Putative Quadruplex Sequences Research Articles

Identification of G-quadruplex DNA sequences in SARS-CoV2.

G-quadruplex structure or Putative Quadruplex Sequences (PQSs) are abundant in human, microbial, DNA, or RNA viral genomes. These sequences in RNA viral genome play critical roles in integration into human genome as LTR (Long Terminal Repeat), genome replication, chromatin rearrangements, gene regulation, antigen variation (Av), and virulence. Here, we investigated whether the genome of SARS-CoV2, an RNA virus, contained such potential G-quadruplex structures. Using bioinformatic tools, we searched for such sequences and found thirty-seven (forward strand (twenty-five) + reverse strand (Twelve)) QGRSs (Quadruplex forming G-Rich Sequences)/PQSs in SARS-CoV2 genome. These sequences are dispersed mainly in the upstream of SARS-CoV2 genes. We discuss whether existing PQS/QGRS ligands could inhibit the SARS-CoV2 replication and gene transcription as has been observed in other RNA viruses. Further experimental validation would determine the role of these G-quadruplex sequences in SARS-CoV2 genome function to survive in the host cells and identify therapeutic agents to destabilize these PQSs/QGRSs.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00251-022-01257-6.

How bioinformatics resources work with G4 RNAs.

Quadruplexes (G4s) are of interest, which increases with the number of identified G4 structures and knowledge about their biomedical potential. These unique motifs form in many organisms, including humans, where their appearance correlates with various diseases. Scientists store and analyze quadruplexes using recently developed bioinformatic tools—many of them focused on DNA structures. With an expanding collection of G4 RNAs, we check how existing tools deal with them. We review all available bioinformatics resources dedicated to quadruplexes and examine their usefulness in G4 RNA analysis. We distinguish the following subsets of resources: databases, tools to predict putative quadruplex sequences, tools to predict secondary structure with quadruplexes and tools to analyze and visualize quadruplex structures. We share the results obtained from processing specially created RNA datasets with these tools. Contact: mszachniuk@cs.put.poznan.pl Supplementary information: Supplementary data are available at Briefings in Bioinformatics online.

Relationship Between G-Quadruplex Sequence Composition in Viruses and Their Hosts.

A subset of guanine-rich nucleic acid sequences has the potential to fold into G-quadruplex (G4) secondary structures, which are functionally important for several biological processes, including genome stability and regulation of gene expression. Putative quadruplex sequences (PQSs) G3+N1–7G3+N1–7G3+N1–7G3+ are widely found in eukaryotic and prokaryotic genomes, but the base composition of the N1-7 loops is biased across species. Since the viruses partially hijack their hosts’ cellular machinery for proliferation, we examined the PQS motif size, loop length, and nucleotide compositions of 7370 viral genome assemblies and compared viral and host PQS motifs. We studied seven viral taxa infecting five distant eukaryotic hosts and created a resource providing a comprehensive view of the viral quadruplex motifs. Overall, short-looped PQSs are predominant and with a similar composition across viral taxonomic groups, albeit subtle trends emerge upon classification by hosts. Specifically, there is a higher frequency of pyrimidine loops in viruses infecting animals irrespective of the viruses’ genome type. This observation is confirmed by an in-depth analysis of the Herpesviridae family of viruses, which showed a distinctive accumulation of thermally stable C-looped quadruplexes in viruses infecting high-order vertebrates. The occurrence of viral C-looped G4s, which carry binding sites for host transcription factors, as well as the high prevalence of viral TTA-looped G4s, which are identical to vertebrate telomeric motifs, provide concrete examples of how PQSs may help viruses impinge upon, and benefit from, host functions. More generally, these observations suggest a co-evolution of virus and host PQSs, thus underscoring the potential functional significance of G4s.

Abstract B04: Direct downregulation of MYC in AML cells using promoter G-quadruplex interacting small molecules

Abstract Acute myeloid leukemia (AML) is a disease that can affect both children and adults and is characterized by complex and heterogeneous genomic changes, including MYC overexpression driven in part by the bromodomain (BRD) and extraterminal (BET) protein family of epigenetic readers. Current limitations in treatment approaches, including protein overexpression and mutations that confer resistance to therapy, lead to a wide range of response variability to conventional therapy, excessive treatment-related toxicity, and an overall poor outcome. A new therapeutic strategy involves stabilizing the MYC promoter G-quadruplex to downregulate expression. We hypothesize that direct downregulation of MYC in AML cells using MYC-promoter G-quadruplex interacting drugs (GQID) is a feasible novel approach in targeted therapy of AML. Treatment of AML cell lines KG-1a and UT-7epo with the MYC-promoter GQID, GQC-05 resulted in decreased MYC expression and induced a more significant decrease in c-Myc protein than treatment with the BET inhibitor (+)-JQ1. GQC-05 also decreased cell viability and increased apoptosis. RNA-seq of GQC-05-treated KG-1a cells identified 947 significantly differentially expressed genes compared to DMSO. The expression of several c-MYC target genes (including E2F1, GNL3, HSPA8, MAT2A, RCC1, SHMT1, and TFRC) was significantly decreased concomitant with MYC downregulation. The 947 genes were compared with genes with putative quadruplex sequences (PQS) from the literature. Of the 17 genes in common, MYC, NOTCH1, PIM1, and RHOU are significantly downregulated following GQC-05 treatment. These findings were validated by Q-RT-PCR in KG-1a and several other AML cell lines. Drug sensitivity and resistance (DSR) screening on a panel of AML cell lines using a library of 55 anticancer and targeted agents identified a range of sensitivities to GQC-05 (AUC range: 1.65-2.99; DMSO AUC ~ 4). These were compared to microarray gene-expression profiles and, as expected, GQC-05 sensitivity was not correlated with MYC expression. Nevertheless, ANOVA identified 1,049 significantly differentially expressed probe sets between the most sensitive vs. the least sensitive cell lines. Functional annotation revealed over-representation of cadherin, Wnt, and p53 signaling pathways as well over genes involved in hemostasis and hematopoietic cell lineage. In conclusion, directly targeting the MYC promoter G-quadruplex in AML cells leads to knockdown of MYC expression and induces apoptosis. These results further support the development of GQID for targeting key genetic drivers in AML, and lay the groundwork for advances in treatment of other cancers driven by G-quadruplex regulated oncogenes. Citation Format: Megan A. Turnidge, Daniel H. Wai, Apurvi Patel, Justin J. Montoya, David W. Lee, Laurence H. Hurley, Vijay Gokhale, Robert J. Arceci, David O. Azorsa. Direct downregulation of MYC in AML cells using promoter G-quadruplex interacting small molecules [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B04.

Machine learning model for sequence-driven DNA G-quadruplex formation

We describe a sequence-based computational model to predict DNA G-quadruplex (G4) formation. The model was developed using large-scale machine learning from an extensive experimental G4-formation dataset, recently obtained for the human genome via G4-seq methodology. Our model differentiates many widely accepted putative quadruplex sequences that do not actually form stable genomic G4 structures, correctly assessing the G4 folding potential of over 700,000 such sequences in the human genome. Moreover, our approach reveals the relative importance of sequence-based features coming from both within the G4 motifs and their flanking regions. The developed model can be applied to any DNA sequence or genome to characterise sequence-driven intramolecular G4 formation propensities.

Genome-wide analysis of G-quadruplexes in herpesvirus genomes.

BackgroundG-quadruplexes are increasingly recognized as regulatory elements in human, animal, bacterial and plant genomes. The presence and function of G-quadruplexes are not well studied among herpesviruses; in particular, there are no systematic genome-wide analysis of these important secondary structures in herpesvirus genomes.ResultsWe performed genome-wide analysis of putative quadruplex sequences (PQS) in human herpesviruses. We found unusually high PQS densities among human herpesviruses. PQS are enriched in the repeat regions and regulatory regions of human herpesviruses. Interestingly, PQS densities are higher in regulatory regions of immediate early genes compared to early and late genes in most herpesviruses. In addition, the majority of genes functionally conserved across human herpesviruses contain one or more PQS within the regulatory regions. We also describe the existence of unique intramolecular PQS repeats or repetitive G-quadruplex motifs in herpesviruses. Functional studies confirm a role for G-quadruplexes in regulating the gene expression of human herpesviruses.ConclusionThe pervasiveness of PQS, their enrichment and conservation at specific genomic locations suggest that these structural entities may represent a novel class of functional elements in herpesviruses. Our findings provide the necessary framework for studies on the biological role of G-quadruplexes in herpesviruses.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3282-1) contains supplementary material, which is available to authorized users.

Recent Advances in Anticancer Drugs Development: G-Quadruplex as New Drug Target

Nucleic Acids are large biomolecules and indispensable for life. They include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids can adopt distinct non-canonical, highly compact secondary structure, G-quadruplex, in the dynamic region of chromosomal DNA and RNA transcripts, predominantly in telomeric sequences, and in promoter region of numerous genes including oncogenes such as Bcl-2 [1,2], VEGF [3,4], c-myc [5]. There are 376,000 putative quadruplex sequences (PQS) in the human genome that have been identified through genome-wide surveys based on a quadruplex folding rule [6], however, all of them may not exist in vivo. Recently, the existence of DNA G-quadruplex has been visualized on chromosomes in human cells [7]. These G-quadruplexes are an active target of drug discovery. The DNA G-quadruplexes formed in the promoter region of oncogene have been shown to be potential targets for anticancer drugs [8,9] and proteins. The formation of these quadruplexes in telomeres has been shown to regulate the activity of the enzyme telomerase, which maintains the length of telomeres and is involved in ~85% of all cancers. For example, Telomestatin [10,11], S2T1-6OTD (telomestatin synthetic Derivative) [12], SYUIQ-5 [13] interact with G-quadruplex formed in telomere and myc sequences and show their inhibitory activity in cancer cell growth. This has become a progressively large field of research. The G-quadruplexes are very condensed structures and formed by the ganosine (G)-rich DNA and RNA sequences and consists of several stacked G-tetrads. Each G-tetrad has four guanine arranged in a square planar arrangement and held together by hoogsteen hydrogen bonding. Besides this, each G-quadruplex structure is further stabilized by the presence of a monovalent cation, mainly potassium, which is localized in the center between each pair of tetrads. The G-quadruplexes could affect gene activity either by upregulation or down regulation, which can be achieved by inducing or stabilizing G-quadruplex formation through a G-quadruplex interacting molecule (small molecule drugs or protein) that can stabilize the G-quadruplex structure and thus perform their desired function [8]. The validation of drug-targeted G-quadruplex DNA and the modulation of cancer genes’ expression has been intensely increased in the recent past, thus opening new avenue for cancer research. Though a lot of research has been focused on DNA G-quadruplexes, there has lately been a rapid advancement in the area of RNA G-quadruplexes, chiefly in the 5 ′-UTRs (untranslated regions) of mRNAs. RNA G-quadruplexes in the 5’-UTRs of mRNAs impact posttranscriptional regulation of gene expression, which affects disruption of normal cell behavior in human diseases, particularly cancers. The recent in-vitro study on small molecule G-quadruplex binding compound that can selectively target RNA G-quadruplexes exposed a new and striking opportunity for RNA-directed drug design [14]. However, there is still a major challenge to understand the systematic effects and selectivity in in-vivo environment. It is distinct that the RNA G-quadruplex motif embodies a structurally attractive scaffold for small molecule targeting and therefore provides a productive area for future research [15].

Validation and Physical Characterization of Ribosomal G-Quadruplexes with MD Simulations

DNA and RNA adopt structures known as G-quadruplexes, and they are typically found in the telomere regions of chromosomes and in promoter regions. G-quadruplexes that form in gene promoter regions are targeted at the nucleolus, the site of ribosome biogenesis, and its formation and stabilization in the nucleolus can act as silencers in gene expression, control cell growth, and thus provide a viable treatment for cancer. Although it is well-known that ribosomal sequences are guanine-rich and could potentially form G-quadruplexes, these putative sequences are seldom validated. In our study, we modeled two putative parallel DNA quadruplex sequences that are based on structures of known stable parallel and antiparallel DNA quadruplexes. We performed 20 ns atomistic molecular dynamic simulations with the CHARMM36 force field and the NAMD simulation suite. We then calculated the base-base distances and pseudo-torsion angles to project these quantities to free energy profiles for the purpose of determining their relative stabilities. Based on these simulations, we created a conformational and structural map based on the free energy stabilities of two putative quadruplex sequences, and they can serve as the basis of developing selective anti-cancer agents.

Transfecting RNA quadruplexes results in few transcriptome perturbations

Guanine-rich nucleic acid sequences can form four-stranded structures called G-quadruplexes. Previous studies showed that transfecting G-quadruplex DNA oligonucleotides inhibits proliferation in many cancer cell lines and can induce apoptosis. However, little is known about the effects of transfecting RNA quadruplexes. In this study, we transfected a G-quadruplex RNA oligonucleotide (GqRNA) into HEK293T cells and observed that it did not alter cell viability. Subsequent transcriptome expression profiling revealed that only two genes, EGR1 and FOS, were significantly altered in the presence of GqRNA (upregulated 2- to 4-fold). Sequence analysis showed that both genes contained putative quadruplex sequences (PQS) in their 3′-UTRs, immediately adjacent to the stop codons. Transfection of the EGR1 PQS as an RNA oligonucleotide also caused an increase in EGR1 expression. Similar motifs are found in a variety of genomes, but are relatively rare and have been missed by previous annotations. A bioinformatic analysis revealed stop codon-proximal enrichment of such motifs compared with the rest of the 3′-UTR, although these genes were not affected by RNA quadruplex transfection, and their function remains unknown. Overall, transfecting RNA quadruplexes results in relatively few alterations in gene expression.

Stability of intramolecular quadruplexes: sequence effects in the central loop.

Hundreds of thousands of putative quadruplex sequences have been found in the human genome. It is important to understand the rules that govern the stability of these intramolecular structures. In this report, we analysed sequence effects in a 3-base-long central loop, keeping the rest of the quadruplex unchanged. A first series of 36 different sequences were compared; they correspond to the general formula GGGTTTGGGHNHGGGTTTGGG. One clear rule emerged from the comparison of all sequence motifs: the presence of an adenine at the first position of the loop was significantly detrimental to stability. In contrast, adenines have no detrimental effect when present at the second or third position of the loop. Cytosines may either have a stabilizing or destabilizing effect depending on their position. In general, the correlation between the Tm or ΔG° in sodium and potassium was weak. To determine if these sequence effects could be generalized to different quadruplexes, specific loops were tested in different sequence contexts. Analysis of 26 extra sequences confirmed the general destabilizing effect of adenine as the first base of the loop(s). Finally, analysis of some of the sequences by microcalorimetry (DSC) confirmed the differences found between the sequence motifs.

Fast detection of quadruplex structure in DNA by the intrinsic fluorescence of a single‐stranded DNA binding protein

Single-stranded guanine-rich (G-rich) DNA can fold into a four-stranded G-quadruplex structure and such structures are implicated in important biological processes and therapeutic applications. So far, bioinformatic analysis has identified up to several hundred thousand of putative quadruplex sequences in the genome of human and other animal. Given such a large number of sequences, a fast assay would be desired to experimentally verify the structure of these sequences. Here we describe a method that identifies the quadruplex structure by a single-stranded DNA binding protein from a thermoautotrophic archaeon. This protein binds single-stranded DNA in the unfolded, but not in the folded form. Upon binding to DNA, its fluorescence can be quenched by up to 70%. Formation of quadruplex greatly reduces fluorescence quenching in a K+-dependent manner. This structure-dependent quenching provides simple and fast detection of quadruplex in DNA at low concentration without DNA labelling.