We demonstrate the assembly of functional hybrid nanopores for single molecule sensing by inserting DNA origami structures into solid-state nanopores. In our experiments, artificial based on are repeatedly inserted in and ejected from with diameters around 15 nm. show that these can be employed detection λ-DNA molecules. Our approach paves way future development adaptable single-molecule nanopore sensors combination self-assembly.

Nanopores can be used to detect and analyze single molecules in solution. We have glass nanopores made by laser-assisted capillary-pulling, as a high-throughput low cost method, range of label-free proteins: lysozyme, avidin, IgG, β-lactoglobulin, ovalbumin, bovine serum albumin (BSA), β-galactosidase Furthermore, we show for the first time solid state nanopore measurements mammalian prion protein, which its abnormal form is associated with transmissible spongiform encephalopathies. Our...

Nanopores are a versatile technique for the detection and characterization of single molecules in solution. An ongoing challenge field is to find methods selectively detect specific biomolecules. In this work we describe new sensing proteins using unmodified solid-state nanopores. We engineered double strand DNA by hybridizing nearly two hundred oligonucleotides linearized version m13mp18 virus genome. This strand, which call carrier, allows positioning protein binding sites at nanometer...

We combine DNA origami structures with glass nanocapillaries to reversibly form hybrid nanopores. Trapping of the onto nanocapillary is proven by imaging fluorescently labeled and simultaneous ionic current measurements trapping events. then show two applications highlighting versatility these First, tuning pore size we can control folding dsDNA molecules ("physical control"). Second, that specific introduction binding sites in nanopore allows selective detection ssDNA as a function sequence...

The condensation of linear diamine and dialdehyde subcomponents around copper(I) templates in the presence bulky trioctylphosphine ancillary ligands gave a linear, conjugated polymeric material DMSO solution. This polymer solution was observed to undergo sol-to-gel transition as temperature raised 140 °C, contrast with behavior most gel-forming polymers, which do so upon cooling. We attribute formation CuIN4 cross-links equilibria 2[CuIN2P2] ⇄ [CuIN4] + [CuPn]+ (4 − n)P favor right-hand side...

Designed "DNA carriers" have been proposed as a new method for nanopore based specific protein detection. In this system, target molecules bind to long DNA strand at defined position creating second level transient current drop against the background translocation. Here, we demonstrate ability of system quantify concentrations in nanomolar range. After incubation with different concentrations, fraction translocations showing secondary spike allows quantification corresponding concentration....

Solid-state nanopores are single-molecule sensors that measure changes in ionic current as charged polymers such DNA pass through. Here, we present comprehensive experiments on the length, voltage, and salt dependence of frequency double-stranded translocations through conical quartz with mean opening diameter 15 nm. We observe an entropic barrier-limited, length-dependent translocation at 4M LiCl concentration a drift-dominated, length-independent 1M KCl concentration. These observations...

In nanopore sensing, changes in ionic current are used to analyse single molecules solution. The translocation dynamics of polyelectrolytes is particular interest given potential applications such as DNA sequencing. this paper, we determine how the voltage driven can be affected by geometry and hence available configurational space for DNA. Using inherent geometrical asymmetry a conically shaped nanopore, examine depends on directionality transport. total time when exiting extended conical...

We report a method for simultaneous ionic current measurements of single molecules across up to 16 solid state nanopore channels. Each device, costing less than $20, contains glass nanopores made by laser assisted capillary pulling. demonstrate multichannel detection double stranded DNA and trapping origami nanostructures form hybrid nanopores.

Nanopore sensors show great potential for rapid, single-molecule determination of DNA sequence information. Here, we develop an ionic current-based method determining the positions short motifs in double-stranded molecules with solid-state nanopores. Using DNA-methyltransferase M.TaqI and a biotinylated S-adenosyl-l-methionine cofactor analogue create covalently attached biotin labels at 5′-TCGA-3′ motifs. Monovalent streptavidin is then added to bind sites giving rise additional current...

A method is established to reliably suspend graphene monolayers across glass nanopores as a simple, low cost platform study ionic transport through membranes. We systematically show that the seals nanopore openings with areas ranging from 180 nm2 20 μm2, allowing detailed measurements of current and graphene. In combination in situ Raman spectroscopy, we characterise defects formed ozone treated graphene, confirming an increase flow defect density. This highlights potential our for studying...

Poly(ADP-ribose) polymerase 1 (PARP1) is an abundant nuclear enzyme that plays important roles in DNA repair, chromatin organization and transcription regulation. Although binding activation of PARP1 by damage sites has been extensively studied, little known about how binds to long stretches undamaged it could shape architecture. Here, using single-molecule techniques, we show condenses undamaged, kilobase-length subject sub-piconewton mechanical forces. Stepwise decondensation at high force...

We demonstrate simultaneous measurements of DNA translocation into glass nanopores using ionic current detection and fluorescent imaging. verify the correspondence between passage a single molecule through nanopore accompanying characteristic blockage. By tracking motion individual molecules in nanocapillary perpendicular to optical axis model, we can extract an effective mobility constant for our geometry under high electric fields.

Poly(ADP-ribose) polymerases (PARPs) play key roles in DNA damage repair pathways eukaryotic cells. Human PARPs 1 and 2 are catalytically activated by the form of both double-strand single-strand breaks. Recent structural work indicates that PARP2 can also bridge two breaks (DSBs), revealing a potential role stabilizing broken ends. In this paper, we have developed magnetic tweezers–based assay order to measure mechanical stability interaction kinetics proteins bridging across ends DSB. We...

We use a resistive-pulse technique to analyze molecular hybrids of single-wall carbon nanotubes (SWNTs) wrapped in either single-stranded DNA or protein. Electric fields confined glass capillary nanopore allow us probe the physical size and surface properties at single-molecule level. find that translocation duration macromolecular hybrid is determined by its hydrodynamic solution mobility. The event current reveals effects ion exclusion rod-shaped possible due temporary polarization SWNT...

The development of catalysts benefits from knowledge the intermediate steps that accelerate transformations substrates into products. However, key transient species are often hidden in ensemble measurements. Here, we show a protein nanoreactor can sample catalytic cycle by continuous single-molecule observation stoichiometric reaction solution. By monitoring changes flow ionic current through an α-hemolysin pore, observed three metal-ligand complexes gold(I)-catalyzed converts acetylenic...

Lysozyme fibrils translocate uncoated glass nanopores at low pH without significant non-specific sticking enabling unprecedented statistics on thousands of fibril translocations and comparison with a simple bulk conductivity model.

We report a simple and efficient way to accomplish the chemical modification of glass nanopores by means lipid self-assembly. Lipid coating improves success rate these as biosensors detect λ-DNA.

Tetrazine- and sydnone-based click reactions have emerged as important bioconjugation strategies with fast kinetics N2 or CO2 the only byproduct. Mechanistic studies of these focused on initial rate-determining cycloaddition steps. The subsequent release from bicyclic intermediates has been approached mainly through computational studies, which predicted lifetimes femtoseconds. In present study, bioorthogonal cycloadditions involving extrusion examined experimentally at single-molecule level...

DNA-protein interactions are vital to cellular function, with key roles in the regulation of gene expression and genome maintenance. Atomic force microscopy (AFM) offers ability visualize at nanometre resolution near-physiological buffers, but it requires that DNA be adhered surface a solid substrate. This presents problem when working biologically relevant protein concentrations, where proteins may present large excess solution; much biophysically information can therefore occluded by...

Abstract Single-molecule techniques such as optical tweezers and fluorescence imaging are powerful tools for probing the biophysics of DNA DNA-protein interactions. The application these methods requires efficient approaches creating designed structures with labels binding to a surface or microscopic beads. In this paper, we develop simple fast technique making diverse range constructs by combining PCR amplicons synthetic oligonucleotides using golden gate assembly rules. We demonstrate high...