Full-length single-molecule protein fingerprinting

Proteins are the primary functional actors of the cell. While proteoform diversity is known to be highly biologically relevant, current protein analysis methods are of limited use for distinguishing proteoforms. Mass spectrometric methods, in particular, often provide only ambiguous information on post-translational modification sites, and...

High throughput single-molecule technology

Graphene nano-electromechanical mass sensor with high resolution at room temperature

The inherent properties of 2D materials—light mass, high out-of-plane flexibility, and large surface area—promise great potential for precise and accurate nanomechanical mass sensing, but their application is often hampered by surface contamination. Here we demonstrate a tri-layer graphene nanomechanical resonant mass sensor with sub-attogram...

Characterization of Noise in a Single-Molecule Fluorescence Signal

Single-molecule fluorescence experiments allow monitoring of the structural change and dynamics of a single biomolecule in real time using dye molecules attached to the molecule. Often, the molecules are immobilized on the surface to observe a longer molecular dynamics, yet the finite photon budget available from an individual dye molecule...

FRETboard: semisupervised classification of FRET traces

Förster resonance energy transfer (FRET) is a useful phenomenon in biomolecular investigations, as it can be leveraged for nanoscale measurements. The optical signals produced by such experiments can be analyzed by fitting a statistical model. Several software tools exist to fit such models in an unsupervised manner but lack the flexibility...

Kinetics of DNA looping by Anabaena sensory rhodopsin transducer (ASRT) by using DNA cyclization assay

DNA cyclization assay together with single-molecule FRET was employed to monitor protein-mediated bending of a short dsDNA (~ 100 bp). This method provides a simple and easy way to monitor the structural change of DNA in real-time without necessitating prior knowledge of the molecular structures for the optimal dye-labeling. This assay was...

The emerging landscape of single-molecule protein sequencing technologies

Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside...

High-Resolution Single-Molecule FRET via DNA eXchange (FRET X)

Single-molecule FRET is a versatile tool to study nucleic acids and proteins at the nanometer scale. However, currently, only a couple of FRET pairs can be reliably measured on a single object, which makes it difficult to apply single-molecule FRET for structural analysis of biomolecules. Here, we present an approach that allows for the...

DNA sequence encodes the position of DNA supercoils

The three-dimensional organization of DNA is increasingly understood to play a decisive role in vital cellular processes. Many studies focus on the role of DNA-packaging proteins, crowding, and confinement in arranging chromatin, but structural information might also be directly encoded in bare DNA itself. Here, we visualize plectonemes ...

RecA filament maintains structural integrity using ATP-driven internal dynamics

At the core of homologous DNA repair, RecA catalyzes the strand exchange reaction. This process is initiated by a RecA loading protein, which nucleates clusters of RecA proteins on single-stranded DNA. Each cluster grows to cover the single-stranded DNA but may leave 1- to 2-nucleotide (nt) gaps between the clusters due to three different...

The supercoiling state of DNA determines the handedness of both H3 and CENP-A nucleosomes

Nucleosomes form the unit structure of the genome in eukaryotes, thereby constituting a fundamental tenet of chromatin biology. In canonical nucleosomes, DNA wraps around the histone octamer in a left-handed toroidal ramp. Here, in single-molecule magnetic tweezers studies of chaperone-assisted nucleosome assembly, we show that the handedness...

Intercalation-based single-molecule fluorescence assay to study DNA supercoil dynamics

DNA supercoiling crucially affects cellular processes such as DNA replication, gene expression, and chromatin organization. However, mechanistic understanding of DNA supercoiling and the related DNA-processing enzymes has remained limited, mainly due to the lack of convenient experimental tools to probe these phenomena. Here, we report a...

CENP-A and H3 Nucleosomes Display a Similar Stability to Force-Mediated Disassembly

Centromere-specific nucleosomes are a central feature of the kinetochore complex during mitosis, in which microtubules exert pulling and pushing forces upon the centromere. CENP-A nucleosomes have been assumed to be structurally unique, thereby providing resilience under tension relative to their H3 canonical counterparts. Here, we directly test...

Dynamic Growth and Shrinkage Govern the pH Dependence of RecA Filament Stability

RecA proteins form a long stable filament on a single-stranded DNA and catalyze strand exchange reaction. The stability of RecA filament changes dramatically with pH, yet its detailed mechanism is not known. Here, using a single molecule assay, we determined the binding and dissociation rates of RecA monomers at the filament ends at various pH....

Evaporation-induced flows in confined droplets of diluted aqueous NaCl solution

The flows in a naturally evaporating liquid droplet confined by two flat plates were investigated experimentally. A diluted sodium-chloride (NaCl) aqueous solution was used to form the confined droplets. The evaporation of water at the free surface of the droplet built up a concentration gradient inside the solution, which induced a natural...

Molecular mechanism of sequence-dependent stability of RecA filament

A is a DNA-dependent ATPase and mediates homologous recombination by first forming a filament on a single-stranded (ss) DNA. RecA binds preferentially to TGG repeat sequence, which resembles the recombination hot spot Chi (5'-GCTGGTGG-3') and is the most frequent pattern (GTG) of the codon usage in Escherichia coli. Because of the highly dynamic...

Design of high-speed planing hulls for the improvement of resistance and seakeeping performance

Seismic behaviors of earth-core, concrete-faced-rock-fill, and composite dams

The investigation on the seismic behavior of dams becomes crucial but is limited due to lack of experimental or field data. This paper aims to experimentally simulate the seismic behavior of three types of dams; earth-core rock-fill dam (ECRD), concrete-faced rock-fill dam (CFRD), and composite dam which combines concrete dam and CFRD. A series...