Biology subjects

Keyser, U. F.

Publications and source records attributed to Keyser, U. F..

At least 19 recordsLinked to original sources

bioRxiv2026

Extreme Slowdown of dsDNA and RNA:DNA Hybrids in Solid-state Nanopores for LiCl concentrations above 4 mol/L

Solid-state nanopores enable single-molecule detection and characterization through ionic-current measurements, but rapid translocation can limit the resolution of molecular features. Here, we investigated the translocation of 7.2 kb dsDNA and 3.3 kb RNA:DNA hybrids at LiCl concentrations extending to 7 mol/L. Both constructs exhibited a substantial increase of {gtrsim} 500% in mean translocation time when increasing LiCl concentration from 2--3 M to 7 M . We also observed a decrease in translocation frequency at increasing LiCl concentration. Both observations indicate a reduction in the effective electric driving force. Further analysis indicates a substantial decrease in the relative width of secondary peaks induced by labels on the molecule. Our results show that increasing salt concentration beyond 4 mol/L is an accessible way for increasing translocation time in solid-state nanopore sensing, with possible implications for barcode sensing resolution.

biophysics↗

bioRxiv2025

Nanopore-based profiling of PEGylation in nucleic acid therapeutics

Nucleic acid therapeutics, including aptamers, offer effective strategies for programmable and targeted disease treatment. To improve their stability and circulation time, oligonucleotides are often conjugated to hydrophilic polymers such as polyethylene glycol (PEG). However, current bulk techniques fail to resolve PEG heterogeneity, especially in complex biological environments. Here, we use nanopore sensing to quantify PEG conjugation efficiency of the FDA-approved RNA aptamer pegaptanib. We assembled DNA nanostructures that bind pegaptanib and then we used solid-state nanopores to quantify pegaptanib PEGylation. We further assess pegaptanib PEGylation and stability in a serum background and demonstrate that nanopore sensing resolves PEG moieties of distinct molecular weights within oligonucleotide conjugates. Single-molecule profiling of polymer-RNA conjugates enables iterative improvements in oligonucleotide design and provides a direct means to assess their stability in complex biological environments, thereby advancing the development of more effective nucleic acid therapeutics.

bioengineering↗

bioRxiv2025

Quantification of disease-associated RNA tandem repeats by nanopore sensing

Short tandem repeat expansions underlie a class of neurological and neuromuscular diseases known as repeat expansion disorders (REDs), yet the precise characterization of these repeats remains technically challenging. Conventional amplification-based methods fail to resolve repeat length accurately due to amplification bias and sequence homogeneity. Here, we present a single-molecule nanopore-based strategy that enables direct quantification of tandem repeats in native RNA. By assembling RNA:DNA nanostructures that encode specific repeat number, we achieve repeat size discrimination with a resolution of 18 nucleotides. Using tandem repeat-containing RNA, we successfully detect and discriminate disease-relevant repeat lengths associated with myotonic dystrophy types 1 (DM1) and 2 (DM2), and congenital central hypoventilation syndrome-1 (CCHS1). Finally, we apply our method to total RNA extracted from a DM1 human cell line model, demonstrating its compatibility with complex biological samples. Our approach offers a platform for studying repeat expansion biology at the single-molecule level, with broad implications for diagnostics, clinical research and multiplexed repeat profiling.

biochemistry↗

bioRxiv2024

One pot RNA:DNA assembly for ribosomal RNA detection of pathogenic bacteria with single-molecule sensitivity

Ribosomal RNAs (rRNAs) serve as species-defining markers and undergo processing steps such as excision of intervening sequences (IVSs). Direct analysis of native rRNAs is hampered by amplification-induced biases and by the high conservation of rRNA sequences, which complicates discrimination of closely related variants. Here, we present modular RNA:DNA nanostructures that enable direct, amplification-free identification of rRNAs and their variants. The approach employs rationally designed RNA:DNA duplexes, named RNA identifiers (IDs), assembled onto native rRNAs via short complementary oligonucleotides bearing programmable coding motifs. We demonstrate that native bacterial 16S rRNAs can be directly converted into RNA IDs and detected with solid-state nanopores. Having established direct rRNA readout, we next show that biologically encoded rRNA processing states, including serovar-specific 23S rRNA fragmentation patterns arising from IVS excision, are resolved using RNA IDs. Finally, to extend discrimination beyond processing-level differences, we incorporate catalytically inactive Cas9 ribonucleoprotein complexes to enable single-nucleotide discrimination of rRNA variants. Our modular RNA ID-nanopore system facilitates studying rRNA processing and rRNA diversity.

biophysics↗

bioRxiv2023

Nanopore translocation reveals electrophoretic force on A- and B-form nucleic acids

Electrophoretic transport plays a pivotal role in advancing sensing technologies. So far, systematic studies have focused on translocation of canonical B-form or A-form nucleic acids, while direct RNA analysis is emerging as the new frontier for nanopore sensing and sequencing. Here, we compare the less-explored dynamics of non-canonical RNA:DNA hybrids in electrophoretic transport with the well-researched transport of B-form DNA. Using DNA/RNA nanotechnology and solid-state nanopores, the translocation of RNA:DNA (RD) and DNA:DNA (DD) duplexes was examined. Notably, RD duplexes were found to translocate through nanopores faster than DD duplexes, despite containing the same number of base pairs. Our experiments reveal that RD duplexes present a non-canonical helix with distinct transport properties from B-form DD molecules. We find RD and DD molecules with the same contour length move with comparable velocity through nanopores. We examined the physical characteristics of both duplex forms using atomic force microscopy, atomistic molecular dynamics simulations, agarose gel electrophoresis, and dynamic light scattering measurements. With the help of coarse-grained and molecular dynamics simulations, we find the effective force per unit length applied by the electric field to a fragment of RD or DD duplex in nanopores with various geometries or shapes to be approximately the same within experimental errors. Our results shed light on the significance of helical form in nucleic acid translocation, with implications for RNA sensing, sequencing, and molecular understanding of electrophoretic transport.

biophysics↗

bioRxiv2023

Multiplexed Digital Characterisation of Misfolded Protein Oligomers via Solid-State Nanopores

Misfolded protein oligomers are of central importance in both the detection and treatment of Alzheimers and Parkinsons diseases. However, accurate high-throughput methods to identify and quantify oligomer populations are currently lacking. We present here a single-molecule approach for the detection of oligomeric species. The approach is based on the use of solid state nanopores and multiplexed DNA barcoding to identify and characterise oligomers from multiple samples. We study -synuclein oligomers in the presence of several small molecule inhibitors of -synuclein aggregation, as an illustration of the applicability of this method to assist the development of diagnostic and therapeutic methods for Parkinsons disease.

biophysics↗

bioRxiv2023

Single-Molecule RNA Sizing Enables Quantitative Analysis of Alternative Transcription Termination

Transcription, a critical process in molecular biology, has found many applications in RNA synthesis, including mRNA vaccines and RNA therapeutics. However, current RNA characterization technologies suffer from amplification and enzymatic biases that lead to loss of native information. Here, we introduce a strategy to quantitatively study both transcription and RNA polymerase behaviour by sizing RNA with RNA nanotechnology and nanopores. To begin, we utilized T7 RNA polymerase to transcribe linear DNA lacking termination sequences. Surprisingly, we discovered alternative transcription termination in the origin of replication sequence. Next, we employed circular DNA without transcription terminators to perform rolling circle transcription. This allowed us to gain valuable insights into the processivity and transcription behaviour of RNA polymerase at the single-molecule level. Our work demonstrates how RNA nanotechnology and nanopores may be used in tandem for the direct and quantitative analysis of RNA transcripts. This methodology provides a promising pathway for accurate RNA structural mapping by enabling the study of full-length RNA transcripts at the single-molecule level.

biophysics↗

bioRxiv2022

Spatially Multiplexed Single-molecule Translocations through a Nanopore at Controlled Speeds

Nanopores are one of the most successful label-free single-molecule techniques with several sensing applications such as biological screening, diagnostics, DNA and protein sequencing1-4. In current nanopore technologies, stochastic processes influence both the selection of the translocating molecule, translocation rate and translocation velocity5,6. As a result, single-molecule translocations are difficult to control spatially and temporally. Here we present a novel method where we engineer precise spatial and temporal control into the single-molecule experiment. We use a glass nanopore mounted on a 3D nanopositioner to spatially select molecules, deterministically tethered on a glass surface, for controlled translocations. By controlling the distance between the nanopore and the glass surface, we can actively select the region of interest on the molecule and scan it a controlled number of times and at controlled velocity. Decreasing the velocity and averaging thousands of consecutive readings of the same molecule increases the signal-to-noise ratio (SNR) by two orders of magnitude compared to free translocations. We applied our method to various DNA constructs, achieving down to single nucleotide gap resolution. The spatial multiplexing combined with the sub-nanometer resolution could be used in conjunction with micro-array technologies to enable screening of DNA, improving point of care devices, or enabling high-density, addressable DNA data storage.

biophysics↗

bioRxiv2021

Single-molecule sizing through nano-cavity confinement

An approach relying on nano-cavity confinement is developed in this paper for the sizing of nanoscale particles and single biomolecules in solution. The approach, termed nano-cavity diffusional sizing (NDS), measures particle residence times within fluidic nano-cavities to determine their hydrodynamic radii. Using theoretical modeling and simulation, we show that the residence time of particles within nano-cavities above a critical timescale depends on the diffusion coefficient of the particle, which allows estimation of the particles size. We demonstrate this approach experimentally through measurement of particle residence times within nano-fluidic cavities using single-molecule confocal microscopy. Our data show that the residence times scale linearly with the sizes of nanoscale colloids, protein aggregates and single DNA oligonucleotides. NDS thus constitutes a new single molecule optofluidic approach that allows rapid and quantitative sizing of nanoscale particles for potential application in nanobiotechnology, biophysics, and clinical diagnostics. Table of content graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/472369v2_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@15abf7eorg.highwire.dtl.DTLVardef@d2180dorg.highwire.dtl.DTLVardef@1290f57org.highwire.dtl.DTLVardef@ae2087_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

2-photon-fabricated nano-fluidic traps for extended detection of single macromolecules and colloids in solution

The analysis of nanoscopic species, such as proteins and colloidal assemblies, at the single-molecule level has become vital in many areas of fundamental and applied research. Approaches to increase the detection timescales for single molecules in solution without immobilising them onto a substrate surface and applying external fields are much sought after. Here we present an easy-to-implement and versatile nanofluidics-based approach that enables increased observational-timescale analysis of single biomacromolecules and nanoscale colloids in solution. We use two-photon-based hybrid lithography in conjunction with soft lithography to fabricate nanofluidic devices with nano-trapping geometries down to 100 nm in height. We provide a rigorous description and characterisation of the fabrication route that enables the writing of nanoscopic 3D structures directly in photoresist and allows for the integration of nano-trapping and nano-channel geometries within micro-channel devices. Using confocal fluorescence burst detection, we validated the functionality of particle confinement in our nano-trap geometries through measurement of particle residence times. All species under study, including nanoscale colloids, -synuclein oligomers, and double-stranded DNA, showed a three to five-fold increase in average residence time in the detection volume of nano-traps, due to the additional local steric confinement, in comparison to free space diffusion in a nearby micro-channel. Our approach thus opens-up the possibility for single-molecule studies at prolonged observational timescales to analyse and detect nanoparticles and protein assemblies in solution without the need for surface immobilisation.

biophysics↗

bioRxiv2021

Effective Cell Membrane Tension is Independent of Substrate Stiffness

Most animal cells are surrounded by a cell membrane and an underlying actomyosin cortex. Both structures are linked, and they are under tension. In-plane membrane tension and cortical tension both influence many cellular processes, including cell migration, division, and endocytosis. However, while actomyosin tension is regulated by substrate stiffness, how membrane tension responds to mechanical substrate properties is currently poorly understood. Here, we probed the effective membrane tension of neurons and fibroblasts cultured on glass and polyacrylamide substrates of varying stiffness using optical tweezers. In contrast to actomyosin-based traction forces, both peak forces and steady state tether forces of cells cultured on hydrogels were independent of substrate stiffness and did not change after blocking myosin II activity using blebbistatin, indicating that tether and traction forces are not directly linked. Peak forces in fibroblasts on hydrogels were about twice as high as those in neurons, indicating stronger membrane-cortex adhesion in fibroblasts. Steady state tether forces were generally higher in cells cultured on hydrogels than on glass, which we explain by a mechanical model. Our results provide new insights into the complex regulation of effective membrane tension and pave the way for a deeper understanding of the biological processes it instructs.

biophysics↗

bioRxiv2021

Nanopore microscope identifies RNA isoforms with structural colors

Identifying RNA transcript isoforms requires intricate protocols that suffer from various enzymatic biases. Here we design three-dimensional molecular constructs that enable identification of transcript isoforms at the single-molecule level using solid-state nanopore microscopy. We refold target RNA into RNA identifiers (IDs) with designed sets of complementary DNA strands. Each reshaped molecule carries a unique sequence of structural (pseudo)colors. Structural colors consist of DNA structures, protein labels, native RNA structures, or a combination of all three. The sequence of structural colors of RNA IDs enables simultaneous identification and relative quantification of multiple RNA targets without prior amplification. Our Amplification-free RNA TargEt Multiplex Isoform Sensing (ARTEMIS) reveals structural arrangements in native transcripts in agreement with published variants. ARTEMIS discriminates circular and linear transcript isoforms in a one step, enzyme-free reaction in a complex human transcriptome using single-molecule readout. One sentence summaryHere we show enzyme-free identification and relative quantification of RNA isoforms using a nanopore microscope and structural colors.

biophysics↗

bioRxiv2021

An ultrasensitive microfluidic approach reveals correlations between the physico-chemical and biological activity of experimental peptide antibiotics

Antimicrobial resistance challenges the ability of modern medicine to contain infections. Given the dire need for new antimicrobials, peptide antibiotics hold particular promise. These agents hit multiple targets in bacteria starting with their most exposed regions - their membranes. However, suitable assays to quantify the efficacy of peptide antibiotics at the membrane and cellular level have been lacking. Here, we employ two complementary microfluidic platforms to probe the structure-activity relationships of two experimental series of peptide antibiotics. We reveal strong correlations between each peptides physicochemical activity at the membrane level and biological activity at the cellular level by assaying the membranolytic activities of the antibiotics on hundreds of individual giant lipid vesicles, and quantifying phenotypic responses within clonal bacterial populations with single-cell resolution. Our strategy proved capable of detecting differential responses for peptides with single amino acid substitutions between them, and can accelerate the rational design and development of peptide antimicrobials.

microbiology↗

bioRxiv2021

DNA Origami Voltage Sensors for Transmembrane Potentials with Single-Molecule Sensitivity

Signal transmission in neurons goes along with changes in the transmembrane potential. To report them, different approaches including optical voltage-sensing dyes and genetically encoded voltage indicators have evolved. Here, we present a DNA nanotechnology-based system. Using DNA origami, we incorporate and optimize different properties such as membrane targeting and voltage sensing modularly. As a sensing unit, we use a hydrophobic red dye anchored to the membrane and an anionic green dye at the DNA connecting the DNA origami and the membrane dye anchor. Voltage-induced displacement of the anionic donor unit is read out by changes of Fluorescence Resonance Energy Transfer (FRET) of single sensors attached to liposomes. They show a FRET change of [~]5% for {Delta}{Psi}=100 mV and allow adapting the potential range of highest sensitivity. Further, the working mechanism is rationalized by molecular dynamics simulations. Our approach holds potential for the application as non-genetically encoded sensors at membranes.

biophysics↗

bioRxiv2021

Measuring thousands of single vesicle leakage events reveals the mode of action of antimicrobial peptides

Host defense or antimicrobial peptides hold promise for providing new pipelines of effective antimicrobial agents. Their activity quantified against model phospholipid membranes is fundamental to a detailed understanding of their structure-activity relationships. However, existing characterization assays lack the resolution necessary to achieve this insight. Leveraging a highly parallelized microfluidic platform for trapping and studying thousands of giant unilamellar vesicles, we conducted quantitative long-term microscopy studies to monitor the membrane-disruptive activity of archetypal antimicrobial peptides with a high spatiotemporal resolution. We described the modes of action of these peptides via measurements of the disruption of the vesicle population under the conditions of continuous peptide dosing using a range of concentrations, and related the observed modes with the molecular activity mechanisms of these peptides. The study offers an effective approach for characterizing membrane-targeting antimicrobial agents in a standardized manner, and for assigning specific modes of action to the corresponding antimicrobial mechanisms.

biophysics↗

bioRxiv2021

Membrane activity of a DNA-based ion channel depends on the stability of its double-stranded structure.

Structural DNA nanotechnology has emerged as a promising method for designing spontaneously-inserting and fully-controllable synthetic ion channels. However, both insertion efficiency and stability of existing DNA-based ion channels leave much room for improvement. Here, we demonstrate an approach to overcoming the unfavorable DNA-lipid interactions that hinder the formation of a stable transmembrane pore. Our all-atom MD simulations and experiments show that the insertion-driving cholesterol modifications, when introduced at an end of a DNA strand, are likely to cause fraying of the terminal base pairs as the DNA nanostructure adopts its energy-minimum configuration in the membrane. We also find that fraying of base pairs distorts nicked DNA constructs when embedded in a lipid bilayer. Here, we show that DNA nanostructures that do not have discontinuities (nicks) in their DNA backbones form considerably more stable DNA-induced conductive pores and insert into lipid membranes with a higher efficiency than the equivalent nicked constructs. Moreover, lack of nicks allows to design and maintain membrane-spanning helices in a tilted orientation within lipid bilayer. Thus, reducing the conformational degrees of freedom of the DNA nanostructures enables better control over their function as synthetic ion channels.

synthetic biology↗

bioRxiv2020

3D DNA structural barcode copying and random access

Three-dimensional (3D) DNA nanostructures built via DNA self-assembly have established recent applications in multiplexed biosensing and storing digital information. However, a key challenge is that 3D DNA structures are not easily copied which is of vital importance for their large-scale production and for access to desired molecules by target-specific amplification. Here, we build 3D DNA structural barcodes and demonstrate the copying and random access of the barcodes from a library of molecules using a modified polymerase chain reaction (PCR). The 3D barcodes were assembled by annealing a single-stranded DNA scaffold with complementary short oligonucleotides containing 3D protrusions at defined locations. DNA nicks in these structures are ligated to facilitate barcode copying using PCR. To randomly access a target from a library of barcodes, we employ a non-complementary end in the DNA construct that serves as a barcode-specific primer template. Readout of the 3D DNA structural barcodes was performed with nanopore measurements. Our study provides a roadmap for convenient production of large quantities of self-assembled 3D DNA nanostructures. In addition, this strategy offers access to specific targets, a crucial capability for multiplexed single-molecule sensing and for DNA data storage.

biophysics↗

bioRxiv2019

Secure data storage on DNA hard drives

DNA is emerging as a novel material for digital data storage. The two main challenges are efficient encoding and data security. Here, we develop an approach that allows for writing and erasing data by relying solely on Watson-Crick base pairing of short oligonucleotides to single-stranded DNA overhangs located along a long double-stranded DNA hard drive (DNA-HD). Our enzyme-free system enables fast synthesis-free data writing with predetermined building blocks. The use of DNA base pairing allows for secure encryption on DNA-HDs that requires a physical key and nanopore sensing for decoding. The system is suitable for miniature integration for an end-to-end DNA storage device. Our study opens a novel pathway for rewritable and secure data storage with DNA. One Sentence SummaryStoring digital information on molecules along DNA hard drives for rewritable and secure data storage.

biophysics↗