Probing Telomeric G-Quadruplex DNA Structures in Cells with In Vitro Generated Single-Chain Antibody Fragments

2009 ◽  
pp. 159-181 ◽  
Author(s):  
Christiane Schaffitzel ◽  
Jan Postberg ◽  
Katrin Paeschke ◽  
Hans J. Lipps
Keyword(s):  
Antibody Fragments ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Quadruplex Dna ◽  
Dna Structures ◽  
G Quadruplex ◽  
In Cells

scholarly journals Llama-Derived Single-Chain Antibody Fragments Directed to Rotavirus VP6 Protein Possess Broad Neutralizing Activity In Vitro and Confer Protection against Diarrhea in Mice

2008 ◽  
Vol 82 (19) ◽  
pp. 9753-9764 ◽  
Author(s):  
Lorena Garaicoechea ◽  
Aurelien Olichon ◽  
Gisela Marcoppido ◽  
Andrés Wigdorovitz ◽  
Marina Mozgovoj ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Binding Capacity ◽  
Antibody Fragments ◽  
Target Antigen ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Group A Rotavirus ◽  
Group A

ABSTRACT Group A rotavirus is one of the most common causes of severe diarrhea in human infants and newborn animals. Rotavirus virions are triple-layered particles. The outer capsid proteins VP4 and VP7 are highly variable and represent the major neutralizing antigens. The inner capsid protein VP6 is conserved among group A rotaviruses, is highly immunogenic, and is the target antigen of most immunodiagnosis tests. Llama-derived single-chain antibody fragments (VHH) are the smallest molecules with antigen-binding capacity and can therefore be expected to have properties different from conventional antibodies. In this study a library containing the VHH genes of a llama immunized with recombinant inner capsid protein VP6 was generated. Binders directed to VP6, in its native conformation within the viral particle, were selected and characterized. Four selected VHH directed to conformational epitopes of VP6 recognized all human and animal rotavirus strains tested and could be engineered for their use in immunodiagnostic tests for group A rotavirus detection. Three of the four VHH neutralized rotavirus in vivo independently of the strain serotype. Furthermore, this result was confirmed by in vivo partial protection against rotavirus challenge in a neonatal mouse model. The present study demonstrates for the first time a broad neutralization activity of VP6 specific VHH in vitro and in vivo. Neutralizing VHH directed to VP6 promise to become an essential tool for the prevention and treatment of rotavirus diarrhea.

scholarly journals Human Rev1 polymerase disrupts G-quadruplex DNA

2013 ◽  
Vol 42 (5) ◽  
pp. 3272-3285 ◽  
Author(s):  
Sarah Eddy ◽  
Amit Ketkar ◽  
Maroof K. Zafar ◽  
Leena Maddukuri ◽  
Jeong-Yun Choi ◽  
...  
Keyword(s):  
Genome Maintenance ◽  
Quadruplex Dna ◽  
Dna Structures ◽  
G4 Dna ◽  
G Quadruplex ◽  
Steady State Rate ◽  
Dna Substrates ◽  
Successful Replication ◽  
Y Family

Abstract The Y-family DNA polymerase Rev1 is required for successful replication of G-quadruplex DNA (G4 DNA) in higher eukaryotes. Here we show that human Rev1 (hRev1) disrupts G4 DNA structures and prevents refolding in vitro. Nucleotidyl transfer by hRev1 is not necessary for mechanical unfolding to occur. hRev1 binds G4 DNA substrates with Kd,DNA values that are 4–15-fold lower than those of non-G4 DNA substrates. The pre-steady-state rate constant of deoxycytidine monophosphate (dCMP) insertion opposite the first tetrad-guanine by hRev1 is ∼56% as fast as that observed for non-G4 DNA substrates. Thus, hRev1 can promote fork progression by either dislodging tetrad guanines to unfold the G4 DNA, which could assist in extension by other DNA polymerases, or hRev1 can prevent refolding of G4 DNA structures. The hRev1 mechanism of action against G-quadruplexes helps explain why replication progress is impeded at G4 DNA sites in Rev1-deficient cells and illustrates another unique feature of this enzyme with important implications for genome maintenance.

scholarly journals Sites of instability in the human TCF3 (E2A) gene adopt G-quadruplex DNA structures in vitro

2015 ◽  
Vol 6 ◽  
Author(s):  
Jonathan D. Williams ◽  
Sara Fleetwood ◽  
Alexandra Berroyer ◽  
Nayun Kim ◽  
Erik D. Larson
Keyword(s):  
Quadruplex Dna ◽  
Dna Structures ◽  
G Quadruplex

scholarly journals Multimeric humanized varicella-zoster virus antibody fragments to gH neutralize virus while monomeric fragments do not

2001 ◽  
Vol 82 (8) ◽  
pp. 1959-1963 ◽  
Author(s):  
P. D. Drew ◽  
M. T. Moss ◽  
T. J. Pasieka ◽  
C. Grose ◽  
W. J. Harris ◽  
...  
Keyword(s):  
Varicella Zoster Virus ◽  
Cultured Cells ◽  
Cross Reactivity ◽  
Antibody Fragments ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Neutralizing Activity ◽  
Varicella Zoster ◽  
Complementarity Determining Region

Murine monoclonal antibody 206 (MAb mu206) binds to gH, the varicella-zoster virus (VZV) fusogen, neutralizing the virus in vitro in the absence of complement and inhibiting cell-to-cell spread and egress of VZV in cultured cells. We have humanized this antibody to generate MAb hu206 by complementarity determining region grafting. MAb hu206 retained binding and in vitro neutralizing activity, as well as cross-reactivity with ten different VZV strains. Single-chain antibody fragments (scAb) derived from MAb hu206 were produced in Escherichia coli. These scAb retained the binding properties of the whole antibody. However, monomeric scAb exhibited markedly reduced neutralizing activity compared to the bivalent parental MAb hu206. Shortening the peptide linker joining the VH to the Vκ domain from 14 to 5 or even 0 residues encouraged multimerization and increased neutralizing efficacy. The fact that Fab fragments enzymatically generated from whole MAb hu206 lost their neutralizing potency lent support to the proposal that valency is important for VZV neutralization at this epitope.

Identification of novel interactors of human telomeric G-quadruplex DNA

2015 ◽  
Vol 51 (14) ◽  
pp. 2964-2967 ◽  
Author(s):  
Bruno Pagano ◽  
Luigi Margarucci ◽  
Pasquale Zizza ◽  
Jussara Amato ◽  
Nunzia Iaccarino ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Dna Structure ◽  
Quadruplex Dna ◽  
G Quadruplex ◽  
In Cells

Starting from a chemoproteomic-driven approach, novel human telomeric G-quadruplex binding proteins were identified that directly bind the DNA structure in vitro and colocalize with such structures in cells.

scholarly journals A red-NIR fluorescent dye detecting nuclear DNA G-quadruplexes: in vitro analysis and cell imaging

2017 ◽  
Vol 53 (14) ◽  
pp. 2268-2271 ◽  
Author(s):  
F. Doria ◽  
M. Nadai ◽  
M. Zuffo ◽  
R. Perrone ◽  
M. Freccero ◽  
...  
Keyword(s):  
Cell Imaging ◽  
Nuclear Dna ◽  
Fluorescent Dye ◽  
Quadruplex Dna ◽  
G Quadruplex ◽  
Vitro Analysis ◽  
In Vitro Analysis ◽  
In Cells

Light-up of nuclear G-quadruplex DNA in cells by an aggregating and red/NIR emitting dye.

scholarly journals Stabilization of G-quadruplex DNA structures in Schizosaccharomyces pombe causes single-strand DNA lesions and impedes DNA replication

2020 ◽  
Vol 48 (19) ◽  
pp. 10998-11015 ◽  
Author(s):  
Ikenna Obi ◽  
Matilda Rentoft ◽  
Vandana Singh ◽  
Jan Jamroskovic ◽  
Karam Chand ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Dna Replication ◽  
Schizosaccharomyces Pombe ◽  
Dna Lesions ◽  
Single Strand ◽  
Quadruplex Dna ◽  
Dna Structures ◽  
Replication Fork Progression ◽  
G Quadruplex

Abstract G-quadruplex (G4) structures are stable non-canonical DNA structures that are implicated in the regulation of many cellular pathways. We show here that the G4-stabilizing compound PhenDC3 causes growth defects in Schizosaccharomyces pombe cells, especially during S-phase in synchronized cultures. By visualizing individual DNA molecules, we observed shorter DNA fragments of newly replicated DNA in the PhenDC3-treated cells, suggesting that PhenDC3 impedes replication fork progression. Furthermore, a novel single DNA molecule damage assay revealed increased single-strand DNA lesions in the PhenDC3-treated cells. Moreover, chromatin immunoprecipitation showed enrichment of the leading-strand DNA polymerase at sites of predicted G4 structures, suggesting that these structures impede DNA replication. We tested a subset of these sites and showed that they form G4 structures, that they stall DNA synthesis in vitro and that they can be resolved by the breast cancer-associated Pif1 family helicases. Our results thus suggest that G4 structures occur in S. pombe and that stabilized/unresolved G4 structures are obstacles for the replication machinery. The increased levels of DNA damage might further highlight the association of the human Pif1 helicase with familial breast cancer and the onset of other human diseases connected to unresolved G4 structures.

Single-chain Antibody Against Reg4 Suppresses Gastric Cancer Cell Growth and Enhances 5-FU-induced Cell Death in vitro

2019 ◽  
Vol 19 (5) ◽  
pp. 610-619 ◽  
Author(s):  
Xue-Qing Zhang ◽  
Lu-Ting Yu ◽  
Pei Du ◽  
Tian-Qi Yin ◽  
Zhi-Yuan Zhang ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cell Proliferation ◽  
Cell Death ◽  
Cancer Cell ◽  
Gastric Cancer Cell ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Ags Cells ◽  
Thiazolyl Blue Tetrazolium Bromide

Background:Regenerating islet-derived gene family member 4 (Reg4), a well-investigated growth factor in the regenerative pancreas, has recently been reported to be highly associated with a majority of gastrointestinal cancers. Pathological hyper-expression or artificial over-expression of Reg4 causes acceleration of tumor growth, migration, and resistance to chemotherapeutic 5-Fluorouracil (5-FU). Until now, no method has been successfully established for eliminating the effects of Reg4 protein.Methods:This study reports the production of an engineered immunoglobin, a single-chain variable fragment (scFv-Reg4), to specifically bind Reg4 and block the bioactivity. The complementary-determining regions (CDRs) against Reg4 were assigned using MOE and ZDOCK servers. The binding affinity (KD) was determined by bio-layer interferometry (BLI). MKN45 and AGS cell proliferation was determined by Thiazolyl blue tetrazolium bromide (MTT) method and the cell apoptosis was detected by flow cytometry assay.Results:The KD of scFv-Reg4 to Reg4 was determined to be 1.91×10-8. In MKN45 and AGS cell lines, scFv- Reg4 depressed Reg4-stimulated cell proliferation and the inhibitory rates were 27.7±1.5% and 17.3±2.6%, respectively. Furthermore, scFv significantly enhanced 5-FU-induced cell death, from 23.0±1.0% to 28.4±1.2% in MKN45 and 28.2±0.7% to 36.6±0.6% in AGS cells. Treatment with scFv alone could lyse cancer cells to a certain extent, but no significance has been observed.Conclusion:The single-chain antibody (scFv-Reg4) significantly inhibited gastric cancer cell proliferation and synergistically enhanced the lethal effect of 5-FU. Thus, traditional chemo-/radio- therapeutics supplemented with scFv-Reg4 may provide advances in the strategy for gastrointestinal cancer treatment.

scholarly journals DNA G-quadruplexes for native mass spectrometry in potassium: a database of validated structures in electrospray-compatible conditions

2021 ◽  
Author(s):  
Anirban Ghosh ◽  
Eric Largy ◽  
Valérie Gabelica
Keyword(s):  
Mass Spectrometry ◽  
Drug Targets ◽  
Native Mass Spectrometry ◽  
Drug Binding ◽  
Quadruplex Dna ◽  
Dna Structures ◽  
Nmr Structures ◽  
G Quadruplex ◽  
Screening Assays

Abstract G-quadruplex DNA structures have become attractive drug targets, and native mass spectrometry can provide detailed characterization of drug binding stoichiometry and affinity, potentially at high throughput. However, the G-quadruplex DNA polymorphism poses problems for interpreting ligand screening assays. In order to establish standardized MS-based screening assays, we studied 28 sequences with documented NMR structures in (usually ∼100 mM) potassium, and report here their circular dichroism (CD), melting temperature (Tm), NMR spectra and electrospray mass spectra in 1 mM KCl/100 mM trimethylammonium acetate. Based on these results, we make a short-list of sequences that adopt the same structure in the MS assay as reported by NMR, and provide recommendations on using them for MS-based assays. We also built an R-based open-source application to build and consult a database, wherein further sequences can be incorporated in the future. The application handles automatically most of the data processing, and allows generating custom figures and reports. The database is included in the g4dbr package (https://github.com/EricLarG4/g4dbr) and can be explored online (https://ericlarg4.github.io/G4_database.html).

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