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. The nanobait approach relies on specific target selection via toehold-mediated strand displacement and rapid readout via nanopore sensing. Here we show that this platform can concurrently identify several common respiratory viruses, detecting a panel of short targets of viral nucleic acids from multiple viruses. Our nanobait can be easily reprogrammed to discriminate viral variants with single-nucleotide resolution, as we demonstrated for several key SARS-CoV-2 variants. Last, we show that the nanobait discriminates between samples extracted from oropharyngeal swabs from negative- and positive-SARS-CoV-2 patients without preamplification. Our system allows for the multiplexed identification of native RNA molecules, providing a new scalable approach for the diagnostics of multiple respiratory viruses in a single assay.

The Chromatin Regulator HMGA1a Undergoes Phase Separation in the Nucleus.

The protein high mobility group A1 (HMGA1) is an important regulator of chromatin organization and function. However, the mechanisms by which it exerts its biological function are not fully understood. Here, we report that the HMGA isoform, HMGA1a, nucleates into foci that display liquid-like properties in the nucleus, and that the protein readily undergoes phase separation to form liquid condensates in vitro. By bringing together machine-leaning modelling, cellular and biophysical experiments and multiscale simulations, we demonstrate that phase separation of HMGA1a is promoted by protein-DNA interactions, and has the potential to be modulated by post-transcriptional effects such as phosphorylation. We further show that the intrinsically disordered C-terminal tail of HMGA1a significantly contributes to its phase separation through electrostatic interactions via AT hooks 2 and 3. Our work sheds light on HMGA1 phase separation as an emergent biophysical factor in regulating chromatin structure.

Image Encoding Using Multi-Level DNA Barcodes with Nanopore Readout.

Deoxyribonucleic acid (DNA) nanostructure-based data encoding is an emerging information storage mode, offering rewritable, editable, and secure data storage. Herein, a DNA nanostructure-based storage method established on a solid-state nanopore sensing platform to save and encrypt a 2D grayscale image is proposed. DNA multi-way junctions of different sizes are attached to a double strand of DNA carriers, resulting in distinct levels of current blockades when passing through a glass nanopore with diameters around 14 nm. The resulting quaternary encoding doubles the capacity relative to a classical binary system. Through toehold-mediated strand displacement reactions, the DNA nanostructures can be precisely added to and removed from the DNA carrier. By encoding the image into 16 DNA carriers using the quaternary barcodes and reading them in one simultaneous measurement, the image is successfully saved, encrypted, and recovered. Avoiding any proteins or enzymatic reactions, the authors thus realize a pure DNA storage system on a nanopore platform with increased capacity and programmability.

Multiplexed DNA Identification Using Site Specific dCas9 Barcodes and Nanopore Sensing.

Decorating double-stranded DNA with dCas9 barcodes to identify characteristic short sequences provides an alternative to fully sequencing DNA samples for rapid and highly specific analysis of a DNA sample. Solid state nanopore sensors are especially promising for this type of single-molecule sensing because of the ability to analyze patterns in the ionic current signatures of DNA molecules. Here, we systematically demonstrate the use of highly specific dCas9 probes to create unique barcodes on the DNA that can be read out using nanopore sensors. Single dCas9 probes are targeted to various positions on DNA strands up to 48 kbp long and are effectively measured in high salt conditions typical of nanopore sensing. Multiple probes bound to the same DNA strand at characteristic target sequences create distinct barcodes of double and triple peaks. Finally, double and triple barcodes are used to simultaneously identify two different DNA targets in a background mixture of bacterial DNA. Our method forms the basis of a fast and versatile assay for multiplexed DNA sensing applications in complex samples.

Current Enhancement in Solid-State Nanopores Depends on Three-Dimensional DNA Structure.

The translocation of double-stranded DNA through a solid-state nanopore may either decrease or increase the ionic current depending on the ionic concentration of the surrounding solution. Below a certain crossover ionic concentration, the current change inverts from a current blockade to current enhancement. In this paper, we show that the crossover concentration for bundled DNA nanostructures composed of multiple connected DNA double-helices is lower than that of double-stranded DNA. Our measurements suggest that counterion mobility in the vicinity of DNA is reduced depending on the three-dimensional structure of the molecule. We further demonstrate that introducing neutral polymers such as polyethylene glycol into the measurement solution reduces electroosmotic outflow from the nanopore, allowing translocation of large DNA structures at low salt concentrations. Our experiments contribute to an improved understanding of ion transport in confined DNA environments, which is critical for the development of nanopore sensing techniques as well as synthetic membrane channels. Our salt-dependent measurements of model DNA nanostructures will guide the development of computational models of DNA translocation through nanopores.

Digital Data Storage Using DNA Nanostructures and Solid-State Nanopores.

Solid-state nanopores are powerful tools for reading the three-dimensional shape of molecules, allowing for the translation of molecular structure information into electric signals. Here, we show a high-resolution integrated nanopore system for identifying DNA nanostructures that has the capability of distinguishing attached short DNA hairpins with only a stem length difference of 8 bp along a DNA double strand named the DNA carrier. Using our platform, we can read up to 112 DNA hairpins with a separating distance of 114 bp attached on a DNA carrier that carries digital information. Our encoding strategy allows for the creation of a library of molecules with a size of up to 5 × 1033 (2112) that is only built from a few hundred types of base molecules for data storage and has the potential to be extended by linking multiple DNA carriers. Our platform provides a nanopore- and DNA nanostructure-based data storage method with convenient access and the potential for miniature-scale integration.

Promoting single-file DNA translocations through nanopores using electro-osmotic flow.

Double-stranded DNA translocates through sufficiently large nanopores either in a linear single-file fashion or in a folded hairpin conformation when captured somewhere along its length. We show that the folding state of DNA can be controlled by changing the electrolyte concentration, pH, and polyethylene glycol content of the measurement buffer. At pH 8 in 1M LiCl or 0.35M KCl, single-file translocations make up more than 90% of the total. We attribute the effect to the onset of electro-osmotic flow from the pore at low ionic strength. Our hypothesis on the critical role of flows is supported by the preferred orientation of entry of a strand that has been folded into a multi-helix structure at one end. Control over DNA folding is critical for nanopore sensing approaches that use modifications along a DNA strand and the associated secondary current drops to encode information.

QuipuNet: Convolutional Neural Network for Single-Molecule Nanopore Sensing.

Nanopore sensing is a versatile technique for the analysis of molecules on the single-molecule level. However, extracting information from data with established algorithms usually requires time-consuming checks by an experienced researcher due to inherent variability of solid-state nanopores. Here, we develop a convolutional neural network (CNN) for the fully automated extraction of information from the time-series signals obtained by nanopore sensors. In our demonstration, we use a previously published data set on multiplexed single-molecule protein sensing. The neural network learns to classify translocation events with greater accuracy than previously possible, while also increasing the number of analyzable events by a factor of 5. Our results demonstrate that deep learning can achieve significant improvements in single molecule nanopore detection with potential applications in rapid diagnostics.

Probing the salt dependence of the torsional stiffness of DNA by multiplexed magnetic torque tweezers.

The mechanical properties of DNA fundamentally constrain and enable the storage and transmission of genetic information and its use in DNA nanotechnology. Many properties of DNA depend on the ionic environment due to its highly charged backbone. In particular, both theoretical analyses and direct single-molecule experiments have shown its bending stiffness to depend on salt concentration. In contrast, the salt-dependence of the twist stiffness of DNA is much less explored. Here, we employ optimized multiplexed magnetic torque tweezers to study the torsional stiffness of DNA under varying salt conditions as a function of stretching force. At low forces (<3 pN), the effective torsional stiffness is ∼10% smaller for high salt conditions (500 mM NaCl or 10 mM MgCl2) compared to lower salt concentrations (20 mM NaCl and 100 mM NaCl). These differences, however, can be accounted for by taking into account the known salt dependence of the bending stiffness. In addition, the measured high-force (6.5 pN) torsional stiffness values of C = 103 ± 4 nm are identical, within experimental errors, for all tested salt concentration, suggesting that the intrinsic torsional stiffness of DNA does not depend on salt.

Probing the mechanical properties, conformational changes, and interactions of nucleic acids with magnetic tweezers.

Nucleic acids are central to the storage and transmission of genetic information. Mechanical properties, along with their sequence, both enable and fundamentally constrain the biological functions of DNA and RNA. For small deformations from the equilibrium conformations, nucleic acids are well described by an isotropic elastic rod model. However, external forces and torsional strains can induce conformational changes, giving rise to a complex force-torque phase diagram. This review focuses on magnetic tweezers as a powerful tool to precisely determine both the elastic parameters and conformational transitions of nucleic acids under external forces and torques at the single-molecule level. We review several variations of magnetic tweezers, in particular conventional magnetic tweezers, freely orbiting magnetic tweezers and magnetic torque tweezers, and discuss their characteristic capabilities. We then describe the elastic rod model for DNA and RNA and discuss conformational changes induced by mechanical stress. The focus lies on the responses to torque and twist, which are crucial in the mechanics and interactions of nucleic acids and can directly be measured using magnetic tweezers. We conclude by highlighting several recent studies of nucleic acid-protein and nucleic acid-small-molecule interactions as further applications of magnetic tweezers and give an outlook of some exciting developments to come.