Binuclear ruthenium complex linker length tunes DNA threading intercalation kinetics.

Binuclear ruthenium complexes have been investigated for potential DNA-targeted therapeutic and diagnostic applications. Studies of DNA threading intercalation, in which DNA base pairs must be broken for intercalation, have revealed means of optimizing a model binuclear ruthenium complex to obtain reversible DNA-ligand assemblies with the desired properties of high affinity and slow kinetics. Here, we used single-molecule force spectroscopy to study a binuclear ruthenium complex with a longer semi-rigid linker relative to the model complex.

Biological Amyloids Chemically Damage DNA.

Amyloid fibrils are protein polymers noncovalently assembled through β-strands arranged in a cross-β structure. Biological amyloids were considered chemically inert until we and others recently demonstrated their ability to catalyze chemical reactions in vitro. To further explore the functional repertoire of amyloids, we here probe if fibrils of α-synuclein (αS) display chemical reactivity toward DNA.

Design and in vitro anticancer assessment of a click chemistry-derived dinuclear copper artificial metallo-nuclease.

Copper compounds with artificial metallo-nuclease (AMN) activity are mechanistically unique compared to established metallodrugs. Here, we describe the development of a new dinuclear copper AMN, Cu2-BPL-C6 (BPL-C6 = bis-1,10-phenanthroline-carbon-6), prepared using click chemistry that demonstrates site-specific DNA recognition with low micromolar cleavage activity. The BPL-C6 ligand was designed to force two redox-active copper centres-central for enhancing AMN activity-to bind DNA, via two phenanthroline ligands separated by an aliphatic linker.

Sequence-Specific Minor Groove Binders in Labeling and Single-Molecule Analysis of DNA.

The ability to address specific sequences within DNA is of tremendous interest in biotechnology and biomedicine. Various technologies have been established over the past few decades, such as nicking enzymes and methyltransferase-directed sequence-specific labeling, transcription activator-like effector nucleases (TALENs), the CRISPR-Cas9 system, and polyamides of heterocycles as sequence-specific DNA minor groove binders. Pyrrole-imidazole polyamides have been reported to recognize predetermined DNA sequences, and some successful attempts have demonstrated their potential in regulating gene expression.

Force-induced melting and S-DNA pathways for DNA overstretching exhibit distinct kinetics.

It is widely appreciated that double stranded DNA (dsDNA) is subjected to strong and dynamic mechanical forces in cells. Under increasing tension B-DNA, the most stable double-stranded (ds) form of DNA, undergoes cooperative elongation into a mixture of S-DNA and single stranded DNA (ssDNA). Despite significant effort, the structure, energetics, kinetics and the biological role of S-DNA remains obscure.

Comparison of CTX-M encoding plasmids present during the early phase of the ESBL pandemic in western Sweden.

Plasmids encoding bla genes have greatly shaped the evolution of E. coli producing extended-spectrum beta-lactamases (ESBL-E. coli) and adds to the global threat of multiresistant bacteria by promoting horizontal gene transfer (HGT).

Photophysical image analysis: Unsupervised probabilistic thresholding for images from electron-multiplying charge-coupled devices.

We introduce the concept photophysical image analysis (PIA) and an associated pipeline for unsupervised probabilistic image thresholding for images recorded by electron-multiplying charge-coupled device (EMCCD) cameras. We base our approach on a closed-form analytic expression for the characteristic function (Fourier-transform of the probability mass function) for the image counts recorded in an EMCCD camera, which takes into account both stochasticity in the arrival of photons at the imaging camera and subsequent noise induced by the detection system of the camera. The only assumption in our method is that the background photon arrival to the imaging system is described by a stationary Poisson process (we make no assumption about the photon statistics for the signal).

Xrs2/NBS1 promote end-bridging activity of the MRE11-RAD50 complex.

DNA double strand breaks (DSBs) can be detrimental to the cell and need to be efficiently repaired. A first step in DSB repair is to bring the free ends in close proximity to enable ligation by non-homologous end-joining (NHEJ), while the more precise, but less available, repair by homologous recombination (HR) requires close proximity of a sister chromatid. The human MRE11-RAD50-NBS1 (MRN) complex, Mre11-Rad50-Xrs2 (MRX) in yeast, is involved in both repair pathways.

Exploring the conformational dynamics of the SARS-CoV-2 SL4 hairpin by combining optical tweezers and base analogues.

The parasitic nature of the SARS-CoV-2 virus demands selective packaging of its RNA genome (gRNA) from the abundance of other nucleic acids present in infected cells. Despite increasing evidence that stem-loop 4 (SL4) of the gRNA 5' UTR is involved in the initiation of this process by binding the nucleocapsid (N) protein, little is known about its conformational dynamics. Here, we unravel the stability, dynamics and (un)folding pathways of SL4 using optical tweezers and a base analogue, tC, that provides a local and subtle increase in base stacking without perturbing hydrogen bonding.

Probing physical properties of single amyloid fibrils using nanofluidic channels.

Amyloid fibril formation is central to the pathology of many diseases, including neurodegenerative disorders such as Alzheimer's and Parkinson's disease. Amyloid fibrils can also have functional and scaffolding roles, for example in bacterial biofilms, and have also been exploited as useful biomaterials. Despite being linear protein homopolymers, amyloid fibrils can exhibit significant structural and morphological polymorphism, making it relevant to study them on the single fibril level.

Label-Free Imaging of Catalytic HO Decomposition on Single Colloidal Pt Nanoparticles Using Nanofluidic Scattering Microscopy.

Single-particle catalysis aims at determining factors that dictate the nanoparticle activity and selectivity. Existing methods often use fluorescent model reactions at low reactant concentrations, operate at low pressures, or rely on plasmonic enhancement effects. Hence, methods to measure single-nanoparticle activity under technically relevant conditions and without fluorescence or other enhancement mechanisms are still lacking.

Fluorescence Microscopy of Nanochannel-Confined DNA.

Stretching of DNA in nanoscale confinement allows for several important studies. The genetic contents of the DNA can be visualized on the single DNA molecule level, and the polymer physics of confined DNA and also DNA/protein and other DNA/DNA-binding molecule interactions can be explored. This chapter describes the basic steps to fabricate the nanostructures, perform the experiments, and analyze the data.

A Click Chemistry-Based Artificial Metallo-Nuclease.

Artificial metallo-nucleases (AMNs) are promising DNA damaging drug candidates. Here, we demonstrate how the 1,2,3-triazole linker produced by the Cu-catalysed azide-alkyne cycloaddition (CuAAC) reaction can be directed to build Cu-binding AMN scaffolds. We selected biologically inert reaction partners tris(azidomethyl)mesitylene and ethynyl-thiophene to develop TC-Thio, a bioactive C -symmetric ligand in which three thiophene-triazole moieties are positioned around a central mesitylene core.

Strain-level bacterial typing directly from patient samples using optical DNA mapping.

Background: Identification of pathogens is crucial to efficiently treat and prevent bacterial infections. However, existing diagnostic techniques are slow or have a too low resolution for well-informed clinical decisions.

Methods: In this study, we have developed an optical DNA mapping-based method for strain-level bacterial typing and simultaneous plasmid characterisation.

Polymer Brushes on Silica Nanostructures Prepared by Aminopropylsilatrane Click Chemistry: Superior Antifouling and Biofunctionality.

In nanobiotechnology, the importance of controlling interactions between biological molecules and surfaces is paramount. In recent years, many devices based on nanostructured silicon materials have been presented, such as nanopores and nanochannels. However, there is still a clear lack of simple, reliable, and efficient protocols for preventing and controlling biomolecule adsorption in such structures.

Identification and characterization of plasmids carrying the mobile colistin resistance gene using optical DNA mapping.

Objectives: Colistin is a last-resort antibiotic, but there has been a rapid increase in colistin resistance, threatening its use in the treatment of infections with carbapenem-resistant (CRE). Plasmid-mediated colistin resistance, in particular the gene, has been identified and WGS is the go-to method in identifying plasmids carrying genes. The goal of this study is to demonstrate the use of optical DNA mapping (ODM), a fast, efficient and amplification-free technique, to characterize plasmids carrying .

Single-Molecule Trapping and Measurement in a Nanostructured Lipid Bilayer System.

The repulsive electrostatic force between a biomolecule and a like-charged surface can be geometrically tailored to create spatial traps for charged molecules in solution. Using a parallel-plate system composed of silicon dioxide surfaces, we recently demonstrated single-molecule trapping and high precision molecular charge measurements in a nanostructured free energy landscape. Here we show that surfaces coated with charged lipid bilayers provide a system with tunable surface properties for molecular electrometry experiments.

DNA in nanochannels: theory and applications.

Nanofluidic structures have over the last two decades emerged as a powerful platform for detailed analysis of DNA on the kilobase pair length scale. When DNA is confined to a nanochannel, the combination of excluded volume and DNA stiffness leads to the DNA being stretched to near its full contour length. Importantly, this stretching takes place at equilibrium, without any chemical modifications to the DNA.

Fecal carriage and clonal dissemination of blaNDM-1 carrying Klebsiella pneumoniae sequence type 147 at an intensive care unit in Lao PDR.

Objectives: Carbapenemase-producing Enterobacterales (CPE) are high priority targets of global antimicrobial surveillance. Herein, we determined the colonization rate of CPE on admission to intensive care units in Vientiane, Lao PDR in August-September 2019.

Methods: Data regarding clinical conditions, infection control, and antibiotic usage were collected during admission.

Nanofluidic Trapping of Faceted Colloidal Nanocrystals for Parallel Single-Particle Catalysis.

Catalyst activity can depend distinctly on nanoparticle size and shape. Therefore, understanding the structure sensitivity of catalytic reactions is of fundamental and technical importance. Experiments with single-particle resolution, where ensemble-averaging is eliminated, are required to study it.

HnRNPK maintains single strand RNA through controlling double-strand RNA in mammalian cells.

Although antisense transcription is a widespread event in the mammalian genome, double-stranded RNA (dsRNA) formation between sense and antisense transcripts is very rare and mechanisms that control dsRNA remain unknown. By characterizing the FGF-2 regulated transcriptome in normal and cancer cells, we identified sense and antisense transcripts IER3 and IER3-AS1 that play a critical role in FGF-2 controlled oncogenic pathways. We show that IER3 and IER3-AS1 regulate each other's transcription through HnRNPK-mediated post-transcriptional regulation.

A simple cut and stretch assay to detect antimicrobial resistance genes on bacterial plasmids by single-molecule fluorescence microscopy.

Antimicrobial resistance (AMR) is a fast-growing threat to global health. The genes conferring AMR to bacteria are often located on plasmids, circular extrachromosomal DNA molecules that can be transferred between bacterial strains and species. Therefore, effective methods to characterize bacterial plasmids and detect the presence of resistance genes can assist in managing AMR, for example, during outbreaks in hospitals.

Label-free nanofluidic scattering microscopy of size and mass of single diffusing molecules and nanoparticles.

Label-free characterization of single biomolecules aims to complement fluorescence microscopy in situations where labeling compromises data interpretation, is technically challenging or even impossible. However, existing methods require the investigated species to bind to a surface to be visible, thereby leaving a large fraction of analytes undetected. Here, we present nanofluidic scattering microscopy (NSM), which overcomes these limitations by enabling label-free, real-time imaging of single biomolecules diffusing inside a nanofluidic channel.

Organic Anisotropic Excitonic Optical Nanoantennas.

Optical nanoantennas provide control of light at the nanoscale, which makes them important for diverse areas ranging from photocatalysis and flat metaoptics to sensors and biomolecular tweezing. They have traditionally been limited to metallic and dielectric nanostructures that sustain plasmonic and Mie resonances, respectively. More recently, nanostructures of organic J-aggregate excitonic materials have been proposed capable of also supporting nanooptical resonances, although their advance has been hampered from difficulty in nanostructuring.