Biology subjects

Reisner, W.

Publications and source records attributed to Reisner, W..

5 recordsLinked to original sources

bioRxiv2026

Non-equilibrium cyclic conformations of DNA under electrokinetic confinement

Classic nanofluidic devices harness geometric confinement to modulate single polymer conformation and control chain dynamics. While classic nanofluidic devices have given rise to a range of applications, they are fundamentally limited by the static nature of their construction, which restricts real time control over molecular conformations. The reversible electrokinetic confinement (RECON) technique enables time-dependent control of DNA conformations in nanowells via dynamic modulation of field-induced confinement. Here, we demonstrate that applying dynamically varying confinement using stochastic and periodic signals can drive the confined polymer to access a range of non-equilibrium states, with the distinct configurations quantified via their radius of gyration tensor. For periodic driving, the device allows controlled cycling across conformational states, with distinct hysteresis observed in the shape anisotropy modulation. We quantify the interplay between the driving frequency, driving amplitude, and resulting hysteresis. Overall, our results establish RECON as a platform for active manipulation of single molecules, opening new avenues for studying non-equilibrium polymer physics and developing high-throughput, dynamically tunable nanofluidic devices.

biophysics↗

bioRxiv2026

Electrokinetic active grooves for liposome capture, confinement, trajectory analysis and controlled release

Extracellular vesicles (EVs) are attractive candidates for minimally invasive disease monitoring. EV diagnostic signals often reside in low-abundant subpopulations, motivating single-vesicle measurements that can report distributions rather than ensemble averages. In practice, such assays rely on transient migration of a mobile vesicle through a detection region or performing cycled processing/measurement steps on vesicles permanently immobilized on surfaces via affinity-based capture. Single vesicle analysis would benefit from technologies that can combine characteristics of transient detection and surface immobilization to enable controlled capture, fixed time analysis and release of vesicles in a marker-neutral way. Here, we present electrokinetically active (RECON) grooves that provides low-voltage, programmable capture, timed retention, and release of nanoscale vesicles within microscopic grooves under continuous optical access. The platform uses a titanium embedded electrode with thin TiO2 passivation, enabling stable actuation over multi-hour experiments. Using fluorescent, size-defined liposomes as standards, we demonstrate (1) reversible, minute-long confinement at <5 V, (2) size-dependent confinement times, and (3) optical quantification of groove-guided drift velocities. Together, these results establish RECON grooves as a reusable, electrically programmable platform for controlled capture, timed retention, and release, while providing quantitative, trajectory-derived measures of confinement and transport.

biophysics↗

bioRxiv2023

Dynamics and quantitative contribution of the aminoglycoside 6'-N-acetyltransferase type Ib to amikacin resistance

Aminoglycosides are essential components in the available armamentarium to treat bacterial infections. The surge and rapid dissemination of resistance genes strongly reduce their efficiency, compromising public health. Among the multitude of modifying enzymes that confer resistance to aminoglycosides, the aminoglycoside acetyltransferase AAC(6')-Ib is the most prevalent and relevant in the clinical setting as it can inactivate numerous aminoglycosides, such as amikacin. Although the mechanism of action, structure, and biochemical properties of the AAC(6')-Ib protein have been extensively studied, the contribution of the intracellular milieu to its activity remains unclear. In this work, we used a fluorescent-based system to quantify the number of AAC(6')-Ib per cell in Escherichia coli, and we modulated this copy number with the CRISPR interference method. These tools were then used to correlate enzyme concentrations with amikacin resistance levels. Our results show that resistance to amikacin increases linearly with a higher concentration of AAC(6')-Ib until it reaches a plateau at a specific protein concentration. In vivo imaging of this protein shows that it diffuses freely within the cytoplasm of the cell, but it tends to form inclusion bodies at higher concentrations in rich culture media. Addition of a chelating agent completely dissolves these aggregates and partially prevents the plateau in the resistance level, suggesting that AAC(6')-Ib aggregation lowers resistance to amikacin. These results provide the first step in understanding the cellular impact of each AAC(6')-Ib molecule on aminoglycoside resistance. They also highlight the importance of studying its dynamic behavior within the cell. ImportanceAntibiotic resistance is a growing threat to human health. Understanding antibiotic resistance mechanisms can serve as foundation for developing innovative treatment strategies to counter this threat. While numerous studies clarified the genetics and dissemination of resistance genes and explored biochemical and structural features of resistance enzymes, their molecular dynamics and individual contribution to resistance within the cellular context remain unknown. Here, we examined this relationship modulating expression levels of AAC(6')-Ib, an enzyme of clinical relevance. We show a linear correlation between copy number of the enzyme per cell and amikacin resistance levels up to a threshold where resistance plateaus. We propose that at concentrations below the threshold, the enzyme diffuses freely in the cytoplasm but aggregates at the cell poles at concentrations over the threshold. This research opens promising avenues for studying enzyme solubilitys impact on resistance, creating opportunities for future approaches to counter resistance.

microbiology↗

bioRxiv2021

Electronic Mapping of a Bacterial Genome with Dual Solid-State Nanopores and Active Single-Molecule Control

We present the first electronic mapping of a bacterial genome using solid-state nanopore technology. A dual-nanopore architecture and active control logic are used to produce single-molecule data that enables estimation of distances between physical tags installed at sequence motifs within double-stranded DNA (dsDNA). Previously developed dual-pore "DNA flossing" control generates multiple scans of tagged regions of each captured DNA. The control logic was extended here in two ways: first, to automate "zooming out" on each molecule to progressively increase the number of tags scanned during DNA flossing; and second, to automate recapture of a molecule that exited flossing to enable interrogation of the same and/or different regions of the molecule. New analysis methods were developed to produce consensus alignments from each multi-scan event. The combined multi-scanning and multi-capture method was applied to the challenge of mapping from a heterogeneous mixture of single-molecule fragments that make up the Escherichia coli (E. coli) chromosome. Coverage of 3.1x across 2,355 resolvable sites (68% of reference sites) of the E. coli genome was achieved after 5.6 hours of recording time. The recapture method showed a 38% increase in the merged-event alignment length compared to single-scan alignments. The observed inter-tag resolution was 150 bp in engineered DNA molecules and 166 bp natively within fragments of E. coli DNA, with detection of 133 inter-site intervals shorter than 200 bp in the E. coli reference map. Proof of concept results on estimating distances in repetitive regions of the E. coli genome are also provided. With an appropriately designed array and future refinements to the control logic, higher throughput implementations can enable human-sized genome and epigenome mapping applications.

biophysics↗

bioRxiv2019

Flossing DNA in a Dual Nanopore Device

Solid-state nanopores are a single-molecule technique that can provide access to biomolecular information that is otherwise masked by ensemble averaging. A promising application uses pores and barcoding chemistries to map molecular motifs along single DNA molecules. Despite recent research breakthroughs, however, it remains challenging to overcome molecular noise to fully exploit single molecule data. Here we present an active control technique termed \"flossing\" that uses a dual nanopore device to trap a protein-tagged DNA molecule and perform up to 100s of back-and-forth electrical scans of the molecule in a few seconds. The protein motifs bound to 48 kb{lambda} DNA are used as detectable features for active triggering of the bidirectional control. Molecular noise is suppressed by averaging the multi-scan data to produce averaged inter-tag distance estimates that are comparable to their known values. Since nanopore feature-mapping applications require DNA linearization when passing through the pore, a key advantage of flossing is that trans-pore linearization is increased to >98% by the second scan, compared to 35% for single nanopore passage of the same set of molecules. In concert with barcoding methods, the dual-pore flossing technique could enable genome mapping and structural variation applications, or mapping loci of epigenetic relevance.

biophysics↗