Characterization of In Vitro G-Quadruplex Formation of Imetelstat Telomerase Inhibitor

2021 ◽  
Author(s):  
Laure-Elie Carloni ◽  
Rainer Wechselberger ◽  
Thomas De Vijlder
Keyword(s):  
Telomerase Inhibitor ◽  
G Quadruplex ◽  
Quadruplex Formation

A multi-functional guanine derivative for studying the DNA G-quadruplex structure

The Analyst ◽  
2017 ◽  
Vol 142 (21) ◽  
pp. 4083-4088 ◽  
Author(s):  
Takumi Ishizuka ◽  
Pei-Yan Zhao ◽  
Hong-Liang Bao ◽  
Yan Xu
Keyword(s):  
Fluorescent Probe ◽  
Living Cells ◽  
Quadruplex Structure ◽  
G Quadruplex ◽  
Quadruplex Formation

A multi-functional guanine derivative, 8FG, as a G-quadruplex stabilizer, a fluorescent probe for the detection of G-quadruplex formation, and a 19F sensor for the observation of the G-quadruplex in vitro and in living cells.

scholarly journals Characterization of human telomere RNA G-quadruplex structures in vitro and in living cells using 19F NMR spectroscopy

2017 ◽  
Vol 45 (9) ◽  
pp. 5501-5511 ◽  
Author(s):  
Hong-Liang Bao ◽  
Takumi Ishizuka ◽  
Takashi Sakamoto ◽  
Kenzo Fujimoto ◽  
Tamayo Uechi ◽  
...  
Keyword(s):  
Nmr Spectroscopy ◽  
Living Cells ◽  
19F Nmr ◽  
19F Nmr Spectroscopy ◽  
G Quadruplex ◽  
Human Telomere

scholarly journals G-quadruplexes in H1N1 influenza genomes

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Václav Brázda ◽  
Otília Porubiaková ◽  
Alessio Cantara ◽  
Natália Bohálová ◽  
Jan Coufal ◽  
...  
Keyword(s):  
H1n1 Virus ◽  
Antiviral Treatment ◽  
H1n1 Influenza ◽  
Influenza Viruses ◽  
Close Relative ◽  
Spectroscopic Methods ◽  
Genotype 4 ◽  
G Quadruplex ◽  
Quadruplex Formation

Abstract Background Influenza viruses are dangerous pathogens. Seventy-Seven genomes of recently emerged genotype 4 reassortant Eurasian avian-like H1N1 virus (G4-EA-H1N1) are currently available. We investigated the presence and variation of potential G-quadruplex forming sequences (PQS), which can serve as targets for antiviral treatment. Results PQS were identified in all 77 genomes. The total number of PQS in G4-EA-H1N1 genomes was 571. Interestingly, the number of PQS per genome in individual close relative viruses varied from 4 to 12. PQS were not randomly distributed in the 8 segments of the G4-EA-H1N1 genome, the highest frequency of PQS being found in the NP segment (1.39 per 1000 nt), which is considered a potential target for antiviral therapy. In contrast, no PQS was found in the NS segment. Analyses of variability pointed the importance of some PQS; even if genome variation of influenza virus is extreme, the PQS with the highest G4Hunter score is the most conserved in all tested genomes. G-quadruplex formation in vitro was experimentally confirmed using spectroscopic methods. Conclusions The results presented here hint several G-quadruplex-forming sequences in G4-EA-H1N1 genomes, that could provide good therapeutic targets.

scholarly journals Characterization of G-quadruplex antibody reveals differential specificity for G4 DNA forms

DNA Research ◽  
2020 ◽  
Vol 27 (5) ◽  
Author(s):  
Saniya M Javadekar ◽  
Namrata M Nilavar ◽  
Amita Paranjape ◽  
Kohal Das ◽  
Sathees C Raghavan
Keyword(s):  
Telomere Maintenance ◽  
Antibody Recognition ◽  
G4 Dna ◽  
Telomere Sequence ◽  
G Quadruplex ◽  
Potential Applications ◽  
Chromosome Fragility ◽  
Mere Presence

Abstract Accumulating evidence suggests that human genome can fold into non-B DNA structures, when appropriate sequence and favourable conditions are present. Among these, G-quadruplexes (G4-DNA) are associated with gene regulation, chromosome fragility and telomere maintenance. Although several techniques are used in detecting such structures in vitro, understanding their intracellular existence has been challenging. Recently, an antibody, BG4, was described to study G4 structures within cells. Here, we characterize BG4 for its affinity towards G4-DNA, using several biochemical and biophysical tools. BG4 bound to G-rich DNA derived from multiple genes that form G-quadruplexes, unlike complementary C-rich or random sequences. BLI studies revealed robust binding affinity (Kd = 17.4 nM). Gel shift assays show BG4 binds to inter- and intramolecular G4-DNA, when it is in parallel orientation. Mere presence of G4-motif in duplex DNA is insufficient for antibody recognition. Importantly, BG4 can bind to G4-DNA within telomere sequence in a supercoiled plasmid. Finally, we show that BG4 binds to form efficient foci in four cell lines, irrespective of their lineage, demonstrating presence of G4-DNA in genome. Importantly, number of BG4 foci within the cells can be modulated, upon knockdown of G4-resolvase, WRN. Thus, we establish specificity of BG4 towards G4-DNA and discuss its potential applications.

scholarly journals A 5′ UTR GGN repeat controls localisation and translation of a potassium leak channel mRNA through G-quadruplex formation

2020 ◽  
Vol 48 (17) ◽  
pp. 9822-9839
Author(s):  
Connor J Maltby ◽  
James P R Schofield ◽  
Steven D Houghton ◽  
Ita O’Kelly ◽  
Mariana Vargas-Caballero ◽  
...  
Keyword(s):  
Hek293 Cells ◽  
Leak Channel ◽  
G Quadruplex ◽  
Rna Immunoprecipitation ◽  
Quadruplex Formation ◽  
Pore Domain ◽  
Normal Cellular Function ◽  
Synaptic Remodelling

Abstract RNA G-quadruplexes (G4s) are secondary structures proposed to function as regulators of post-transcriptional mRNA localisation and translation. G4s within some neuronal mRNAs are known to control distal localisation and local translation, contributing to distinct local proteomes that facilitate the synaptic remodelling attributed to normal cellular function. In this study, we characterise the G4 formation of a (GGN)13 repeat found within the 5′ UTR of the potassium 2-pore domain leak channel Task3 mRNA. Biophysical analyses show that this (GGN)13 repeat forms a parallel G4 in vitro exhibiting the stereotypical potassium specificity of G4s, remaining thermostable under physiological ionic conditions. Through mouse brain tissue G4-RNA immunoprecipitation, we further confirm that Task3 mRNA forms a G4 structure in vivo. The G4 is inhibitory to translation of Task3 in vitro and is overcome through activity of a G4-specific helicase DHX36, increasing K+ leak currents and membrane hyperpolarisation in HEK293 cells. Further, we observe that this G4 is fundamental to ensuring delivery of Task3 mRNA to distal primary cortical neurites. It has been shown that aberrant Task3 expression correlates with neuronal dysfunction, we therefore posit that this G4 is important in regulated local expression of Task3 leak channels that maintain K+ leak within neurons.

scholarly journals A novel G-quadruplex motif in the Human MET promoter region

2017 ◽  
Vol 37 (6) ◽  
Author(s):  
Jing Yan ◽  
Deming Zhao ◽  
Liping Dong ◽  
Shuang Pan ◽  
Fengjin Hao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Regulation ◽  
Luciferase Assay ◽  
Klenow Fragment ◽  
Regulate Gene Expression ◽  
Physiological Conditions ◽  
Quadruplex Structure ◽  
G Quadruplex ◽  
Quadruplex Formation

It is known that the guanine-rich strands in proto-oncogene promoters can fold into G-quadruplex structures to regulate gene expression. An intramolecular parallel G-quadruplex has been identified in MET promoter. It acts as a repressor in regulating MET expression. However, the full guanine-rich region in MET promoter forms a hybrid parallel/antiparallel G-quadruplex structure under physiological conditions, which means there are some antiparallel and hybrid parallel/antiparallel G-quadruplex structures in this region. In the present study, our data indicate that g3-5 truncation adopts an intramolecular hybrid parallel/antiparallel G-quadruplex under physiological conditions in vitro. The g3-5 G-quadruplex structure significantly stops polymerization by Klenow fragment in K+ buffer. Furthermore, the results of circular dichroism (CD) spectra and polymerase stop assay directly demonstrate that the G-quadruplex structure in g3-5 fragment can be stabilized by the G-quadruplex ligand TMPyP4 (5,10,15,20-tetra-(N-methyl-4-pyridyl) porphine). But the dual luciferase assay indicates TMPyP4 has no effect on the formation of g3-5 G-quadruplex in HepG2 cells. The findings in the present study will enrich our understanding of the G-quadruplex formation in proto-oncogene promoters and the mechanisms of gene expression regulation.

scholarly journals RNA G-quadruplex structures exist and function in vivo

2019 ◽  
Author(s):  
Xiaofei Yang ◽  
Jitender Cheema ◽  
Yueying Zhang ◽  
Hongjing Deng ◽  
Susan Duncan ◽  
...  
Keyword(s):  
Direct Evidence ◽  
Rna Structures ◽  
Specific Sequence ◽  
Form Complex ◽  
Folding Potential ◽  
G Quadruplex ◽  
Quadruplex Formation ◽  
And Function

AbstractGuanine-rich sequences are able to form complex RNA structures termed RNA G-quadruplexes in vitro. Because of their high stability, RNA G-quadruplexes are proposed to exist in vivo and are suggested to be associated with important biological relevance. However, there is a lack of direct evidence for RNA G-quadruplex formation in living cells. Therefore, it is unclear whether any purported functions are associated with the specific sequence content or the formation of an RNA G-quadruplex structure. Here, we profiled the landscape of those guanine-rich regions with the in vitro folding potential in the Arabidopsis transcriptome. We found a global enrichment of RNA G-quadruplexes with two G-quartets whereby the folding potential is strongly influenced by RNA secondary structures. Using in vitro and in vivo RNA chemical structure profiling, we determined that hundreds of RNA G-quadruplex structures are strongly folded in both Arabidopsis and rice, providing direct evidence of RNA G-quadruplex formation in living eukaryotic cells. Subsequent genetic and biochemical analysis showed that RNA G-quadruplex folding was sufficient to regulate translation and modulate plant growth. Our study reveals the existence of RNA G-quadruplex in vivo, and indicates that RNA G-quadruplex structures act as important regulators of plant development and growth.

scholarly journals Towards Profiling of the G-Quadruplex Targeting Drugs in the Living Human Cells Using NMR Spectroscopy

2021 ◽  
Vol 22 (11) ◽  
pp. 6042
Author(s):  
Daniel Krafčík ◽  
Eva Ištvánková ◽  
Šimon Džatko ◽  
Pavlína Víšková ◽  
Silvie Foldynová-Trantírková ◽  
...  
Keyword(s):  
Nmr Spectroscopy ◽  
Human Cells ◽  
Complex Environment ◽  
Promising Strategy ◽  
Anti Cancer ◽  
G Quadruplex ◽  
Ligand Interactions ◽  
Application Potential

Recently, the 1H-detected in-cell NMR spectroscopy has emerged as a unique tool allowing the characterization of interactions between nucleic acid-based targets and drug-like molecules in living human cells. Here, we assess the application potential of 1H and 19F-detected in-cell NMR spectroscopy to profile drugs/ligands targeting DNA G-quadruplexes, arguably the most studied class of anti-cancer drugs targeting nucleic acids. We show that the extension of the original in-cell NMR approach is not straightforward. The severe signal broadening and overlap of 1H in-cell NMR spectra of polymorphic G-quadruplexes and their complexes complicate their quantitative interpretation. Nevertheless, the 1H in-cell NMR can be used to identify drugs that, despite strong interaction in vitro, lose their ability to bind G-quadruplexes in the native environment. The in-cell NMR approach is adjusted to a recently developed 3,5-bis(trifluoromethyl)phenyl probe to monitor the intracellular interaction with ligands using 19F-detected in-cell NMR. The probe allows dissecting polymorphic mixture in terms of number and relative populations of individual G-quadruplex species, including ligand-bound and unbound forms in vitro and in cellulo. Despite the probe’s discussed limitations, the 19F-detected in-cell NMR appears to be a promising strategy to profile G-quadruplex–ligand interactions in the complex environment of living cells.

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