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 Single-molecule visualization of human BLM helicase as it acts upon double- and single-stranded DNA substrates

2019 ◽  
Vol 47 (21) ◽  
pp. 11225-11237 ◽  
Author(s):  
Chaoyou Xue ◽  
James M Daley ◽  
Xiaoyu Xue ◽  
Justin Steinfeld ◽  
Youngho Kwon ◽  
...  
Keyword(s):  
Single Molecule ◽  
Molecular Mechanisms ◽  
Genetic Disorder ◽  
Protein A ◽  
Base Pairs ◽  
Single Stranded Dna ◽  
Double Stranded Dna ◽  
Blm Helicase ◽  
Dna Replication And Repair ◽  
Regulatory Effects

Abstract Bloom helicase (BLM) and its orthologs are essential for the maintenance of genome integrity. BLM defects represent the underlying cause of Bloom Syndrome, a rare genetic disorder that is marked by strong cancer predisposition. BLM deficient cells accumulate extensive chromosomal aberrations stemming from dysfunctions in homologous recombination (HR). BLM participates in several HR stages and helps dismantle potentially harmful HR intermediates. However, much remains to be learned about the molecular mechanisms of these BLM-mediated regulatory effects. Here, we use DNA curtains to directly visualize the activity of BLM helicase on single molecules of DNA. Our data show that BLM is a robust helicase capable of rapidly (∼70–80 base pairs per second) unwinding extensive tracts (∼8–10 kilobases) of double-stranded DNA (dsDNA). Importantly, we find no evidence for BLM activity on single-stranded DNA (ssDNA) that is bound by replication protein A (RPA). Likewise, our results show that BLM can neither associate with nor translocate on ssDNA that is bound by the recombinase protein RAD51. Moreover, our data reveal that the presence of RAD51 also blocks BLM translocation on dsDNA substrates. We discuss our findings within the context of potential regulator roles for BLM helicase during DNA replication and repair.

scholarly journals Single-molecule visualization of RecQ helicase reveals DNA melting, nucleation, and assembly are required for processive DNA unwinding

2015 ◽  
Vol 112 (50) ◽  
pp. E6852-E6861 ◽  
Author(s):  
Behzad Rad ◽  
Anthony L. Forget ◽  
Ronald J. Baskin ◽  
Stephen C. Kowalczykowski
Keyword(s):  
Single Molecule ◽  
Fluorescent Sensor ◽  
Holliday Junctions ◽  
Dna Unwinding ◽  
Recq Helicase ◽  
Dna Breaks ◽  
Dna Helicases ◽  
Double Stranded Dna ◽  
Functional Forms ◽  
And Migration

DNA helicases are motor proteins that unwind double-stranded DNA (dsDNA) to reveal single-stranded DNA (ssDNA) needed for many biological processes. The RecQ helicase is involved in repairing damage caused by DNA breaks and stalled replication forks via homologous recombination. Here, the helicase activity of RecQ was visualized on single molecules of DNA using a fluorescent sensor that directly detects ssDNA. By monitoring the formation and progression of individual unwinding forks, we observed that both the frequency of initiation and the rate of unwinding are highly dependent on RecQ concentration. We establish that unwinding forks can initiate internally by melting dsDNA and can proceed in both directions at up to 40–60 bp/s. The findings suggest that initiation requires a RecQ dimer, and that continued processive unwinding of several kilobases involves multiple monomers at the DNA unwinding fork. We propose a distinctive model wherein RecQ melts dsDNA internally to initiate unwinding and subsequently assembles at the fork into a distribution of multimeric species, each encompassing a broad distribution of rates, to unwind DNA. These studies define the species that promote resection of DNA, proofreading of homologous pairing, and migration of Holliday junctions, and they suggest that various functional forms of RecQ can be assembled that unwind at rates tailored to the diverse biological functions of RecQ helicase.

Investigation of the binding modes between AIE-active molecules and dsDNA by single molecule force spectroscopy

Nanoscale ◽  
2015 ◽  
Vol 7 (19) ◽  
pp. 8939-8945 ◽  
Author(s):  
Ying Chen ◽  
Ke Ma ◽  
Ting Hu ◽  
Bo Jiang ◽  
Bin Xu ◽  
...  
Keyword(s):  
Single Molecule ◽  
Force Spectroscopy ◽  
Binding Modes ◽  
Single Molecule Force Spectroscopy ◽  
Aggregation Induced Emission ◽  
Double Stranded Dna

The binding modes between double-stranded DNA (dsDNA) and typical AIE (aggregation-induced emission)-active molecules were investigated using AFM-based single molecule force spectroscopy.

Single molecule detection of double-stranded DNA in poly(methylmethacrylate) and polycarbonate microfluidic devices

2001 ◽  
Vol 22 (18) ◽  
pp. 3939-3948 ◽  
Author(s):  
Musundi B. Wabuyele ◽  
Sean M. Ford ◽  
Wieslaw Stryjewski ◽  
James Barrow ◽  
Steven A. Soper
Keyword(s):  
Single Molecule ◽  
Microfluidic Devices ◽  
Single Molecule Detection ◽  
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.

scholarly journals Mechanism of RPA-Facilitated Processive DNA Unwinding by the Eukaryotic CMG Helicase

2019 ◽  
Author(s):  
Hazal B. Kose ◽  
Sherry Xie ◽  
George Cameron ◽  
Melania S. Strycharska ◽  
Hasan Yardimci
Keyword(s):  
Single Molecule ◽  
Replication Fork ◽  
Double Helix ◽  
Dna Unwinding ◽  
Helicase Activity ◽  
Replicative Helicase ◽  
Double Stranded Dna ◽  
Order Of Magnitude ◽  
Dna Strand ◽  
Dna Double Helix

AbstractThe DNA double helix is unwound by the Cdc45/Mcm2-7/GINS (CMG) complex at the eukaryotic replication fork. While isolated CMG unwinds duplex DNA very slowly, its fork unwinding rate is stimulated by an order of magnitude by single-stranded DNA binding protein, RPA. However, the molecular mechanism by which RPA enhances CMG helicase activity remained elusive. Here, we demonstrate that engagement of CMG with parental double-stranded DNA (dsDNA) at the replication fork impairs its helicase activity, explaining the slow DNA unwinding by isolated CMG. Using single-molecule and ensemble biochemistry, we show that binding of RPA to the excluded DNA strand prevents duplex engagement by the helicase and speeds up CMG-mediated DNA unwinding. When stalled due to dsDNA interaction, DNA rezipping-induced helicase backtracking re-establishes productive helicase-fork engagement underscoring the significance of plasticity in helicase action. Together, our results elucidate the dynamics of CMG at the replication fork and reveal how other replisome components can mediate proper DNA engagement by the replicative helicase to achieve efficient fork progression.

scholarly journals Single-molecule manipulation of double-stranded DNA using optical tweezers: Interaction studies of DNA with RecA and YOYO-1

Cytometry ◽  
1999 ◽  
Vol 36 (3) ◽  
pp. 200-208 ◽  
Author(s):  
Martin L. Bennink ◽  
Orlando D. Sch�rer ◽  
Roland Kanaar ◽  
Kumiko Sakata-Sogawa ◽  
Juleon M. Schins ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Double Stranded Dna ◽  
Interaction Studies ◽  
Single Molecule Manipulation

scholarly journals γ-Hemolysin Nanopore Is Sensitive to Guanine-to-Inosine Substitutions in Double-Stranded DNA at the Single-Molecule Level

2018 ◽  
Vol 140 (43) ◽  
pp. 14224-14234 ◽  
Author(s):  
Cherie S. Tan ◽  
Aaron M. Fleming ◽  
Hang Ren ◽  
Cynthia J. Burrows ◽  
Henry S. White
Keyword(s):  
Single Molecule ◽  
Single Molecule Level ◽  
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
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