scholarly journals Bisulfite treatment and single-molecule real-time sequencing reveal D-loop length, position, and distribution

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Shanaya Shital Shah ◽  
Stella R Hartono ◽  
Frédéric Chédin ◽  
Wolf-Dietrich Heyer
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Detection Methods ◽  
Loop Formation ◽  
Vitro Assay ◽  
Bisulfite Treatment ◽  
Loop Detection ◽  
D Loop ◽  
Donor Choice

Displacement loops (D-loops) are signature intermediates formed during homologous recombination. Numerous factors regulate D-loop formation and disruption, thereby influencing crucial aspects of DNA repair, including donor choice and the possibility of crossover outcome. While D-loop detection methods exist, it is currently unfeasible to assess the relationship between D-loop editors and D-loop characteristics such as length and position. Here, we developed a novel in vitro assay to characterize the length and position of individual D-loops with near base-pair resolution and deep coverage, while also revealing their distribution in a population. Non-denaturing bisulfite treatment modifies the cytosines on the displaced strand of the D-loop to uracil, leaving a permanent signature for the displaced strand. Subsequent single-molecule real-time sequencing uncovers the cytosine conversion patch as a D-loop footprint. The D-loop Mapping Assay is widely applicable with different substrates and donor types and can be used to study factors that influence D-loop properties.

scholarly journals Non-denaturing bisulfite treatment and single-molecule real-time sequencing reveals d-loop footprints, their length, position and distribution

2020 ◽  
Author(s):  
Shanaya Shital Shah ◽  
Stella Hartono ◽  
Frédéric Chédin ◽  
Wolf-Dietrich Heyer
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Detection Methods ◽  
Loop Formation ◽  
Bisulfite Treatment ◽  
Loop Detection ◽  
D Loop ◽  
Donor Choice ◽  
The Relationship

ABSTRACTDisplacement loops (D-loops) are signature intermediates formed during homologous recombination. Numerous factors regulate D-loop formation and disruption, thereby influencing crucial aspects of DNA repair, including donor choice and the possibility of a crossover outcome. While D-loop detection methods exist, it is currently unfeasible to assess the relationship between D-loop editors and D-loop characteristics such as length and position. Here, we developed a novel in vitro assay to characterize the length and position of individual D-loop with base-pair resolution and deep coverage, while also revealing their distribution in a population. Non-denaturing bisulfite treatment modifies the cytosines on the displaced strand of the D-loop to uracil, leaving a permanent signature for the displaced strand. Subsequent single-molecule real-time sequencing uncovers the cytosine conversion patch as a D-loop footprint, revealing D-loop characteristics at unprecedented resolution. The D-loop Mapping Assay is widely applicable with different substrates and donor types and can be used to study factors that influence D-loop properties.

scholarly journals Protein binding to a single termination-associated sequence in the mitochondrial DNA D-loop region.

1993 ◽  
Vol 13 (4) ◽  
pp. 2162-2171 ◽  
Author(s):  
C S Madsen ◽  
S C Ghivizzani ◽  
W W Hauswirth
Keyword(s):  
Mitochondrial Dna ◽  
Protein Binding ◽  
Loop Formation ◽  
Sequence Element ◽  
Conserved Sequence ◽  
Loop Region ◽  
In Organello ◽  
Close Proximity ◽  
D Loop

A methylation protection assay was used in a novel manner to demonstrate a specific bovine protein-mitochondrial DNA (mtDNA) interaction within the organelle (in organello). The protected domain, located near the D-loop 3' end, encompasses a conserved termination-associated sequence (TAS) element which is thought to be involved in the regulation of mtDNA synthesis. In vitro footprinting studies using a bovine mitochondrial extract and a series of deleted mtDNA templates identified a approximately 48-kDa protein which binds specifically to a single TAS element also protected within the mitochondrion. Because other TAS-like elements located in close proximity to the protected region did not footprint, protein binding appears to be highly sequence specific. The in organello and in vitro data, together, provide evidence that D-loop formation is likely to be mediated, at least in part, through a trans-acting factor binding to a conserved sequence element located 58 bp upstream of the D-loop 3' end.

scholarly journals Single-molecule Mechanical Analysis of Strand Invasion in Human Telomere DNA

2021 ◽  
Author(s):  
Terren Chang ◽  
Xi Long ◽  
Shankar Shastry ◽  
Joseph William Parks ◽  
Michael D Stone
Keyword(s):  
Single Molecule ◽  
Mechanical Analysis ◽  
Magnetic Tweezers ◽  
Repeat Sequence ◽  
Single Stranded Dna ◽  
Telomere Repeat ◽  
D Loop ◽  
Human Telomere ◽  
Strand Invasion

Telomeres are essential chromosome end capping structures that safeguard the genome from dangerous DNA processing events. DNA strand invasion occurs during vital transactions at telomeres, including telomere length maintenance by the alternative lengthening of telomeres (ALT) pathway. During telomeric strand invasion, a single stranded guanine-rich (G-rich) DNA invades at a complimentary duplex telomere repeat sequence forming a displacement loop (D-loop) in which the displaced DNA consists of the same G-rich sequence as the invading single stranded DNA. Single stranded G-rich telomeric DNA readily folds into stable, compact, structures called G-quadruplexes (GQ) in vitro, and is anticipated to form within the context of a D-loop; however, evidence supporting this hypothesis is lacking. Here we report a magnetic tweezers assay that permits the controlled formation of telomeric D-loops (TDLs) within uninterrupted duplex human telomere DNA molecules of physiologically relevant lengths. Our results are consistent with a model wherein the displaced single stranded DNA of a TDL folds into a GQ. This study provides new insight into telomere structure and establishes a framework for development of novel therapeutics designed to target GQs at telomeres in cancer cells.

scholarly journals 5’ modifications to CRISPR Cas9 gRNA can change the dynamics and size of R-loops and inhibit DNA cleavage

2020 ◽  
Author(s):  
Grace Mullally ◽  
Kara van Aelst ◽  
Mohsin M. Naqvi ◽  
Fiona M. Diffin ◽  
Tautvydas Karvelis ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Cleavage ◽  
Rna Aptamers ◽  
Loop Formation ◽  
Cleavage Activity ◽  
Dna Cleavage Activity ◽  
Rnp Complex ◽  
Target Binding ◽  
The Impact

A key aim in exploiting CRISPR-Cas is the engineering of gRNA to introduce additional functionalities, ranging from small nucleotide changes that increase efficiency of on-target binding to the inclusion of large functional RNA aptamers and ribonucleoproteins (RNPs. Interactions between gRNA and Cas9 are crucial for RNP complex assembly but several distinct regions of the gRNA are amenable to modification. Using a library of modified gRNAs, we used in vitro ensemble and single-molecule assays to assess the impact of RNA structural alterations on RNP complex formation, R-loop dynamics, and endonuclease activity. Our results indicate that R-loop formation and DNA cleavage activity are essentially unaffected by gRNA modifications of the Upper Stem, first Hairpin and 3’ end. In contrast, 5’ additions of only two or three nucleotides reduced R-loop formation and cleavage activity of the RuvC domain relative to a single nucleotide addition. Such gRNA modifications are a common by-product of in vitro transcribed gRNA. We also observed that addition of a 20 nt RNA hairpin to the 5’ end supported formation of a stable ~9 bp R-loop that could not activate DNA cleavage. These observations will assist in successful gRNA design.

Single Molecule Force Spectroscopy Maps to Study Receptors Clustering

2001 ◽  
Vol 7 (S2) ◽  
pp. 860-861
Author(s):  
R. Bhatia ◽  
N. Almqvist ◽  
S. Banerjee ◽  
G. Primbs ◽  
N. Desai ◽  
...  
Keyword(s):  
Cell Surface ◽  
Real Time ◽  
Single Molecule ◽  
Regional Distribution ◽  
Cellular Membrane ◽  
Angiogenic Factor ◽  
Extracellular Region ◽  
Force Volume ◽  
Endothelial Growth Factor

An atomic force microscope (AFM) allows molecular resolution imaging of hydrated specimens. However, it is often limited in providing identity of the imaged structures, especially in a complex system such as a cellular membrane. Cell surface macromolecules such as ion channels and receptors serve as the interface between the cytoplasm and the extracellular region and toward which many regulatory signals are directed. Their density, distribution and clustering are key spatial features influencing effective and proper physiological responses. We used a method that uses AFM “force-volume maps” to identify and map regional distribution as well as ligand-, or antibody-induced real-time clustering of receptors on the cell surface. This technique also allows simultaneous imaging of the resultant changes in cellular micromechanical properties, such as elasticity and cytoskeletal reorganization of the cell. As an appropriate physiological sample, we have examined spatial distribution and real-time clustering of VEGFR, the receptor for vascular endothelial growth factor which is an important angiogenic factor in human and animal tissues.We have used AFM probes conjugated with anti-VEGFR-antibody (anti-Flk-1 antibody) to examine binding (or unbinding) forces between VEGF-R2 (Flk-1) in both in vitro as well as in live endothelial cells. A quantal set of binding and unbinding forces was measured between the antibody conjugated to the AFM tip and purified VEGFRs adsorbed on to a mica surface (Fig 1). The unbinding force varied between 60 and 240 pN and was a multiple of discrete quantized strength of approximately 60 pN (Figure 1B).

Detection of alpha-methylacyl-CoA racemase (AMACR), a biomarker of prostate cancer, in patient blood samples using a nanoparticle electrochemical biosensor.

2012 ◽  
Vol 30 (30_suppl) ◽  
pp. 41-41
Author(s):  
Matthew M. Cooney ◽  
Cheryl L. Thompson ◽  
Po-Yuan Lin ◽  
Kai-Lun Cheng ◽  
James D. McGuffin-Cawley ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cancer Patients ◽  
High Sensitivity ◽  
Intraepithelial Neoplasia ◽  
Detection Methods ◽  
Assay Method ◽  
Vitro Assay ◽  
Single Use ◽  
Detection And Diagnosis

41 Background: A promising prostate cancer biomarker, alpha-methylacyl-CoA racemase (AMACR), has demonstrated the ability to distinguish cancer from healthy and benign cells with high sensitivity and specificity. However the lack of a good clinical assay has limited its translation into the clinic. Here we report on the development of a single use disposable biosensor for AMACR detection. Methods: This is a very inexpensive, small, single-use disposable sensor that requires only a drop of plasma and connects to a portable device. The biosensor utilizes the reaction of pristanic acid with a substrate that includes AMACR to produce Trans-2,3-dehydropritanayl-CoA plus H2O2. The biosensor utilizes iridium oxide nanoparticle catalyst to oxidize the H2O2 produced in the above metabolic pathway. Thus the oxidation of H2O2 yields a measurable current to quantify AMACR in the sample. This is the first in vitro assay method for AMACR based on this reaction mechanism. Results: In our study including plasma from 9 healthy males, 10 patients with high grade prostatic intraepithelial neoplasia and 5 prostate cancer patients we show 100% accuracy in separating prostate cancer patients from controls as well as those with benign prostate conditions. Conclusions: Our data provides strong evidence for the ability of this biosensor to perform as a reliable assay for prostate cancer detection and diagnosis.

Evaluation of an In Vitro Bioassay for the Detection of Purified Ricin and Castor Bean in Beverages and Liquid Food Matrices

2007 ◽  
Vol 70 (10) ◽  
pp. 2377-2382 ◽  
Author(s):  
JENNIFER L. BRZEZINSKI ◽  
DAVID L. CRAFT
Keyword(s):  
Castor Bean ◽  
Colorimetric Assay ◽  
Cell Damage ◽  
Jurkat Cells ◽  
Biologically Active ◽  
Detection Methods ◽  
Mouse Bioassay ◽  
Animal Testing ◽  
Vitro Assay

The potential use of ricin as a biological weapon in food highlights the necessity for the development of food-specific detection methods. Current methods for the detection of ricin consist of various immunoassays, which detect only one subunit of the ricin toxin and therefore may not be indicative of a biologically active molecule. An in vivo assay, such as a mouse bioassay, can indicate the biological activity of the toxin; however, this method is not feasible for laboratories that do not have animal testing facilities. The purpose of this study was to develop an in vitro assay for the detection of biologically active ricin in beverages and liquid foods. Acidic and high-protein beverages were spiked with either purified ricin or ground castor beans and added to cultured human Jurkat cells. After an overnight incubation, the supernatant was tested for lactate dehydrogenase (LDH) activity with a colorimetric assay. LDH was released from the cytosol upon cell damage and was positively correlated with cell death. Ricin was detectable in all the matrices tested, with a sensitivity of 10 to 100 pg/ml. Biologically active ricin was detectable in all the matrices incubated with ground castor bean material. This method provides a confirmatory way to detect biologically active ricin that can be utilized by laboratories lacking animal facilities.

scholarly journals A new in vitro assay measuring direct interaction of nonsense suppressors with the eukaryotic protein synthesis machinery

2018 ◽  
Author(s):  
Martin Y. Ng ◽  
Haibo Zhang ◽  
Amy Weil ◽  
Vijay Singh ◽  
Ryan Jamiolkowski ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Single Molecule ◽  
Direct Interaction ◽  
Vitro Assay ◽  
Premature Termination Codons ◽  
Protein Synthesis Machinery ◽  
Nonsense Suppressor ◽  
Nonsense Suppressors ◽  
Selection Of

ABSTRACTNonsense suppressors (NonSups) induce “readthrough”, i.e., the selection of near cognate tRNAs at premature termination codons and insertion of the corresponding amino acid into nascent polypeptide. Prior readthrough measurements utilized contexts in which NonSups can promote readthrough directly, by binding to one or more of the components of the protein synthesis machinery, or indirectly, by several other mechanisms. Here we utilize a new, highly-purified in vitro assay to measure exclusively direct nonsense suppressor-induced readthrough. Of 16 NonSups tested, 12 display direct readthrough, with results suggesting that such NonSups act by at least two different mechanisms. In preliminary work we demonstrate the potential of single molecule fluorescence energy transfer measurements to elucidate mechanisms of NonSup-induced direct readthrough, which will aid efforts to identify NonSups having improved clinical efficacy.Table of Contents artwork

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