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. Nanopore-based sensing of DNA-barcoded nanostructures may help to improve the design of efficient and specific ribonucleoproteins for biomedical applications, and could be developed into sensitive protein-sensing assays.

Current Fluctuations in Nanopores Reveal the Polymer-Wall Adsorption Potential.

Modification of surface properties by polymer adsorption is a widely used technique to tune interactions in molecular experiments such as nanopore sensing. Here, we investigate how the ionic current noise through solid-state nanopores reflects the adsorption of short, neutral polymers to the pore surface. The power spectral density of the noise shows a characteristic change upon adsorption of polymer, the magnitude of which is strongly dependent on both polymer length and salt concentration. In particular, for short polymers at low salt concentrations no change is observed, despite the verification of comparable adsorption in these systems using quartz crystal microbalance measurements. We propose that the characteristic noise is generated by the movement of polymers on and off the surface and perform simulations to assess the feasibility of this model. Excellent agreement with experimental data is obtained using physically motivated simulation parameters, providing deep insight into the shape of the adsorption potential and underlying processes. This paves the way toward using noise spectral analysis for in situ characterization of functionalized nanopores.

Minimally instrumented SHERLOCK (miSHERLOCK) for CRISPR-based point-of-care diagnosis of SARS-CoV-2 and emerging variants.

The COVID-19 pandemic highlights the need for diagnostics that can be rapidly adapted and deployed in a variety of settings. Several SARS-CoV-2 variants have shown worrisome effects on vaccine and treatment efficacy, but no current point-of-care (POC) testing modality allows their specific identification. We have developed miSHERLOCK, a low-cost, CRISPR-based POC diagnostic platform that takes unprocessed patient saliva; extracts, purifies, and concentrates viral RNA; performs amplification and detection reactions; and provides fluorescent visual output with only three user actions and 1 hour from sample input to answer out. miSHERLOCK achieves highly sensitive multiplexed detection of SARS-CoV-2 and mutations associated with variants B.1.1.7, B.1.351, and P.1. Our modular system enables easy exchange of assays to address diverse user needs and can be rapidly reconfigured to detect different viruses and variants of concern. An adjunctive smartphone application enables output quantification, automated interpretation, and the possibility of remote, distributed result reporting.

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.

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.

Comparison of the specificity and affinity of surface immobilised Affimer binders using the quartz crystal microbalance.

To enable multiplexed protein analysis through the use of microarrays, reliable molecules capable of specifically binding to a protein of interest with high affinity are required. Further, this specificity and affinity must be retained upon immobilization to the microarray surface. This study investigates the performance of surface bound Affimer proteins, comparing the affinity and specificity of different binders for closely related immunoglobulin molecules using the quartz crystal microbalance with dissipation monitoring (QCM-D). It is demonstrated that the surface bound Affimer proteins are highly specific, differentiating between their target IgG and other closely related IgG subclasses. The binding affinities of the protein aptamers for their target IgG molecules are determined to be in the nanomolar range, comparable to typical antibody-antigen binding affinities. While measurements herein are done using QCM-D, the high specificity and binding affinities of the surface bound Affimer proteins opens applications in a range of microarray biosensors.

Evaluating the binding of selected biomolecules to cranberry derived proanthocyanidins using the quartz crystal microbalance.

Despite cranberry being associated with the prevention of bacterial infections for over a century, our understanding of the bioavailability and mechanisms by which cranberry prevents infection is limited. This study investigates the interactions between cranberry proanthocyanidins (CPAC) and human serum proteins (albumin, α-1-acid glycoprotein, and fibrinogen) that may be encountered during CPAC metabolism following ingestion. To better understand how CPAC might interfere with bacterial infection, we also examined the interactions between CPAC and selected bacterial virulence factors; namely, lipopolysaccharide (LPS) and rhamnolipid. The binding of CPAC to the serum proteins, rhamnolipids and LPS from Escherichia coli O111:B4 can be described by Langmuir-type isotherms, allowing the determination of the apparent adsorption affinity constants, with CPAC interacting most strongly with fibrinogen with a binding constant of 2.2 × 10(8) M(-1). These binding interactions will limit the bioavailability of the CPAC at the site of action, an important consideration in designing further clinical trials. Furthermore, CPAC interacts with Pseudomonas aeruginosa 10 LPS, E. coli O111:B4 LPS, and P. aeruginosa rhamnolipids in fundamentally different manners, supporting the theory that cranberry prevents bacterial infections via multiple mechanisms.