Browse
Submit Data
Databases
API
Help

Dataset Information

18 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Enzymatic digestion of single DNA molecules anchored on nanogold-modified surfaces.

ABSTRACT: To study enzyme-DNA interactions at single molecular level, both the attachment points and the immediate surroundings of surfaces must be carefully considered such that they do not compromise the structural information and biological properties of the sample under investigation. The present work demonstrates the feasibility of enzymatic digestion of single DNA molecules attached to nanoparticle-modified surfaces. With Nanogold linking DNA to the mica surface by electrostatic interactions, advantageous conditions with fewer effects on the length and topography of DNA are obtained, and an appropriate environment for the activities of DNA is created. We demonstrate that by using Dip-Pen Nanolithography, individual DNA molecules attached to modified mica surfaces can be efficiently digested by DNase I.

SUBMITTER: Lu J

PROVIDER: S-EPMC2893842 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Electrocatalytic amplification of DNA-modified nanoparticle collisions <i>via</i> enzymatic digestion.

Project description:We report a new and general approach that will be useful for adapting the method of electrocatalytic amplification (ECA) to biosensing applications. In ECA, individual collisions of catalytic nanoparticles with a noncatalytic electrode surface lead to bursts of current. In the work described here, the current arises from catalytic electrooxidation of N2H4 at the surface of platinum nanoparticles (PtNPs). The problem with using ECA for biosensing applications heretofore, is that it is necessary to immobilize a receptor, such as DNA (as in the case here) or an antibody on the PtNP surface. This inactivates the colliding NP, however, and leads to very small collision signatures. In the present article, we show that single-stranded DNA (ssDNA) present on the PtNP surface can be detected by selectively removing a fraction of the ssDNA using the enzyme Exonuclease I (Exo I). About half of the current associated with collisions of naked PtNPs can be recovered from fully passivated PtNPs after exposure to Exo I. Experiments carried out using both Au and Hg ultramicroelectrodes reveal some mechanistic aspects of the collision process before and after treatment of the ssDNA-modified PtNPs with Exo I.

| S-EPMC5356041 | biostudies-literature

Single-molecule enzymatic conformational dynamics: spilling out the product molecules.

Project description:Product releasing is an essential step of an enzymatic reaction, and a mechanistic understanding primarily depends on the active-site conformational changes and molecular interactions that are involved in this step of the enzymatic reaction. Here we report our work on the enzymatic product releasing dynamics and mechanism of an enzyme, horseradish peroxidase (HRP), using combined single-molecule time-resolved fluorescence intensity, anisotropy, and lifetime measurements. Our results have shown a wide distribution of the multiple conformational states involved in active-site interacting with the product molecules during the product releasing. We have identified that there is a significant pathway in which the product molecules are spilled out from the enzymatic active site, driven by a squeezing effect from a tight active-site conformational state, although the conventional pathway of releasing a product molecule from an open active-site conformational state is still a primary pathway. Our study provides new insight into the enzymatic reaction dynamics and mechanism, and the information is uniquely obtainable from our combined time-resolved single-molecule spectroscopic measurements and analyses.

| S-EPMC4126733 | biostudies-literature

High efficiency acetylcholinesterase immobilization on DNA aptamer modified surfaces.

Project description:We report here the in vitro selection of DNA aptamers for electric eel acetylcholinesterase (AChE). One selected aptamer sequence (R15/19) has a high affinity towards the enzyme (Kd=157±42 pM). Characterization of the aptamer showed its binding is not affected by low ionic strength (~20 mM), however significant reduction in affinity occurred at high ionic strength (~1.2 M). In addition, this aptamer does not inhibit the catalytic activity of AChE that we exploit through immobilization of the DNA on a streptavidin-coated surface. Subsequent immobilization of AChE by the aptamer results in a 4-fold higher catalytic activity when compared to adsorption directly on to plastic.

| S-EPMC6271157 | biostudies-literature

Piezoresistivity in single DNA molecules.

Project description:Piezoresistivity is a fundamental property of materials that has found many device applications. Here we report piezoresistivity in double helical DNA molecules. By studying the dependence of molecular conductance and piezoresistivity of single DNA molecules with different sequences and lengths, and performing molecular orbital calculations, we show that the piezoresistivity of DNA is caused by force-induced changes in the π-π electronic coupling between neighbouring bases, and in the activation energy of hole hopping. We describe the results in terms of thermal activated hopping model together with the ladder-based mechanical model for DNA proposed by de Gennes.

| S-EPMC4569700 | biostudies-literature

Genipin Delays Corneal Stromal Enzymatic Digestion.

Project description:PurposeTo evaluate the use of genipin in delaying enzymatic digestion of corneal stroma.MethodsHuman corneal stromal tissue was treated with genipin, a known chemical crosslinker, and then along with control tissue was subjected to enzymatic digestion with collagenase. The effects of genipin treatment in retarding stromal digestion were analyzed with phase contrast microscopy, a protein quantification assay, second harmonic generation imaging, and transmission electron microscopy.ResultsGenipin increased stromal resistance to enzymatic digestion when compared with untreated stroma. A morphologic analysis and protein quantification showed increased stromal resistance to enzymatic digestion once stromal tissue was treated with genipin. Second harmonic generation imaging revealed persistent fibrillar collagen signaling in genipin-treated tissue in contrast with untreated tissue suggesting that genipin retards collagenolysis.ConclusionsGenipin increases stromal resistance to enzymatic digestion in controlled experiments as demonstrated by protein quantification studies and through morphologic imaging.Translational relevanceThis study explores the novel use of genipin in delaying enzymatic stromal digestion. Delaying stromal melting in the setting of corneal infectious or autoimmune keratitis can potentially decrease clinical morbidity.

| S-EPMC8394563 | biostudies-literature

Enzymatic Polymerization on DNA Modified Gold Nanowire for Label-Free Detection of Pathogen DNA.

Project description:This paper presents a label-free biosensor for the detection of single-stranded pathogen DNA through the target-enhanced gelation between gold nanowires (AuNW) and the primer DNAs branched on AuNW. The target DNA enables circularization of the linear DNA template, and the primer DNA is elongated continuously via rolling circle amplification. As a result, in the presence of the target DNA, a macroscopic hydrogel was fabricated by the entanglement of the elongated DNA with AuNWs as a scaffold fiber for effective gelation. In contrast, very small separate particles were generated in the absence of the target DNA. This label-free biosensor might be a promising tool for the detection of pathogen DNAs without any devices for further analysis. Moreover, the biosensor based on the weaving of AuNW and DNAs suggests a novel direction for the applications of AuNWs in biological engineering.

| S-EPMC4490515 | biostudies-literature

A bio-pen for direct writing of single molecules on user-functionalized surfaces.

Project description:Advancing ultrahigh resolution (below 10 nm) direct writing technologies could lead to impacts in areas as diverse as disease detection, genetic analysis and nanomanufacturing. Current methods based on electron-beams and photo- or dip-pen nanolithography are laborious and lack flexibility when aiming to create single molecule patterns for application specific integration. We hypothesize that a novel strategy could be developed to allow for writing of parallel and yet individually addressable patterns of single molecules on user-controlled surfaces. The strategy is based on using in vitro self-recognition of tubulin protein to assemble rigid protofilaments of microtubules, with one such microtubule to be subsequently used as a "bio-pen" capable of writing "inks" of single kinesin molecules in user-defined environments. Our results show that single kinesin inks could be written under the energy of adenosine triphosphate hydrolysis and observed by both atomic force and optical microscopy. Upon extending ink functionalities, the integration of soft and hard materials for nanostructure assembly and complex single molecule pattern formation is envisioned.

| S-EPMC9417116 | biostudies-literature

Displacement Statistics of Unhindered Single Molecules Show no Enhanced Diffusion in Enzymatic Reactions.

Project description:Recent studies have sparked debate over whether catalytic reactions enhance the diffusion coefficients D of enzymes. Through high statistics of the transient (600 μs) displacements of unhindered single molecules freely diffusing in common buffers, we here quantify D for four enzymes under catalytic turnovers. We thus formulate how ∼ ±1% precisions may be achieved for D, and show no changes in diffusivity for catalase, urease, aldolase, and alkaline phosphatase under the application of wide concentration ranges of substrates. Our single-molecule approach thus overcomes potential limitations and artifacts underscored by recent studies to show no enhanced diffusion in enzymatic reactions.

| S-EPMC8975259 | biostudies-literature

Bacterial DNA Recognition by SERS Active Plasma-Coupled Nanogold.

Project description:It is shown that surface-enhanced Raman spectroscopy (SERS) can identify bacteria based on their genomic DNA composition, acting as a "sample-distinguishing marker". Successful spectral differentiation of bacterial species was accomplished with nanogold aggregates synthesized through single-step plasma reduction of the ionic gold-containing vapored precursor. A high enhancement factor (EF = 107) in truncated coupled plasmonic particulates allowed SERS-probing at nanogram sample quantities. Simulations confirmed the occurrence of the strongest electric field confinement within nanometric gaps between gold dimers/chains from where the molecular fingerprints of bacterial DNA fragments gained photon scattering enhancement. The most prominent Raman modes linked to fundamental base-pair molecular vibrations were deconvoluted and used to proceed with nitrogenous base content estimation. The genomic composition (percentage of guanine-cytosine and adenine-thymine) was successfully validated by third-generation sequencing using nanopore technology, further proving that the SERS technique can be employed to swiftly specify bioentities by the discriminative principal-component statistical approach.

| S-EPMC9756328 | biostudies-literature

Efficient amplification of self-gelling polypod-like structured DNA by rolling circle amplification and enzymatic digestion.

Project description:The application of DNA as a functional material such as DNA hydrogel has attracted much attention. Despite an increasing interest, the high cost of DNA synthesis is a limiting factor for its utilization. To reduce the cost, we report here a highly efficient amplification technique for polypod-like structured DNA (polypodna) with adhesive ends that spontaneously forms DNA hydrogel. Two types of polypodna with three (tripodna) and four (tetrapodna) pods were selected, and a template oligodeoxynucleotide, containing a tandem sequence of a looped tripodna or tetrapodna, respectively, along with restriction enzyme (TspRI) sites, was designed. The template was circularized using T4 DNA ligase, and amplified by rolling circle amplification (RCA). The RCA product was highly viscous and resistant to restriction digestion. Observation under an electron microscope revealed microflower-like structures. These structures were composed of long DNA and magnesium pyrophosphate, and their treatment with EDTA followed by restriction digestion with TspRI resulted in numerous copies of polypodna with adhesive ends, which formed a DNA hydrogel. Thus, we believe this technique provides a new approach to produce DNA nanostructures, and helps in expanding their practical applications.

| S-EPMC4604513 | biostudies-other

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data