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 utilize T7 RNA polymerase to transcribe linear DNA lacking termination sequences. Surprisingly, we discover alternative transcription termination in the origin of replication sequence. Next, we employ circular DNA without transcription terminators to perform rolling circle transcription. This allows 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.

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. The DNA nanotechnology-based sensor switches from an "open" into a "closed" state when a target strand hybridizes to two sequence-specific sensing overhangs. The loop in the DNA pulls two groups of dumbbells together. The change in topology results in an easily recognized peak in the current trace. Simultaneous detection of four different sequences was achieved by assembling four DNA dumbbell nanoswitches on one carrier. The high specificity of the dumbbell nanoswitch was verified by distinguishing single base variants in DNA and RNA targets using four barcoded carriers in multiplexed measurements. By combining multiple dumbbell nanoswitches with barcoded DNA carriers, we identified different bacterial species even with high sequence similarity by detecting strain specific 16S ribosomal RNA (rRNA) fragments.

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.

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. Each reshaped molecule carries a unique sequence of structural (pseudo)colours. Structural colours consist of DNA structures, protein labels, native RNA structures or a combination of all three. The sequence of structural colours of RNA identifiers enables simultaneous identification and relative quantification of multiple RNA targets without prior amplification. Our Amplification-free RNA TargEt Multiplex Isoform Sensing (ARTEMIS) method 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 read-out.

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. Specifically, we split a GQ into two asymmetric parts extended with sequences complementary to the target. The longer G-segment is in solution, and the shorter one is on a DNA carrier. If the target is present, the two separate GQ parts will be brought together to facilitate the split GQ formation and enhance the nanopore signal. We demonstrated detection of multiple target sequences from different viruses with low crosstalk. Given the programmability of this DNA based nanopore sensing platform, it is promising in biosensing.

Lifetime of glass nanopores in a PDMS chip for single-molecule sensing.

Nanopore sensing is an emerging technology that has many biosensing applications ranging from DNA sequencing using biological pores to biomolecular analysis using solid-state pores. Solid-state nanopores that are more stable are an attractive choice for biosensing applications. Still, biomolecule interactions with the nanopore surface reduce nanopore stability and increase usage costs. In this study, we investigated the biosensing capability for 102 quartz glass nanopores with a diameter of 11-18 nm that were fabricated using laser-assisted capillary pulling. Nanopores were assembled into multiple microfluidic chips that were repeatedly used for up to 19 weeks. We find that using vacuum storage combined with minimal washing steps improved the number of use cycles for nanopores. The single-molecule biosensing capability over repeated use cycles was demonstrated by quantitative analysis of a DNA carrier designed for detection of short single-stranded DNA oligonucleotides.

Toward single-molecule proteomics.

Nanopore rereading of single proteins opens a pathway to next-generation proteomics.

FeII 4L4 tetrahedron binds and aggregates DNA G-quadruplexes.

Since the discovery of the G-quadruplex (G4) structure in telomeres in 1980s, studies have established the role it plays in various biological processes. Here we report binding between DNA G4 and a self-assembled tetrahedral metal-organic cage 1 and consequent formation of aggregates, whereby the cage protects the DNA G4 from cleavage by S1 nuclease. We monitor DNA-cage interaction using fluorescence spectroscopy, firstly by quenching of a fluorescent label appended to the 5' end of G4. Secondly, we detect the decrease in fluorescence of the G4-selective dyes thioflavin-T and Zn-PPIX bound to various DNA G4 sequences following the addition of cage 1. Our results demonstrate that 1 interacts with a wide range of G4s. Moreover, gel electrophoresis, circular dichroism and dynamic light scattering measurements establish the binding of 1 to G4 and indicate the formation of aggregate structures. Finally, we find that DNA G4 contained in an aggregate of cage 1 is protected from cleavage by S1 nuclease.

Kinetics of Toehold-Mediated DNA Strand Displacement Depend on FeII4L4 Tetrahedron Concentration.

The toehold-mediated strand displacement reaction (SDR) is a powerful enzyme-free tool for molecular manipulation, DNA computing, signal amplification, etc. However, precise modulation of SDR kinetics without changing the original design remains a significant challenge. We introduce a new means of modulating SDR kinetics using an external stimulus: a water-soluble FeII4L4 tetrahedral cage. Our results show that the presence of a flexible phosphate group and a minimum toehold segment length are essential for FeII4L4 binding to DNA. SDRs mediated by toehold ends in different lengths (3-5) were investigated as a function of cage concentration. Their reaction rates all first increased and then decreased as cage concentration increased. We infer that cage binding on the toehold end slows SDR, whereas the stabilization of intermediates that contain two overhangs accelerates SDR. The tetrahedral cage thus serves as a versatile tool for modulation of SDR kinetics.

Nanopore-Based DNA Hard Drives for Rewritable and Secure Data Storage.

Nanopores are powerful single-molecule tools for label-free sensing of nanoscale molecules including DNA that can be used for building designed nanostructures and performing computations. Here, DNA hard drives (DNA-HDs) are introduced based on DNA nanotechnology and nanopore sensing as a rewritable molecular memory system, allowing for storing, operating, and reading data in the changeable three-dimensional structure of DNA. Writing and erasing data are significantly improved compared to previous molecular storage systems by employing controllable attachment and removal of molecules on a long double-stranded DNA. Data reading is achieved by detecting the single molecules at the millisecond time scale using nanopores. The DNA-HD also ensures secure data storage where the data can only be read after providing the correct physical molecular keys. Our approach allows for easy-writing and easy-reading, rewritable, and secure data storage toward a promising miniature scale integration for molecular data storage and computation.

A pseudomolecule-scale genome assembly of the liverwort Marchantia polymorpha.

Marchantia polymorpha has recently become a prime model for cellular, evo-devo, synthetic biological, and evolutionary investigations. We present a pseudomolecule-scale assembly of the M. polymorpha genome, making comparative genome structure analysis and classical genetic mapping approaches feasible. We anchored 88% of the M. polymorpha draft genome to a high-density linkage map resulting in eight pseudomolecules. We found that the overall genome structure of M. polymorpha is in some respects different from that of the model moss Physcomitrella patens. Specifically, genome collinearity between the two bryophyte genomes and vascular plants is limited, suggesting extensive rearrangements since divergence. Furthermore, recombination rates are greatest in the middle of the chromosome arms in M. polymorpha like in most vascular plant genomes, which is in contrast with P. patens where recombination rates are evenly distributed along the chromosomes. Nevertheless, some other properties of the genome are shared with P. patens. As in P. patens, DNA methylation in M. polymorpha is spread evenly along the chromosomes, which is in stark contrast with the angiosperm model Arabidopsis thaliana, where DNA methylation is strongly enriched at the centromeres. Nevertheless, DNA methylation and recombination rate are anticorrelated in all three species. Finally, M. polymorpha and P. patens centromeres are of similar structure and marked by high abundance of retroelements unlike in vascular plants. Taken together, the highly contiguous genome assembly we present opens unexplored avenues for M. polymorpha research by linking the physical and genetic maps, making novel genomic and genetic analyses, including map-based cloning, feasible.

Monitoring G-Quadruplex Formation with DNA Carriers and Solid-State Nanopores.

G-quadruplexes (Gqs) are guanine-rich DNA structures formed by single-stranded DNA. They are of paramount significance to gene expression regulation, but also drug targets for cancer and human viruses. Current ensemble and single-molecule methods require fluorescent labels, which can affect Gq folding kinetics. Here we introduce, a single-molecule Gq nanopore assay (smGNA) to detect Gqs and kinetics of Gq formation. We use ∼5 nm solid-state nanopores to detect various Gq structural variants attached to designed DNA carriers. Gqs can be identified by localizing their positions along designed DNA carriers, establishing smGNA as a tool for Gq mapping. In addition, smGNA allows for discrimination of (un)folded Gq structures, provides insights into single-molecule kinetics of Gq folding, and probes quadruplex-to-duplex structural transitions. smGNA can elucidate the formation of Gqs at the single-molecule level without labeling and has potential implications on the study of these structures both in single-stranded DNA and in genomic samples.

Nanomechanics of Extracellular Vesicles Reveals Vesiculation Pathways.

Extracellular vesicles (EVs) are emerging as important mediators of cell-cell communication as well as potential disease biomarkers and drug delivery vehicles. However, the mechanical properties of these vesicles are largely unknown, and processes leading to microvesicle-shedding from the plasma membrane are not well understood. Here an in depth atomic force microscopy force spectroscopy study of the mechanical properties of natural EVs is presented. It is found that several natural vesicles of different origin have a different composition of lipids and proteins, but similar mechanical properties. However, vesicles generated by red blood cells (RBC) at different temperatures/incubation times are different mechanically. Quantifying the lipid content of EVs reveals that their stiffness decreases with the increase in their protein/lipid ratio. Further, by maintaining RBC at "extreme" nonphysiological conditions, the cells are pushed to utilize different vesicle generation pathways. It is found that RBCs can generate protein-rich soft vesicles, possibly driven by protein aggregation, and low membrane-protein content stiff vesicles, likely driven by cytoskeleton-induced buckling. Since similar cortical cytoskeleton to that of the RBC exists on the membranes of most mammalian cells, our findings help advancing the understanding of the fundamental process of vesicle generation.

Supported Planar Mammalian Membranes as Models of in Vivo Cell Surface Architectures.

Emerging technologies use cell plasma membrane vesicles or "blebs" as an intermediate to form molecularly complete, planar cell surface mimetics that are compatible with a variety of characterization tools and microscopy methods. This approach enables direct incorporation of membrane proteins into supported lipid bilayers without using detergents and reconstitution and preserves native lipids and membrane species. Such a system can be advantageous as in vitro models of in vivo cell surfaces for study of the roles of membrane proteins as drug targets in drug delivery, host-pathogen interactions, tissue engineering, and many other bioanalytical and sensing applications. However, the impact of methods used to induce cell blebbing (vesiculation) on protein and membrane properties is still unknown. This study focuses on characterization of cell blebs created under various bleb-inducing conditions and the result on protein properties (orientation, mobility, activity, etc.) and lipid scrambling in this platform. The orientation of proteins in the cell blebs and planar bilayers is revealed using a protease cleavage assay. Lipid scrambling in both cell blebs and planar bilayers is indicated through an annexin V binding assay. To quantify protein confinement, immobility, etc., incorporation of GPI-linked yellow fluorescent protein (GPI-YFP) was used in conjunction with single-molecule tracking (SMT) microscopy. Finally, to investigate the impact of the bleb induction method on protein activity and expression level, cell blebs expressing human aminopeptidase N (hAPN) were analyzed by an enzyme activity assay and immunoblotting. This work enriches our understanding of cell plasma membrane bleb bilayers as a biomimetic platform, reveals conditions under which specific properties are met, and represents one of the few ways to make molecularly complete supported bilayers directly from cell plasma membranes.