Identification and characterization of two conserved G-quadruplex forming motifs in the Nipah virus genome and their interaction with G-quadruplex specific ligands.
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ABSTRACT:
SUBMITTER: Majee P
PROVIDER: S-EPMC6992748 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundThe Plasmodium genus of malaria parasites encodes several families of antigen-encoding genes. These genes tend to be hyper-variable, highly recombinogenic and variantly expressed. The best-characterized family is the var genes, exclusively found in the Laveranian subgenus of malaria parasites infecting humans and great apes. Var genes encode major virulence factors involved in immune evasion and the maintenance of chronic infections. In the human parasite P. falciparum, var gene recombination and diversification appear to be promoted by G-quadruplex (G4) DNA motifs, which are strongly associated with var genes in P. falciparum. Here, we investigated how this association might have evolved across Plasmodium species - both Laverania and also more distantly related species which lack vars but encode other, more ancient variant gene families.ResultsThe association between var genes and G4-forming motifs was conserved across Laverania, spanning ~ 1 million years of evolutionary time, with suggestive evidence for evolution of the association occurring within this subgenus. In rodent malaria species, G4-forming motifs were somewhat associated with pir genes, but this was not conserved in the Laverania, nor did we find a strong association of these motifs with any gene family in a second outgroup of avian malaria parasites. Secondly, we compared two different G4 prediction algorithms in their performance on extremely A/T-rich Plasmodium genomes, and also compared these predictions with experimental data from G4-seq, a DNA sequencing method for identifying G4-forming motifs. We found a surprising lack of concordance between the two algorithms and also between the algorithms and G4-seq data.ConclusionsG4-forming motifs are uniquely strongly associated with Plasmodium var genes, suggesting a particular role for G4s in recombination and diversification of these genes. Secondly, in the A/T-rich genomes of Plasmodium species, the choice of prediction algorithm may be particularly influential when studying G4s in these important protozoan pathogens.
| S-EPMC7077173 | biostudies-literature
Project description:Several G-quadruplex forming motifs have been reported to be highly conserved in the regulatory regions of the genome of different organisms and influence various biological processes like DNA replication, recombination and gene expression. Here, we report the highly conserved and three potentially G-quadruplex forming motifs (SP-PGQs) in the essential genes (hsdS, recD, and pmrA) of the Streptococcus pneumoniae genome. These genes were previously observed to play a vital role in providing the virulence to the bacteria, by participating in the host-pathogen interaction, drug-efflux system and recombination- repair system. However, the presence and importance of highly conserved G-quadruplex motifs in these genes have not been previously recognized. We employed the CD spectroscopy, NMR spectroscopy, and electrophoretic mobility shift assay to confirm the adaptation of the G-quadruplex structure by the SP-PGQs. Further, ITC and CD melting analysis revealed the energetically favorable and thermodynamically stable interaction between a candidate G4 binding small molecule TMPyP4 and SP-PGQs. Next, TFP reporter based assay confirmed the regulatory role of SP-PGQs in the expression of PGQ harboring genes. All these experiments together characterized the SP-PGQs as a promising drug target site for combating the Streptococcus pneumoniae infection.
| S-EPMC6370756 | biostudies-literature
Project description:Genomic DNA methylation is involved in many diseases and is expected to be a specific biomarker for even the pre-symptomatic diagnosis of many diseases. Thus, a rapid and inexpensive detection method is required for disease diagnosis. We have previously reported that cytosine methylation in G-quadruplex (G4)-forming oligonucleotides develops different G4 topologies. In this study, we developed a method for detecting CpG methylation in G4-forming oligonucleotides based on the structural differences between methylated and unmethylated G4 DNAs. The differences in G4 topologies due to CpG methylation can be discriminated by G4 ligands. We performed a binding assay between methylated or unmethylated G4 DNAs and G4 ligands. The binding abilities of fluorescent G4 ligands to BCL-2, HRAS1, HRAS2, VEGF G4-forming sequences were examined by fluorescence-based microtiter plate assay. The differences in fluorescence intensities between methylated and unmethylated G4 DNAs were statistically significant. In addition to fluorescence detection, the binding of G4 ligand to DNA was detected by chemiluminescence. A significant difference was also detected in chemiluminescence intensity between methylated and unmethylated DNA. This is the first study on the detection of CpG methylation in G4 structures, focusing on structural changes using G4 ligands.
| S-EPMC8658440 | biostudies-literature
Project description:SH3 domains are evolutionarily conserved protein interaction domains that control nearly all cellular processes in eukaryotes. The current model is that most SH3 domains bind discreet PxxPxR motifs with weak affinity and relatively low selectivity. However, the interactions of full-length SH3 domain-containing proteins with ligands are highly specific and have much stronger affinity. This suggests that regions outside of PxxPxR motifs drive these interactions. In this study, we observed that PxxPxR motifs were required for the binding of the adaptor protein GRB2 to short peptides from its ligand SOS1. Surprisingly, PxxPxR motifs from the proline rich region of SOS1 or CBL were neither necessary nor sufficient for the in vitro or in vivo interaction with full-length GRB2. Together, our findings show that regions outside of the consensus PxxPxR sites drive the high affinity association of GRB2 with SH3 domain ligands, suggesting that the binding mechanism for this and other SH3 domain interactions may be more complex than originally thought.
| S-EPMC4558342 | biostudies-literature
Project description:Human enteroviruses cause many diseases; however, there is no specific therapeutic drug. G-quadruplex is an atypical secondary structure formed in the guanine rich region of DNA or RNA, which can exist in the viral genome. The different positions of G-quadruplex play an important role in the regulation of virus replication and infection. Whether G-quadruplexes are present in human enteroviruses is unknown. In current study, we analyzed the potential quadruplex forming sequences of human enteroviruses, especially EV-A71 virus, which causes hand, foot, and mouth disease. The results showed that there were a certain number of potential quadruplex-forming sequences in human enteroviruses. Through a variety of experimental methods, we evaluated the formation potential of EV-A71 encoded G-quadruplex and analyzed the binding ability of G-quadruplex ligands, including BRACO-19, pyridostatin and TMPyP4 to virus encoded G-quadruplexes. G-quadruplex ligands BRACO-19, PDS and TMPyP4 could inhibit the transcription of constructs containing EV-A71 G-quadruplex sequences. Moreover, we found that BRACO-19 was able to inhibit the replication of EV-A71, suggesting that targeting G-quadruplexes in EV-A71 genome by G-quadruplex ligands could be a novel antiviral way against EV-A71. Our finding not only uncovered the G-quadruplexes in human enteroviruses, but also would provide a new strategy for human enteroviruses therapy. IMPORTANCE G-quadruplex is a stable nucleic acid secondary structure formed by the folding of guanine rich nucleic acid. The important regulatory function of G-quadruplex makes it an attractive target of antiviral effect. Human enteroviruses cause a variety of human diseases, including common cold, nervous system diseases, cardiovascular damage, and diabetes. Enterovirus A71 (EV-A71) is one of pathogens causing hand, foot, and mouth disease; however, whether G-quadruplexes are present in the genomes of human enteroviruses is unknown. The function of G-quadruplexes in the EV-A71 genomes is not clear. We predicted and characterized G-quadruplex sequences in EV-A71. G-quadruplex ligands were identified to stabilize EV-A71 G-quadruplexes with high affinities. We also demonstrated G-quadruplex ligand BRACO-19 inhibited EV-A71 replication. Our studies provide a framework for targeting G-quadruplexes in the enteroviruses genome, which will be a new way to develop antiviral agents against human enteroviruses.
| S-EPMC9241713 | biostudies-literature
Project description:BackgroundNucleic acids containing guanine tracts can form quadruplex structures via non-Watson-Crick base pairing. Formation of G-quadruplexes is associated with the regulation of important biological functions such as transcription, genetic instability, DNA repair, DNA replication, epigenetic mechanisms, regulation of translation, and alternative splicing. G-quadruplexes play important roles in human diseases and are being considered as targets for a variety of therapies. Identification of functional G-quadruplexes and the study of their overall distribution in genomes and transcriptomes is an important pursuit. Traditional computational methods map sequence motifs capable of forming G-quadruplexes but have difficulty in distinguishing motifs that occur by chance from ones which fold into G-quadruplexes.ResultsWe present Quadruplex forming 'G'-rich sequences (QGRS)-Conserve, a computational method for calculating motif conservation across exomes and supports filtering to provide researchers with more precise methods of studying G-quadruplex distribution patterns. Our method quantitatively evaluates conservation between quadruplexes found in homologous nucleotide sequences based on several motif structural characteristics. QGRS-Conserve also efficiently manages overlapping G-quadruplex sequences such that the resulting datasets can be analyzed effectively.ConclusionsWe have applied QGRS-Conserve to identify a large number of G-quadruplex motifs in the human exome conserved across several mammalian and non-mammalian species. We have successfully identified multiple homologs of many previously published G-quadruplexes that play post-transcriptional regulatory roles in human genes. Preliminary large-scale analysis identified many homologous G-quadruplexes in the 5'- and 3'-untranslated regions of mammalian species. An expectedly smaller set of G-quadruplex motifs was found to be conserved across larger phylogenetic distances. QGRS-Conserve provides means to build datasets that can be filtered and categorized in a variety of biological dimensions for more targeted studies in order to better understand the roles that G-quadruplexes play.
| S-EPMC4017754 | biostudies-literature
Project description:Approaches to characterize the nucleic acid-binding properties of drugs and druglike small molecules are crucial to understanding the behavior of these compounds in cellular systems. Here, we use a Small Molecule Microarray (SMM) profiling approach to identify the preferential interaction between chlorhexidine, a widely used oral antiseptic, and the G-quadruplex (G4) structure in the KRAS oncogene promoter. The interaction of chlorhexidine and related drugs to the KRAS G4 is evaluated using multiple biophysical methods, including thermal melt, fluorescence titration and surface plasmon resonance (SPR) assays. Chlorhexidine has a specific low micromolar binding interaction with the G4, while related drugs have weaker and/or less specific interactions. Through NMR experiments and docking studies, we propose a plausible binding mode driven by both aromatic stacking and groove binding interactions. Additionally, cancer cell lines harbouring oncogenic mutations in the KRAS gene exhibit increased sensitivity to chlorhexidine. Treatment of breast cancer cells with chlorhexidine decreases KRAS protein levels, while a KRAS gene transiently expressed by a promoter lacking a G4 is not affected. This work confirms that known ligands bind broadly to G4 structures, while other drugs and druglike compounds can have more selective interactions that may be biologically relevant.
| S-EPMC5888870 | biostudies-other
Project description:G-quadruplexes (G4s) are secondary structures of nucleic acids that epigenetically regulate cellular processes. In the human immunodeficiency lentivirus 1 (HIV-1), dynamic G4s are located in the unique viral LTR promoter. Folding of HIV-1 LTR G4s inhibits viral transcription; stabilization by G4 ligands intensifies this effect. Cellular proteins modulate viral transcription by inducing/unfolding LTR G4s. We here expanded our investigation on the presence of LTR G4s to all lentiviruses. G4s in the 5'-LTR U3 region were completely conserved in primate lentiviruses. A G4 was also present in a cattle-infecting lentivirus. All other non-primate lentiviruses displayed hints of less stable G4s. In primate lentiviruses, the possibility to fold into G4s was highly conserved among strains. LTR G4 sequences were very similar among phylogenetically related primate viruses, while they increasingly differed in viruses that diverged early from a common ancestor. A strong correlation between primate lentivirus LTR G4s and Sp1/NFκB binding sites was found. All LTR G4s folded: their complexity was assessed by polymerase stop assay. Our data support a role of the lentiviruses 5'-LTR G4 region as control centre of viral transcription, where folding/unfolding of G4s and multiple recruitment of factors based on both sequence and structure may take place.
| S-EPMC5435695 | biostudies-literature
Project description:Sequences with the potential to form RNA G-quadruplexes (G4s) are common in mammalian introns, especially in the proximity of the 5' splice site (5'SS). However, the difficulty of demonstrating that G4s form in pre-mRNA in functional conditions has meant that little is known about their effects or mechanisms of action. We have shown previously that two G4s form in Bcl-X pre-mRNA, one close to each of the two alternative 5'SS. If these G4s affect splicing but are in competition with other RNA structures or RNA binding proteins, then ligands that stabilize them would increase the proportion of Bcl-X pre-mRNA molecules in which either or both G4s had formed, shifting Bcl-X splicing. We show here that a restricted set of G4 ligands do affect splicing, that their activity and specificity are strongly dependent on their structures and that they act independently at the two splice sites. One of the ligands, the ellipticine GQC-05, antagonizes the major 5'SS that expresses the anti-apoptotic isoform of Bcl-X and activates the alternative 5'SS that expresses a pro-apoptotic isoform. We propose mechanisms that would account for these see-saw effects and suggest that these effects contribute to the ability of GQC-05 to induce apoptosis.
| S-EPMC5778605 | biostudies-literature
Project description:HIV is primarily transmitted by sexual intercourse and predominantly infects people in Third World countries. Here an important medical need is self-protection for women, particularly in societies where condoms are not widely accepted. Therefore, availability of antiviral microbicides may significantly reduce sexual HIV transmission in such environments.Here, we investigated structural characteristics and the antiviral activity of the polypurine tract (PPT)-specific ODN A, a 54-mer oligodeoxynucleotide (ODN) that has been previously shown to trigger the destruction of viral RNA genomes by prematurely activating the retroviral RNase H. The stability of ODN A and mutants thereof was tested at various storage conditions. Furthermore, antiviral effects of ODN A were analyzed in various tissue culture HIV-1 infection models. Finally, circular dichroism spectroscopy was employed to gain insight into the structure of ODN A.We show here that ODN A is a powerful tool to abolish HIV-1 particle infectivity, as required for a candidate compound in vaginal microbicide applications. We demonstrate that ODN A is not only capable to prematurely activate the retroviral RNase H, but also prevents HIV-1 from entering host cells. ODN A also exhibited extraordinary stability lasting several weeks. Notably, ODN A is biologically active under various storage conditions, as well as in the presence of carboxymethylcellulose CMC (K-Y Jelly), a potential carrier for application as a vaginal microbicide. ODN A's remarkable thermostability is apparently due to its specific, guanosine-rich sequence. Interestingly, these residues can form G-quadruplexes and may lead to G-based DNA hyperstructures. Importantly, the pronounced antiviral activity of ODN A is maintained in the presence of human semen or semen-derived enhancer of virus infection (SEVI; i.e. amyloid fibrils), both known to enhance HIV infectivity and reduce the efficacy of some antiviral microbicides.Since ODN A efficiently inactivates HIV-1 and also displays high stability and resistance against semen, it combines unique and promising features for its further development as a vaginal microbicide against HIV.
| S-EPMC4957839 | biostudies-other