytoplasm. This new labeling approach is evaluated against various conditions and it is shown that applying Shielded DNA-PAINT can reduce non-specific events [~]5 fold within the nucleus. This marked reduction in non-specific binding of probes during the labeling procedure is comparable to results obtained with unnatural left-handed DNA albeit at a fraction of the cost. Shielded DNA-PAINT is a straightforward adaption of current DNA-PAINT protocols and enables nanometer precision imaging of nuclear targets with low non-specific background.

biophysics↗

bioRxiv2020

Molecular fluctuations as a ruler of force-induced protein conformations

Molecular fluctuations directly reflect the underlying energy landscape. Variance analysis can probe protein dynamics in several biochemistry-driven approaches, yet measurement of probe-independent fluctuations in proteins exposed to mechanical forces remains only accessible through steered molecular dynamics simulations. Using single molecule magnetic tweezers, here we conduct variance analysis to show that individual unfolding and refolding transitions occurring in dynamic equilibrium in a single protein under force are hallmarked by a change in the protein's end-to-end fluctuations, revealing a change in protein stiffness. By unfolding and refolding three structurally distinct proteins under a wide range of constant forces, we demonstrate that the associated change in protein compliance to reach force-induced thermodynamically-stable states scales with the protein's contour length, in agreement with the sequence-independent FJC model of polymer physics. Our findings will help probe the conformational dynamics of proteins exposed to mechanical force at high resolution, of central importance in mechanosensing and mechanotransduction.

biophysics↗