Nanopore Translocation Reveals Electrophoretic Force on Noncanonical RNA:DNA Double Helix.

Electrophoretic transport plays a pivotal role in advancing sensing technologies. So far, systematic studies have focused on the 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 noncanonical RNA:DNA hybrids in electrophoretic transport to the well-researched transport of B-form DNA.

Chemical Annealing Restructures RNA for Nanopore Detection.

RNA is a key biochemical marker, yet its chemical instability and complex secondary structure hamper its integration into DNA nanotechnology-based sensing platforms. Relying on the denaturation of the native RNA structure using urea, we show that restructured DNA/RNA hybrids can readily be prepared at room temperature. Using solid-state nanopore sensing, we demonstrate that the structures of our DNA/RNA hybrids conform to the design at the single-molecule level.

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.

DNA Carrier-Assisted Molecular Ping-Pong in an Asymmetric Nanopore.

Nanopore analysis relies on ensemble averaging of translocation signals obtained from numerous molecules, requiring a relatively high sample concentration and a long turnaround time from the sample to results. The recapture and subsequent re-reading of the same molecule is a promising alternative that enriches the signal information from a single molecule. Here, we describe how an asymmetric nanopore improves molecular ping-pong by promoting the recapture of the molecule in the trans reservoir.

Multiplexed Digital Characterization of Misfolded Protein Oligomers via Solid-State Nanopores.

Misfolded protein oligomers are of central importance in both the diagnosis and treatment of Alzheimer's and Parkinson's diseases. However, accurate high-throughput methods to detect and quantify oligomer populations are still needed. We present here a single-molecule approach for the detection and quantification of oligomeric species.

Replication-induced DNA secondary structures drive fork uncoupling and breakage.

Sequences that form DNA secondary structures, such as G-quadruplexes (G4s) and intercalated-Motifs (iMs), are abundant in the human genome and play various physiological roles. However, they can also interfere with replication and threaten genome stability. Multiple lines of evidence suggest G4s inhibit replication, but the underlying mechanism remains unclear.

Nanopore translocation reveals electrophoretic force on non-canonical RNA:DNA double helix.

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.

Sensing the DNA-mismatch tolerance of catalytically inactive Cas9 via barcoded DNA nanostructures in solid-state nanopores.

Single-molecule quantification of the strength and sequence specificity of interactions between proteins and nucleic acids would facilitate the probing of protein-DNA binding. Here we show that binding events between the catalytically inactive Cas9 ribonucleoprotein and any pre-defined short sequence of double-stranded DNA can be identified by sensing changes in ionic current as suitably designed barcoded linear DNA nanostructures with Cas9-binding double-stranded DNA overhangs translocate through solid-state nanopores. We designed barcoded DNA nanostructures to study the relationships between DNA sequence and the DNA-binding specificity, DNA-binding efficiency and DNA-mismatch tolerance of Cas9 at the single-nucleotide level.

DNA Volume, Topology, and Flexibility Dictate Nanopore Current Signals.

Nanopores have developed into powerful single-molecule sensors capable of identifying and characterizing small polymers, such as DNA, by electrophoretically driving them through a nanoscale pore and monitoring temporary blockades in the ionic pore current. However, the relationship between nanopore signals and the physical properties of DNA remains only partly understood. Herein, we introduce a programmable DNA carrier platform to capture carefully designed DNA nanostructures.

Diversity of dynamic voltage patterns in neuronal dendrites revealed by nanopipette electrophysiology.

Dendrites and dendritic spines are the essential cellular compartments in neuronal communication, conveying information through transient voltage signals. Our understanding of these compartmentalized voltage dynamics in fine, distal neuronal dendrites remains poor due to the difficulties inherent to accessing and stably recording from such small, nanoscale cellular compartments for a sustained time. To overcome these challenges, we use nanopipettes that permit long and stable recordings directly from fine neuronal dendrites.

Amplified Plasmonic Forces from DNA Origami-Scaffolded Single Dyes in Nanogaps.

Developing highly enhanced plasmonic nanocavities allows direct observation of light-matter interactions at the nanoscale. With DNA origami, the ability to precisely nanoposition single-quantum emitters in ultranarrow plasmonic gaps enables detailed study of their modified light emission. By developing protocols for creating nanoparticle-on-mirror constructs in which DNA nanostructures act as reliable and customizable spacers for nanoparticle binding, we reveal that the simple picture of Purcell-enhanced molecular dye emission is misleading.

Multiplexed Nanopore-Based Nucleic Acid Sensing and Bacterial Identification Using DNA Dumbbell Nanoswitches.

Multiplexed nucleic acid sensing methods with high specificity are vital for clinical diagnostics and infectious disease control, especially in the postpandemic era. Nanopore sensing techniques have developed in the past two decades, offering versatile tools for biosensing while enabling highly sensitive analyte measurements at the single-molecule level. Here, we establish a nanopore sensor based on DNA dumbbell nanoswitches for multiplexed nucleic acid detection and bacterial identification.

Single-Molecule Sizing through Nanocavity Confinement.

An approach relying on nanocavity confinement is developed in this paper for the sizing of nanoscale particles and single biomolecules in solution. The approach, termed nanocavity diffusional sizing (NDS), measures particle residence times within nanofluidic cavities to determine their hydrodynamic radii. Using theoretical modeling and simulations, we show that the residence time of particles within nanocavities above a critical time scale depends on the diffusion coefficient of the particle, which allows the estimation of the particle's size.

Effective cell membrane tension is independent of polyacrylamide 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.

Simultaneous identification of viruses and viral variants with programmable DNA nanobait.

Respiratory infections are the major cause of death from infectious disease worldwide. Multiplexed diagnostic approaches are essential as many respiratory viruses have indistinguishable symptoms. We created self-assembled DNA nanobait that can simultaneously identify multiple short RNA targets.

Super-Resolution Detection of DNA Nanostructures Using a Nanopore.

High-resolution analysis of biomolecules has brought unprecedented insights into fundamental biological processes and dramatically advanced biosensing. Notwithstanding the ongoing resolution revolution in electron microscopy and optical imaging, only a few methods are presently available for high-resolution analysis of unlabeled single molecules in their native states. Here, label-free electrical sensing of structured single molecules with a spatial resolution down to single-digit nanometers is demonstrated.

Emerging Approaches to DNA Data Storage: Challenges and Prospects.

With the total amount of worldwide data skyrocketing, the global data storage demand is predicted to grow to 1.75 × 10 GB by 2025. Traditional storage methods have difficulties keeping pace given that current storage media have a maximum density of 10 GB/mm.

Parallel DNA circuits by autocatalytic strand displacement and nanopore readout.

DNA nanotechnology provides a unique opportunity for molecular computation, with strand displacement reactions enabling controllable reorganization of nanostructures. Additional DNA strand exchange strategies with high selectivity for input will enable novel complex systems including biosensing applications. Herein, we propose an autocatalytic strand displacement (ACSD) circuit: initiated by DNA breathing and accelerated by a seesaw catalytic reaction, ACSD ensures that only the correct base sequence starts the catalytic cycle.

DNA-Based Optical Quantification of Ion Transport across Giant Vesicles.

Accurate measurements of ion permeability through cellular membranes remains challenging due to the lack of suitable ion-selective probes. Here we use giant unilamellar vesicles (GUVs) as membrane models for the direct visualization of mass translocation at the single-vesicle level. Ion transport is indicated with a fluorescently adjustable DNA-based sensor that accurately detects sub-millimolar variations in K concentration.

Nanopore microscope identifies RNA isoforms with structural colours.

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 with designed sets of complementary DNA strands.

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, classical characterization assays often lack the ability to achieve this insight.

Split G-Quadruplexes Enhance Nanopore Signals for Simultaneous Identification of Multiple Nucleic Acids.

Assembly of DNA structures based on hybridization like split G-quadruplex (GQ) have great potential for the base-pair specific identification of nucleic acid targets. Herein, we combine multiple split G-quadruplex (GQ) assemblies on designed DNA nanostructures (carrier) with a solid-state nanopore sensing platform. The split GQ probes recognize various nucleic acid sequences in a parallel assay that is based on glass nanopore analysis of molecular structures.

Observing capture with a colloidal model membrane channel.

We use video microscopy to study the full capture process for colloidal particles transported through microfluidic channels by a pressure-driven flow. In particular, we obtain trajectories for particles as they move from the bulk into confinement, using these to map in detail the spatial velocity and concentration fields for a range of different flow velocities. Importantly, by changing the height profiles of our microfluidic devices, we consider systems for which flow profiles in the channel are the same, but flow fields in the reservoir differ with respect to the quasi-2D monolayer of particles.

3D flow field measurements outside nanopores.

We demonstrate a non-stereoscopic, video-based particle tracking system with optical tweezers to study fluid flow in 3D in the vicinity of glass nanopores. In particular, we used the quadrant interpolation algorithm to extend our video-based particle tracking to displacements out of the trapping plane of the tweezers. This permitted the study of flow from nanopores oriented at an angle to the trapping plane, enabling the mounting of nanopores on a micromanipulator with which it was then possible to automate the mapping procedure.