Circular Dichroism of G-Quadruplex: a Laboratory Experiment for the Study of Topology and Ligand Binding

Carvalho, Josué; Queiroz, João A.; Cruz, Carla

doi:10.1021/acs.jchemed.7b00160

Cited by 61 publications

(48 citation statements)

References 19 publications

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“…To gain ab etter understanding of the potentialc hanges in G4 DNA topology upon bindingt oTPA-PY ligands, we carried out circulard ichroism (CD) studies with the following G4 structures:H Te lo22-K (hybrid topology), 22CTA( anti-parallel) and c-Myc (parallel). [31] The 22CTAs equence shows the characteristic bands of an anti-parallel conformation:apositive band centred at 295 nm and an egative band at 265 nm. Although addition of the TPA-PY ligandsl eads to ad ecreasei nt he intensity of the original bands, the overall shape of the spectrum is retained, which indicatest hat the G4 largely maintainsi ts conformationu pon bindingt oTPA-PY ligands ( Figure 12 ca nd Figures S21 ca nd S22 ci nt he Supporting Information).…”

Section: Circular Dichroism Spectroscopic Studiesmentioning

confidence: 99%

Aza‐Macrocyclic Triphenylamine Ligands for G‐Quadruplex Recognition

Pont

González‐García

Inclán

et al. 2018

Chemistry A European J

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A new series of triphenylamine-based ligands with one (TPA1PY), two (TPA2PY) or three pendant aza-macrocycle(s) (TPA3PY) has been synthesised and studied by means of pH-metric titrations, UV/Vis spectroscopy and fluorescence experiments. The affinity of these ligands for G-quadruplex (G4) DNA and the selectivity they show for G4s over duplex DNA were investigated by Förster resonance energy transfer (FRET) melting assays, fluorimetric titrations and circular dichroism spectroscopy. Interestingly, the interactions of the bi- and especially the tri-branched ligands with G4s lead to a very intense redshifted fluorescence emission band that may be associated with intermolecular aggregation between the molecule and DNA. This light-up effect allows the application of the ligands as fluorescence probes to selectively detect G4s.

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Section: Circular Dichroism Spectroscopic Studiesmentioning

confidence: 99%

Aza‐Macrocyclic Triphenylamine Ligands for G‐Quadruplex Recognition

Pont

González‐García

Inclán

et al. 2018

Chemistry A European J

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“…PQS found in smp_319480 ( myb like protein ) and smp_127680 ( rab connectin ) displayed a positive peak at 263 nm, a negative peak at 238 nm and a shoulder at 293 nm, mirroring the spectra observed for smTelo (a negative peak at 238 nm, shoulder peak at 263 nm and positive peak at 293 nm) suggesting their formation of an antiparallel hybrid quadruplex conformation (Fig. 4B) [42,46]. In contrast, the CD spectrum of the PQS identified in smp_196840 (collagen α 1 chain ) could not be associated with any known G4-CD profiles, leading us to exclude its folding into any G4 structure in the applied experimental conditions (Fig.…”

Section: Resultsmentioning

confidence: 75%

“…To assess the role of a cation for proper folding [46], oligonucleotides containing G4 sequences were annealed in the absence of K + and compared to those achieved in presence of 50 mM K + (Fig 5). Interestingly, these CD spectra showed differences in folding profiles and ellipticity in all cases, not corresponding to any type of G4-folding.…”

Section: Resultsmentioning

confidence: 99%

Identifying and validating the presence of Guanine-Quadruplexes (G4) within the blood fluke parasiteSchistosoma mansoni

Craven

Bonsignore

Lenis

et al. 2020

Preprint

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BackgroundSchistosomiasis is a neglected tropical disease that currently affects over 250 million individuals worldwide. In the absence of an immunoprophylactic vaccine and the recognition that mono-chemotherapeutic control of schistosomiasis by praziquantel has limitations, new strategies for managing disease burden are urgently needed. A better understanding of schistosome biology could identify previously undocumented areas suitable for the development of novel interventions.Methodology/Principal findingsHere, for the first time, we detail the presence of G-quadruplexes (G4) and putative quadruplex forming sequences (PQS) within the Schistosoma mansoni genome. We find that G4 are present in both intragenic and intergenic regions of the seven autosomes as well as the sex- defining allosome pair. Amongst intragenic regions, G4 are particularly enriched in 3’ UTR regions. Gene Ontology (GO) term analysis evidenced significant G4 enrichment in the wnt signalling pathway (p<0.05) and PQS oligonucleotides synthetically derived from wnt-related genes resolve into parallel and hybrid G4 motifs as elucidated by circular dichroism (CD) spectroscopy. Finally, utilising a single chain anti-G4 antibody called BG4, we confirm the in situ presence of G4 within both adult female and male worm nuclei.Conclusion/SignificanceThese results collectively suggest that G4-targeted compounds could be tested as novel anthelmintic agents and highlights the possibility that G4-stabilizing molecules could be progressed as candidates for the treatment of schistosomiasis.Author SummarySchistosoma mansoni causes schistosomiasis, a parasitic disease that affects millions of people living in resource-deprived areas of developing countries. No vaccine exists and the current drug treatment has limitations, notably inefficacy against the larval stages of the parasite. New drugs are, therefore, needed to sustainably control schistosomiasis. A further understanding of parasite biology will uncover new targets and lead to the development of novel therapies. Here, we identify the presence of G-Quadruplexes (G4s) in S. mansoni. G4s are four-stranded DNA structures that can affect gene function and, to date, have not been previously found in any parasitic helminth. Computational analysis predicted potential G4 folding sequences within the S. mansoni genome, several of which were confirmed to fold by circular dichroism spectroscopy. Analysis of G4-containing protein coding genes found an enrichment within the wnt signalling pathway, a developmental pathway crucial for axial development in the parasite. Additionally, G4s could be detected within adult worms using a fluorescent antibody that selectively recognises quadruplex structures in nucleic acids. This research describes the presence of a previously unknown structure within the parasite, which could present a new target for developing novel treatments.

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“…First, we performed circular dichroism spectropolarimetry (CD) on the samples to investigate whether the wildtype and mutant oligomers form a G-quadruplex structure in solution. We observed a peak in ellipticity upon CD analysis (θ at λ≈263 nm and a minimum at λ≈242 (as detailed in Table S1), Figure 2B, suggesting that the wt oligomer adopts a parallel G-quadruplex structure in solution (49)(50)(51). Similarly, the G1738A mutant showed a comparable quadruplex CD profile.…”

Section: Figure 1 (A)mentioning

confidence: 82%

Identification and characterization of a G-quadruplex structure in the pre-core promoter region of hepatitis B virus

Meier-Stephenson

Badmalia

Mrozowich

et al. 2021

Preprint

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Worldwide, ∼250 million people are chronically infected with the hepatitis B virus (HBV) and are at increased risk of cirrhosis and hepatocellular carcinoma. The HBV persists as covalently closed circular DNA (cccDNA), which acts as the template for all HBV mRNA transcripts. Nucleos(t)ide analogs do not directly target the HBV cccDNA and cannot eradicate the HBV. We have discovered a unique structural motif, a G-quadruplex in HBV’s pre-core promoter region that is conserved amongst nearly all genotypes, and is central to critical steps in the viral life-cycle including the production of pre-genomic RNA, core and polymerase proteins, and encapsidation. Thus, an increased understanding of the HBV pre-core may lead to the development of novel anti-HBV cccDNA targets. We utilized biophysical methods to characterize the presence of the G-quadruplex, employed assays using a known quadruplex-binding protein (DHX36) to pull-down HBV cccDNA, and compared HBV infection in HepG2 cells transfected with wild-type and mutant HBV plasmids. This study provides insights into the presence of a G-quadruplex in the HBV pre-core promoter region essential for HBV replication. The evaluation of this critical host-protein interaction site in the HBV cccDNA may ultimately facilitate the development of novel anti-HBV therapeutics against the resilient cccDNA template.

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Circular Dichroism of G-Quadruplex: a Laboratory Experiment for the Study of Topology and Ligand Binding

Abstract: Notes for the tutor, detailed instructions for sample preparation, data acquisition and T m determination, and additional material for students (PDF, DOCX)

Cited by 61 publications

References 19 publications

Aza‐Macrocyclic Triphenylamine Ligands for G‐Quadruplex Recognition

Aza‐Macrocyclic Triphenylamine Ligands for G‐Quadruplex Recognition

Identifying and validating the presence of Guanine-Quadruplexes (G4) within the blood fluke parasiteSchistosoma mansoni

Identification and characterization of a G-quadruplex structure in the pre-core promoter region of hepatitis B virus

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