scholarly journals A Unified Sensor Architecture for Isothermal Detection of Double-Stranded DNA, Oligonucleotides, and Small Molecules

ChemBioChem ◽  
2015 ◽  
Vol 16 (5) ◽  
pp. 725-730 ◽  
Author(s):  
Carl W. Brown ◽  
Matthew R. Lakin ◽  
Aurora Fabry-Wood ◽  
Eli K. Horwitz ◽  
Nicholas A. Baker ◽  
...  
Keyword(s):  
Small Molecules ◽  
Dna Oligonucleotides ◽  
Double Stranded Dna

scholarly journals Cover Picture: A Unified Sensor Architecture for Isothermal Detection of Double-Stranded DNA, Oligonucleotides, and Small Molecules (ChemBioChem 5/2015)

ChemBioChem ◽  
2015 ◽  
Vol 16 (5) ◽  
pp. 697-697
Author(s):  
Carl W. Brown ◽  
Matthew R. Lakin ◽  
Aurora Fabry-Wood ◽  
Eli K. Horwitz ◽  
Nicholas A. Baker ◽  
...  
Keyword(s):  
Small Molecules ◽  
Dna Oligonucleotides ◽  
Double Stranded Dna

Single-Molecule Conductance Measurements of Single- and Double-Stranded DNA Oligonucleotides

ChemPhysChem ◽  
2006 ◽  
Vol 7 (1) ◽  
pp. 94-98 ◽  
Author(s):  
Harm van Zalinge ◽  
David J. Schiffrin ◽  
Andrew D. Bates ◽  
Wolfgang Haiss ◽  
Jens Ulstrup ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Oligonucleotides ◽  
Double Stranded Dna

scholarly journals Phosphorothioate-modified DNA oligonucleotides inactivate CRISPR-Cpf1 mediated genome editing

2018 ◽  
Author(s):  
Bin Li ◽  
Chunxi Zeng ◽  
Wenqing Li ◽  
Xinfu Zhang ◽  
Xiao Luo ◽  
...  
Keyword(s):  
Genome Editing ◽  
Adaptive Immune System ◽  
Dependent Manner ◽  
Dna Breaks ◽  
Dna Oligonucleotides ◽  
Double Stranded Dna ◽  
Modified Dna ◽  
Target Dna ◽  
Editing Activity ◽  
Dose Dependent

CRISPR-Cpf1, a microbial adaptive immune system discovered from Prevotella and Francisella 1, employs a single-stranded CRISPR RNA (crRNA) to induce double stranded DNA breaks1. To modulate genome editing activity of Cpf1 in human cells, we designed a series of crRNA variants including DNA-crRNA and RNA-crRNA duplexes, and identified that phosphorothioate (PS)-modified DNA-crRNA duplex completely blocked the function of Cpf1 mediated gene editing. More importantly, without prehybridization, this PS-modified DNA was able to regulate Cpf1 activity in a time-and dose-dependent manner. Mechanistic studies indicate that PS-modified DNA oligonucleotides hinder the binding between Cpf1-crRNA complex and target DNA substrate. Consequently, phosphorothioate-modified DNA oligonucleotides provide a tunable platform to inactivate Cpf1 mediated genome editing.

Interaction of oligonucleotides with benzo[c]phenanthridine alkaloid sanguilutine

2013 ◽  
Vol 67 (5) ◽  
Author(s):  
Stanislava Rybáková ◽  
Michal Rájecký ◽  
Jana Urbanová ◽  
Kristýna Pěnčíková ◽  
Eva Táborská ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Luminescence Spectroscopy ◽  
Base Pairs ◽  
Dna Oligonucleotides ◽  
Sanguinaria Canadensis ◽  
Double Stranded Dna ◽  
Low Concentrations ◽  
Intensity Response ◽  
Heterogeneous Base ◽  
Positively Charged

AbstractBenzo[c]phenanthridine alkaloid sanguilutine was extracted and purified from the dried roots of Sanguinaria canadensis. The interaction of the positively charged iminium form of alkaloid with double-stranded DNA oligonucleotides was studied using luminescence spectroscopy. The results showed that the interaction with various double-stranded oligonucleotides was not specific to A-T or G-C base pairs; also, no preference was found for either homogeneous or heterogeneous base composition of strands. The association constants were calculated to be in the range of (1.31–14.36) × 105 M−1. The luminescence intensity response at 610 nm to low concentrations of double-stranded DNA was found to be linear and can potentially be used for the fluorometric quantification of DNA. The limit of detection was estimated to be 120 ng mL−1 of DNA (calculated by 3σ method).

scholarly journals Recombineering Using RecTE from Pseudomonas syringae

2010 ◽  
Vol 76 (15) ◽  
pp. 4960-4968 ◽  
Author(s):  
Bryan Swingle ◽  
Zhongmeng Bao ◽  
Eric Markel ◽  
Alan Chambers ◽  
Samuel Cartinhour
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Syringae ◽  
Recombination Frequency ◽  
Quantitative Assay ◽  
Tomato Dc3000 ◽  
Linear Dna ◽  
Dna Oligonucleotides ◽  
Double Stranded Dna ◽  
Genes Encoding ◽  
Genomic Recombination

ABSTRACT In this report, we describe the identification of functions that promote genomic recombination of linear DNA introduced into Pseudomonas cells by electroporation. The genes encoding these functions were identified in Pseudomonas syringae pv. syringae B728a based on similarity to the lambda Red Exo/Beta and RecET proteins encoded by the lambda and Rac bacteriophages of Escherichia coli. The ability of the pseudomonad-encoded proteins to promote recombination was tested in P. syringae pv. tomato DC3000 using a quantitative assay based on recombination frequency. The results show that the Pseudomonas RecT homolog is sufficient to promote recombination of single-stranded DNA oligonucleotides and that efficient recombination of double-stranded DNA requires the expression of both the RecT and RecE homologs. Additionally, we illustrate the utility of this recombineering system to make targeted gene disruptions in the P. syringae chromosome.

Combined fluorescent and gold nucleic acid probes

1994 ◽  
Vol 52 ◽  
pp. 176-177
Author(s):  
Richard D. Powell ◽  
James F. Hainfeld ◽  
Mair E. A. Churchill ◽  
Andrew S. Belmont
Keyword(s):  
Fluorescence Microscopy ◽  
Small Molecules ◽  
Membrane Separation ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Specific Staining ◽  
Nucleic Acid Probes ◽  
Ovary Cells ◽  
Double Stranded Dna ◽  
Amine Groups

Site-specific probes which incorporate both fluorophores and the 1.4 nm “Nanogold” gold particle have been prepared (Fig. 1). Specimens labeled with these probes may be imaged by fluorescence microscopy, then examined at higher resolution in the electron microscope without an additional gold-labeling step: this allows better specimen preservation and unprecedented correlation between the two sets of complementary data. In addition, the covalent gold attachment procedure allows the preparation of electron microscopy probes using small molecules, which cannot be coupled to colloidal gold.Two types of probes have been prepared. In the first, the Nanogold particle was covalently attached, via peripheral primary amine groups, to Hoescht-33258, a fluorescent dye which is specific for AT-rich regions of double-stranded DNA. Excess Hoescht-33258 was removed by membrane separation. The resulting reagent was tested in Chinese hamster ovary cells: results are shown in Figs. 2 and 3. Specific staining of the nucleus was observed both by fluorescence microscopy and in the light microscope, using silver enhancement (Danscher's procedure) to visualize the gold-labeled regions.

scholarly journals Specificity and Efficiency of the Uracil DNA Glycosylase-Mediated Strand Cleavage Surveyed on Large Sequence Libraries

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Kathrin Hölz ◽  
Angelina Pavlic ◽  
Jory Lietard ◽  
Mark M. Somoza
Keyword(s):  
Large Scale ◽  
Life Forms ◽  
Dna Glycosylase ◽  
Detailed Knowledge ◽  
Sequence Specificity ◽  
Uracil Dna Glycosylase ◽  
Cytosine Deamination ◽  
Dna Oligonucleotides ◽  
Double Stranded Dna ◽  
Molecular Biotechnology

AbstractUracil-DNA glycosylase (UDG) is a critical DNA repair enzyme that is well conserved and ubiquitous in nearly all life forms. UDG protects genomic information integrity by catalyzing the excision from DNA of uracil nucleobases resulting from misincorporation or spontaneous cytosine deamination. UDG-mediated strand cleavage is also an important tool in molecular biotechnology, allowing for controlled and location-specific cleavage of single- and double DNA chemically or enzymatically synthesized with single or multiple incorporations of deoxyuridine. Although the cleavage mechanism is well-understood, detailed knowledge of efficiency and sequence specificity, in both single and double-stranded DNA contexts, has so far remained incomplete. Here we use an experimental approach based on the large-scale photolithographic synthesis of uracil-containing DNA oligonucleotides to comprehensively probe the context-dependent uracil excision efficiency of UDG.

Label-free detection of double-stranded DNA molecules with polyelectrolyte-modified capacitive field-effect sensors

2017 ◽  
Vol 84 (10) ◽  
Author(s):  
Thomas S. Bronder ◽  
Arshak Poghossian ◽  
Michael Keusgen ◽  
Michael J. Schöning
Keyword(s):  
Field Effect ◽  
Deoxyribonucleic Acid ◽  
Label Free ◽  
Electrical Detection ◽  
Dna Oligonucleotides ◽  
Double Stranded Dna ◽  
Polyelectrolyte Layer ◽  
Target Dna ◽  
Label Free Detection ◽  
Positively Charged

AbstractIn this study, polyelectrolyte-modified field-effect-based electrolyte-insulator-semiconductor (EIS) devices have been used for the label-free electrical detection of double-stranded deoxyribonucleic acid (dsDNA) molecules. The sensor-chip functionalization with a positively charged polyelectrolyte layer provides the possibility of direct adsorptive binding of negatively charged target DNA oligonucleotides onto the SiO

scholarly journals Integration of diverse DNA substrates by a casposase can be targeted to R-loops in vitro by its fusion to Cas9

2021 ◽  
Vol 41 (1) ◽  
Author(s):  
Chun Hang Lau ◽  
Edward L. Bolt
Keyword(s):  
Protein Complexes ◽  
Dna Integration ◽  
Synthetic Dna ◽  
Methanosarcina Mazei ◽  
Dna Oligonucleotides ◽  
Double Stranded Dna ◽  
Dna Insertion ◽  
Dna Capture ◽  
Target Molecules

Abstract CRISPR systems build adaptive immunity against mobile genetic elements by DNA capture and integration catalysed by Cas1–Cas2 protein complexes. Recent studies suggested that CRISPR repeats and adaptation module originated from a novel type of DNA transposons called casposons. Casposons encode a Cas1 homologue called casposase that alone integrates into target molecules single and double-stranded DNA containing terminal inverted repeats (TIRs) from casposons. A recent study showed Methanosarcina mazei casposase is able to integrate random DNA oligonucleotides, followed up in this work using Acidoprofundum boonei casposase, from which we also observe promiscuous substrate integration. Here we first show that the substrate flexibility of Acidoprofundum boonei casposase extends to random integration of DNA without TIRs, including integration of a functional gene. We then used this to investigate targeting of the casposase-catalysed DNA integration reactions to specific DNA sites that would allow insertion of defined DNA payloads. Casposase–Cas9 fusions were engineered that were catalytically proficient in vitro and generated RNA-guided DNA integration products from short synthetic DNA or a gene, with or without TIRs. However, DNA integration could still occur unguided due to the competing background activity of the casposase moiety. Expression of Casposase-dCas9 in Escherichia coli cells effectively targeted chromosomal and plasmid lacZ revealed by reduced β-galactosidase activity but DNA integration was not detected. The promiscuous substrate integration properties of casposases make them potential DNA insertion tools. The Casposase–dCas9 fusion protein may serves as a prototype for development in genetic editing for DNA insertion that is independent of homology-directed DNA repair.

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