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1.
ACS Sens ; 7(2): 453-459, 2022 02 25.
Article in English | MEDLINE | ID: covidwho-1655454

ABSTRACT

Unravelling unique molecular targets specific to viruses is challenging yet critical for diagnosing emerging viral diseases. Nucleic acids and proteins are the major targets in diagnostic assays of viral pathogens. Identification of novel sequences and conformations of nucleic acids as targets is desirable for developing diagnostic assays specific to a virus of interest. Here, we disclose the identification and characterization of a highly conserved antiparallel G-quadruplex (GQ)-forming DNA sequence present within the SARS-CoV-2 genome. The two-quartet GQ with unique loop compositions formed a distinct recognition motif. Design, synthesis, and fine tuning of structure-activity of a set of small molecules led to the identification of a benzobisthiazole-based fluorogenic probe which unambiguously recognizes the target SARS-CoV-2 GQ DNA. A robust cost-effective assay was developed through thermal cycler PCR-based amplification of the antiparallel GQ-forming ORF1ab region of the SARS-CoV-2 genome and endpoint fluorescence detection with the probe. An exclusive pH window (3.5-4) helped trigger reliable conformational polymorphism (RCP) involving DNA duplex to GQ transformation, which aided the development of a GQ-RCP platform for the diagnosis of SARS-CoV-2 clinical samples. This general strategy can be adapted for the development of specific diagnostic assays targeting different noncanonical nucleic acid sequences.


Subject(s)
COVID-19 , G-Quadruplexes , COVID-19/diagnosis , Humans , Hydrogen-Ion Concentration , Nucleic Acid Amplification Techniques , SARS-CoV-2/genetics
2.
ACS Synth Biol ; 11(1): 317-324, 2022 01 21.
Article in English | MEDLINE | ID: covidwho-1586042

ABSTRACT

Current tools for detecting transgenic crops, such as polymerase chain reaction (PCR), require professional equipment and complex operation. Herein, we introduce a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system to analyze transgenes by designing an isothermal amplification to serve as the amplified reporter, allowing an isothermal and label-free detection of transgenic crops. The use of Cas12a allowed direct and specific recognition of transgenes. To enhance the sensitivity of the assay, we used rolling circle amplification (RCA) to monitor the recognition of transgenes by designing the RCA primer as the cleavage substrate of Cas12a. The presence of transgenes can be detected by monitoring the G-quadruplex in RCA amplicon using a G-quadruplex binding dye, N-methyl mesoporphyrin IX (NMM). We termed the assay as isoCRISPR and showed that the assay allowed distinguishing transgenic corn cultivars ("Bt11" and "MON89034") from nontransgenic corn cultivars ("yellow", "shenyu", "xianyu", and "jingke"). The isoCRISPR assay will enrich the toolbox for transgenic crop identification and broaden the application of CRISPR/Cas in food authenticity and safety.


Subject(s)
Biosensing Techniques , G-Quadruplexes , CRISPR-Cas Systems/genetics , Nucleic Acid Amplification Techniques , Polymerase Chain Reaction
3.
Viruses ; 13(11)2021 11 04.
Article in English | MEDLINE | ID: covidwho-1538544

ABSTRACT

Pseudorabies virus (PRV) is the causative agent of Aujeszky's disease, which still causes large economic losses for the swine industry. Therefore, it is urgent to find a new strategy to prevent and control PRV infection. Previous studies have proven that guanine (G)-rich DNA or RNA sequences in some other viruses' genomes have the potential to form G-quadruplex (G4), which serve as promising antivirus targets. In this study, we identified two novel G4-forming sequences, OriL-A and OriL-S, which are located at the upstream origin of replication (OriL) in the PRV genome and conserved across 32 PRV strains. Circular dichroism (CD) spectroscopy and a gel electrophoresis assay showed that the two G-rich sequences can fold into parallel G4 structures in vitro. Moreover, fluorescence resonance energy transfer (FRET) melting and a Taq polymerase stop assay indicated that the G4 ligand PhenDC3 has the capacity to bind and stabilize the G4. Notably, the treatment of PRV-infected cells with G4-stabilizer PhenDC3 significantly inhibited PRV DNA replication in host cells but did not affect PRV's attachment and entry. These results not only expand our knowledge about the G4 characteristics in the PRV genome but also suggest that G4 may serve as an innovative therapeutic target against PRV.


Subject(s)
Antiviral Agents/pharmacology , G-Quadruplexes , Herpesvirus 1, Suid/genetics , Replication Origin/genetics , Animals , Antiviral Agents/chemistry , Cell Line , DNA Replication/drug effects , DNA, Viral/biosynthesis , DNA, Viral/chemistry , DNA, Viral/drug effects , Fused-Ring Compounds/chemistry , Fused-Ring Compounds/pharmacology , G-Quadruplexes/drug effects , Genome, Viral/drug effects , Genome, Viral/genetics , Herpesvirus 1, Suid/drug effects , Herpesvirus 1, Suid/physiology , Replication Origin/drug effects , Swine , Virus Replication/drug effects
4.
J Phys Chem Lett ; 12(42): 10277-10283, 2021 Oct 28.
Article in English | MEDLINE | ID: covidwho-1469948

ABSTRACT

Guanine quadruplex (G4) structures in the viral genome have a key role in modulating viruses' biological activity. While several DNA G4 structures have been experimentally resolved, RNA G4s are definitely less explored. We report the first calculated G4 structure of the RG-1 RNA sequence of SARS-CoV-2 genome, obtained by using a multiscale approach combining quantum and classical molecular modeling and corroborated by the excellent agreement between the corresponding calculated and experimental circular dichroism spectra. We prove the stability of the RG-1 G4 arrangement as well as its interaction with G4 ligands potentially inhibiting viral protein translation.


Subject(s)
COVID-19/genetics , G-Quadruplexes , Genome, Viral , RNA, Viral/chemistry , RNA, Viral/genetics , SARS-CoV-2/genetics , COVID-19/virology , Humans , Models, Molecular , Nucleic Acid Conformation
5.
Int J Mol Sci ; 22(20)2021 Oct 12.
Article in English | MEDLINE | ID: covidwho-1463715

ABSTRACT

G-quadruplexes (G4s) are noncanonical nucleic acid structures involved in the regulation of key cellular processes, such as transcription and replication. Since their discovery, G4s have been mainly investigated for their role in cancer and as targets in anticancer therapy. More recently, exploration of the presence and role of G4s in viral genomes has led to the discovery of G4-regulated key viral pathways. In this context, employment of selective G4 ligands has helped to understand the complexity of G4-mediated mechanisms in the viral life cycle, and highlighted the possibility to target viral G4s as an emerging antiviral approach. Research in this field is growing at a fast pace, providing increasing evidence of the antiviral activity of old and new G4 ligands. This review aims to provide a punctual update on the literature on G4 ligands exploited in virology. Different classes of G4 binders are described, with emphasis on possible antiviral applications in emerging diseases, such as the current COVID-19 pandemic. Strengths and weaknesses of G4 targeting in viruses are discussed.


Subject(s)
Antiviral Agents/chemistry , G-Quadruplexes , Antiviral Agents/therapeutic use , COVID-19/drug therapy , COVID-19/virology , DNA, Viral/chemistry , DNA, Viral/metabolism , Humans , Ligands , MicroRNAs/antagonists & inhibitors , MicroRNAs/metabolism , RNA, Viral/chemistry , RNA, Viral/metabolism , SARS-CoV-2/isolation & purification , Virus Diseases/drug therapy , Virus Diseases/pathology
6.
Phys Chem Chem Phys ; 23(40): 22957-22971, 2021 Oct 20.
Article in English | MEDLINE | ID: covidwho-1462045

ABSTRACT

The identification of chemical compounds able to bind specific sites of the human/viral proteins involved in the SARS-CoV-2 infection cycle is a prerequisite to design effective antiviral drugs. Here we conduct a molecular dynamics study with the aim to assess the interactions of ivermectin, an antiparasitic drug with broad-spectrum antiviral activity, with the human Angiotensin-Converting Enzyme 2 (ACE2), the viral 3CLpro and PLpro proteases, and the viral SARS Unique Domain (SUD). The drug/target interactions have been characterized in silico by describing the nature of the non-covalent interactions found and by measuring the extent of their time duration along the MD simulation. Results reveal that the ACE2 protein and the ACE2/RBD aggregates form the most persistent interactions with ivermectin, while the binding with the remaining viral proteins is more limited and unspecific.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Antiviral Agents/metabolism , Coronavirus 3C Proteases/metabolism , Coronavirus Papain-Like Proteases/metabolism , Ivermectin/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Antiviral Agents/chemistry , Binding Sites , Coronavirus 3C Proteases/chemistry , Coronavirus Papain-Like Proteases/chemistry , G-Quadruplexes , Humans , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Ivermectin/chemistry , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Binding , Protein Domains , RNA/genetics , RNA/metabolism , SARS-CoV-2
7.
J Phys Chem Lett ; 11(14): 5661-5667, 2020 Jul 16.
Article in English | MEDLINE | ID: covidwho-1387115

ABSTRACT

Coronaviruses may produce severe acute respiratory syndrome (SARS). As a matter of fact, a new SARS-type virus, SARS-CoV-2, is responsible for the global pandemic in 2020 with unprecedented sanitary and economic consequences for most countries. In the present contribution we study, by all-atom equilibrium and enhanced sampling molecular dynamics simulations, the interaction between the SARS Unique Domain and RNA guanine quadruplexes, a process involved in eluding the defensive response of the host thus favoring viral infection of human cells. Our results evidence two stable binding modes involving an interaction site spanning either the protein dimer interface or only one monomer. The free energy profile unequivocally points to the dimer mode as the thermodynamically favored one. The effect of these binding modes in stabilizing the protein dimer was also assessed, being related to its biological role in assisting the SARS viruses to bypass the host protective response. This work also constitutes a first step in the possible rational design of efficient therapeutic agents aiming at perturbing the interaction between SARS Unique Domain and guanine quadruplexes, hence enhancing the host defenses against the virus.


Subject(s)
Betacoronavirus/chemistry , Betacoronavirus/genetics , Coronavirus Infections/virology , G-Quadruplexes/drug effects , Pneumonia, Viral/virology , RNA, Viral/chemistry , RNA, Viral/genetics , Betacoronavirus/drug effects , COVID-19 , Dimerization , Humans , Models, Molecular , Molecular Dynamics Simulation , Pandemics , Protein Binding , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics
8.
Molecules ; 26(16)2021 Aug 19.
Article in English | MEDLINE | ID: covidwho-1376915

ABSTRACT

G-quadruplexes (G4s) are higher-order supramolecular structures, biologically important in the regulation of many key processes. Among all, the recent discoveries relating to RNA-G4s, including their potential involvement as antiviral targets against COVID-19, have triggered the ever-increasing need to develop selective molecules able to interact with parallel G4s. Naphthalene diimides (NDIs) are widely exploited as G4 ligands, being able to induce and strongly stabilize these structures. Sometimes, a reversible NDI-G4 interaction is also associated with an irreversible one, due to the cleavage and/or modification of G4s by functional-NDIs. This is the case of NDI-Cu-DETA, a copper(II) complex able to cleave G4s in the closest proximity to the target binding site. Herein, we present two original Cu(II)-NDI complexes, inspired by NDI-Cu-DETA, differently functionalized with 2-(2-aminoethoxy)ethanol side-chains, to selectively drive redox-catalyzed activity towards parallel G4s. The selective interaction toward parallel G4 topology, controlled by the presence of 2-(2-aminoethoxy)ethanol side chains, was already firmly demonstrated by us using core-extended NDIs. In the present study, the presence of protonable moieties and the copper(II) cavity, increases the binding affinity and specificity of these two NDIs for a telomeric RNA-G4. Once defined the copper coordination relationship and binding constants by competition titrations, ability in G4 stabilization, and ROS-induced cleavage were analyzed. The propensity in the stabilization of parallel topology was highlighted for both of the new compounds HP2Cu and PE2Cu. The results obtained are particularly promising, paving the way for the development of new selective functional ligands for binding and destructuring parallel G4s.


Subject(s)
Coordination Complexes/chemistry , Copper/chemistry , G-Quadruplexes , Imides/chemistry , Naphthalenes/chemistry , Binding Sites , DEET/chemistry , Ligands , Oxidation-Reduction , Polyethylene Glycols/chemistry , Structure-Activity Relationship
9.
Brief Bioinform ; 22(2): 1150-1160, 2021 03 22.
Article in English | MEDLINE | ID: covidwho-1352102

ABSTRACT

The outbreak caused by the novel coronavirus SARS-CoV-2 has been declared a global health emergency. G-quadruplex structures in genomes have long been considered essential for regulating a number of biological processes in a plethora of organisms. We have analyzed and identified 25 four contiguous GG runs (G2NxG2NyG2NzG2) in the SARS-CoV-2 RNA genome, suggesting putative G-quadruplex-forming sequences (PQSs). Detailed analysis of SARS-CoV-2 PQSs revealed their locations in the open reading frames of ORF1 ab, spike (S), ORF3a, membrane (M) and nucleocapsid (N) genes. Identical PQSs were also found in the other members of the Coronaviridae family. The top-ranked PQSs at positions 13385 and 24268 were confirmed to form RNA G-quadruplex structures in vitro by multiple spectroscopic assays. Furthermore, their direct interactions with viral helicase (nsp13) were determined by microscale thermophoresis. Molecular docking model suggests that nsp13 distorts the G-quadruplex structure by allowing the guanine bases to be flipped away from the guanine quartet planes. Targeting viral helicase and G-quadruplex structure represents an attractive approach for potentially inhibiting the SARS-CoV-2 virus.


Subject(s)
COVID-19/virology , G-Quadruplexes , SARS-CoV-2/chemistry , Humans , Molecular Docking Simulation , Open Reading Frames
10.
Int J Mol Sci ; 22(15)2021 Jul 27.
Article in English | MEDLINE | ID: covidwho-1335095

ABSTRACT

G-quadruplexes are the non-canonical nucleic acid structures that are preferentially formed in G-rich regions. This structure has been shown to be associated with many biological functions. Regardless of the broad efforts on DNA G-quadruplexes, we still have limited knowledge on RNA G-quadruplexes, especially in a transcriptome-wide manner. Herein, by integrating the DMS-seq and the bioinformatics pipeline, we profiled and depicted the RNA G-quadruplexes in the human transcriptome. The genes that contain RNA G-quadruplexes in their specific regions are significantly related to immune pathways and the COVID-19-related gene sets. Bioinformatics analysis reveals the potential regulatory functions of G-quadruplexes on miRNA targeting at the scale of the whole transcriptome. In addition, the G-quadruplexes are depleted in the putative, not the real, PAS-strong poly(A) sites, which may weaken the possibility of such sites being the real cleaved sites. In brief, our study provides insight into the potential function of RNA G-quadruplexes in post-transcription.


Subject(s)
G-Quadruplexes , Transcriptome/genetics , COVID-19/genetics , Cell Line , Computational Biology , Gene Expression Profiling , Humans , MicroRNAs/chemistry , MicroRNAs/metabolism , Poly A/genetics , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Untranslated Regions/genetics
11.
Trends Biochem Sci ; 46(4): 270-283, 2021 04.
Article in English | MEDLINE | ID: covidwho-958915

ABSTRACT

RNA G-quadruplexes (RG4s) are four-stranded structures known to control gene expression mechanisms, from transcription to protein synthesis, and DNA-related processes. Their potential impact on RNA biology allows these structures to shape cellular processes relevant to disease development, making their targeting for therapeutic purposes an attractive option. We review here the current knowledge on RG4s, focusing on the latest breakthroughs supporting the notion of transient structures that fluctuate dynamically in cellulo, their interplay with RNA modifications, their role in cell compartmentalization, and their deregulation impacting the host immune response. We emphasize RG4-binding proteins as determinants of their transient conformation and effectors of their biological functions.


Subject(s)
G-Quadruplexes , Biology , DNA , Protein Biosynthesis , RNA/metabolism
12.
Int J Mol Sci ; 22(7)2021 Mar 26.
Article in English | MEDLINE | ID: covidwho-1299435

ABSTRACT

The importance of gene expression regulation in viruses based upon G-quadruplex may point to its potential utilization in therapeutic targeting. Here, we present analyses as to the occurrence of putative G-quadruplex-forming sequences (PQS) in all reference viral dsDNA genomes and evaluate their dependence on PQS occurrence in host organisms using the G4Hunter tool. PQS frequencies differ across host taxa without regard to GC content. The overlay of PQS with annotated regions reveals the localization of PQS in specific regions. While abundance in some, such as repeat regions, is shared by all groups, others are unique. There is abundance within introns of Eukaryota-infecting viruses, but depletion of PQS in introns of bacteria-infecting viruses. We reveal a significant positive correlation between PQS frequencies in dsDNA viruses and corresponding hosts from archaea, bacteria, and eukaryotes. A strong relationship between PQS in a virus and its host indicates their close coevolution and evolutionarily reciprocal mimicking of genome organization.


Subject(s)
Computational Biology/methods , DNA/genetics , G-Quadruplexes , Genome, Viral , Viral Proteins/genetics , Archaea/virology , Bacteria/virology , Gene Expression Regulation , Genome , Humans , Viruses/genetics
13.
Nucleic Acids Res ; 49(13): 7695-7712, 2021 07 21.
Article in English | MEDLINE | ID: covidwho-1298980

ABSTRACT

The multidomain non-structural protein 3 (Nsp3) is the largest protein encoded by coronavirus (CoV) genomes and several regions of this protein are essential for viral replication. Of note, SARS-CoV Nsp3 contains a SARS-Unique Domain (SUD), which can bind Guanine-rich non-canonical nucleic acid structures called G-quadruplexes (G4) and is essential for SARS-CoV replication. We show herein that the SARS-CoV-2 Nsp3 protein also contains a SUD domain that interacts with G4s. Indeed, interactions between SUD proteins and both DNA and RNA G4s were evidenced by G4 pull-down, Surface Plasmon Resonance and Homogenous Time Resolved Fluorescence. These interactions can be disrupted by mutations that prevent oligonucleotides from folding into G4 structures and, interestingly, by molecules known as specific ligands of these G4s. Structural models for these interactions are proposed and reveal significant differences with the crystallographic and modeled 3D structures of the SARS-CoV SUD-NM/G4 interaction. Altogether, our results pave the way for further studies on the role of SUD/G4 interactions during SARS-CoV-2 replication and the use of inhibitors of these interactions as potential antiviral compounds.


Subject(s)
COVID-19/virology , Coronavirus Papain-Like Proteases/metabolism , G-Quadruplexes , Protein Interaction Domains and Motifs , SARS-CoV-2 , Amino Acid Sequence , Coronavirus Papain-Like Proteases/chemistry , Humans , Ligands , Models, Molecular , Protein Binding , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Spectrum Analysis , Structure-Activity Relationship , Virus Replication
14.
Anal Chem ; 93(28): 9933-9938, 2021 07 20.
Article in English | MEDLINE | ID: covidwho-1297285

ABSTRACT

Sensitive detection of the SARS-CoV-2 protein remains a great research interest in clinical screening and diagnosis owing to the coronavirus epidemic. Here, an ultrasensitive chemiluminescence (CL) imaging strategy was developed through proximity hybridization to trigger the formation of a rolling circle-amplified G-quadruplex/hemin DNAzyme for the detection of the SARS-CoV-2 protein. The target protein was first recognized by a pair of DNA-antibody conjugates, Ab-1 and Ab-2, to form a proximity-ligated complex, Ab-1/SARS-CoV-2/Ab-2, which contained a DNA sequence complemental to block DNA and thus induced a strand displacement reaction to release the primer from a block/primer complex. The released primer then triggered a rolling circle amplification to form abundant DNAzyme units in the presence of hemin, which produced a strong chemiluminescent signal for the detection of the target protein by catalyzing the oxidation of luminol by hydrogen peroxide. The proposed assay showed a detectable concentration range over 5 orders of magnitude with the detection limit down to 6.46 fg/mL. The excellent selectivity, simple procedure, acceptable accuracy, and intrinsic high throughput of the imaging technique for analysis of serum samples demonstrated the potential applicability of the proposed detection method in clinical screening and diagnosis.


Subject(s)
Biosensing Techniques , COVID-19 , DNA, Catalytic , G-Quadruplexes , DNA, Catalytic/metabolism , Hemin , Humans , Immunoassay , Limit of Detection , Luminescence , SARS-CoV-2
15.
PLoS One ; 16(6): e0250654, 2021.
Article in English | MEDLINE | ID: covidwho-1261292

ABSTRACT

Quadruplex structures have been identified in a plethora of organisms where they play important functions in the regulation of molecular processes, and hence have been proposed as therapeutic targets for many diseases. In this paper we report the extensive bioinformatic analysis of the SARS-CoV-2 genome and related viruses using an upgraded version of the open-source algorithm G4-iM Grinder. This version improves the functionality of the software, including an easy way to determine the potential biological features affected by the candidates found. The quadruplex definitions of the algorithm were optimized for SARS-CoV-2. Using a lax quadruplex definition ruleset, which accepts amongst other parameters two residue G- and C-tracks, 512 potential quadruplex candidates were discovered. These sequences were evaluated by their in vitro formation probability, their position in the viral RNA, their uniqueness and their conservation rates (calculated in over seventeen thousand different COVID-19 clinical cases and sequenced at different times and locations during the ongoing pandemic). These results were then compared subsequently to other Coronaviridae members, other Group IV (+)ssRNA viruses and the entire viral realm. Sequences found in common with other viral species were further analyzed and characterized. Sequences with high scores unique to the SARS-CoV-2 were studied to investigate the variations amongst similar species. Quadruplex formation of the best candidates were then confirmed experimentally. Using NMR and CD spectroscopy, we found several highly stable RNA quadruplexes that may be suitable therapeutic targets for the SARS-CoV-2.


Subject(s)
G-Quadruplexes , Genome, Viral , Nucleotide Motifs , RNA, Viral/genetics , SARS-CoV-2/genetics , Computational Biology , Guanine
16.
BMC Genomics ; 22(1): 77, 2021 Jan 23.
Article in English | MEDLINE | ID: covidwho-1045026

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.


Subject(s)
G-Quadruplexes , Influenza A Virus, H1N1 Subtype , Influenza, Human , Genome, Viral , Genotype , Humans , Influenza A Virus, H1N1 Subtype/genetics , Reassortant Viruses/genetics
17.
Int J Mol Sci ; 22(5)2021 Mar 05.
Article in English | MEDLINE | ID: covidwho-1129734

ABSTRACT

The Coronavirus Disease 2019 (COVID-19) pandemic has become a global health emergency with no effective medical treatment and with incipient vaccines. It is caused by a new positive-sense RNA virus called severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). G-quadruplexes (G4s) are nucleic acid secondary structures involved in the control of a variety of biological processes including viral replication. Using several G4 prediction tools, we identified highly putative G4 sequences (PQSs) within the positive-sense (+gRNA) and negative-sense (-gRNA) RNA strands of SARS-CoV-2 conserved in related betacoronaviruses. By using multiple biophysical techniques, we confirmed the formation of two G4s in the +gRNA and provide the first evidence of G4 formation by two PQSs in the -gRNA of SARS-CoV-2. Finally, biophysical and molecular approaches were used to demonstrate for the first time that CNBP, the main human cellular protein bound to SARS-CoV-2 RNA genome, binds and promotes the unfolding of G4s formed by both strands of SARS-CoV-2 RNA genome. Our results suggest that G4s found in SARS-CoV-2 RNA genome and its negative-sense replicative intermediates, as well as the cellular proteins that interact with them, are relevant factors for viral genes expression and replication cycle, and may constitute interesting targets for antiviral drugs development.


Subject(s)
G-Quadruplexes , RNA-Binding Proteins/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Circular Dichroism , Computational Biology/methods , Databases, Genetic , Electrophoretic Mobility Shift Assay , Genome, Viral/physiology , Humans , Proton Magnetic Resonance Spectroscopy , Virus Replication/physiology
18.
Can J Physiol Pharmacol ; 99(5): 449-460, 2021 May.
Article in English | MEDLINE | ID: covidwho-1125455

ABSTRACT

Ribavirin is a guanosine analog with broad-spectrum antiviral activity against RNA viruses. Based on this, we aimed to show the anti-SARS-CoV-2 activity of this drug molecule via in vitro, in silico, and molecular techniques. Ribavirin showed antiviral activity in Vero E6 cells following SARS-CoV-2 infection, whereas the drug itself did not show any toxic effect over the concentration range tested. In silico analysis suggested that ribavirin has a broad-spectrum impact on SARS-CoV-2, acting at different viral proteins. According to the detailed molecular techniques, ribavirin was shown to decrease the expression of TMPRSS2 at both mRNA and protein levels 48 h after treatment. The suppressive effect of ribavirin in ACE2 protein expression was shown to be dependent on cell types. Finally, proteolytic activity assays showed that ribavirin also showed an inhibitory effect on the TMPRSS2 enzyme. Based on these results, we hypothesized that ribavirin may inhibit the expression of TMPRSS2 by modulating the formation of inhibitory G-quadruplex structures at the TMPRSS2 promoter. As a conclusion, ribavirin is a potential antiviral drug for the treatment against SARS-CoV-2, and it interferes with the effects of TMPRSS2 and ACE2 expression.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Antiviral Agents/pharmacology , Down-Regulation/drug effects , Ribavirin/pharmacology , SARS-CoV-2/drug effects , Serine Endopeptidases/metabolism , Animals , Caco-2 Cells , Chlorocebus aethiops , G-Quadruplexes/drug effects , Humans , Promoter Regions, Genetic/genetics , SARS-CoV-2/physiology , Serine Endopeptidases/genetics , Vero Cells
19.
Angew Chem Int Ed Engl ; 60(1): 432-438, 2021 01 04.
Article in English | MEDLINE | ID: covidwho-774564

ABSTRACT

The COVID-19 pandemic caused by SARS-CoV-2 has become a global threat. Understanding the underlying mechanisms and developing innovative treatments are extremely urgent. G-quadruplexes (G4s) are important noncanonical nucleic acid structures with distinct biofunctions. Four putative G4-forming sequences (PQSs) in the SARS-CoV-2 genome were studied. One of them (RG-1), which locates in the coding sequence region of SARS-CoV-2 nucleocapsid phosphoprotein (N), has been verified to form a stable RNA G4 structure in live cells. G4-specific compounds, such as PDP (pyridostatin derivative), can stabilize RG-1 G4 and significantly reduce the protein levels of SARS-CoV-2 N by inhibiting its translation both in vitro and in vivo. This result is the first evidence that PQSs in SARS-CoV-2 can form G4 structures in live cells, and that their biofunctions can be regulated by a G4-specific stabilizer. This finding will provide new insights into developing novel antiviral drugs against COVID-19.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , G-Quadruplexes/drug effects , RNA, Viral/drug effects , SARS-CoV-2/drug effects , Drug Evaluation, Preclinical , Gene Expression Regulation, Viral/drug effects , Genome, Viral , Humans , Nucleocapsid Proteins/chemistry , Nucleocapsid Proteins/drug effects , Protein Folding , SARS-CoV-2/genetics , Small Molecule Libraries , Temperature
20.
Biosens Bioelectron ; 167: 112494, 2020 Nov 01.
Article in English | MEDLINE | ID: covidwho-694826

ABSTRACT

G-quadruplex is a non-canonical nucleic acid structure formed by the folding of guanine rich DNA or RNA. The conformation and function of G-quadruplex are determined by a number of factors, including the number and polarity of nucleotide strands, the type of cations and the binding targets. Recent studies led to the discovery of additional advantageous attributes of G-quadruplex with the potential to be used in novel biosensors, such as improved ligand binding and unique folding properties. G-quadruplex based biosensor can detect various substances, such as metal ions, organic macromolecules, proteins and nucleic acids with improved affinity and specificity compared to standard biosensors. The recently developed G-quadruplex based biosensors include electrochemical and optical biosensors. A novel G-quadruplex based biosensors also show better performance and broader applications in the detection of a wide spectrum of pathogens, including SARS-CoV-2, the causative agent of COVID-19 disease. This review highlights the latest developments in the field of G-quadruplex based biosensors, with particular focus on the G-quadruplex sequences and recent applications and the potential of G-quadruplex based biosensors in SARS-CoV-2 detection.


Subject(s)
Betacoronavirus , Biosensing Techniques/methods , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Coronavirus Infections/virology , G-Quadruplexes , Pneumonia, Viral/diagnosis , Pneumonia, Viral/virology , Betacoronavirus/genetics , Betacoronavirus/isolation & purification , Biosensing Techniques/trends , COVID-19 , COVID-19 Testing , Clinical Laboratory Techniques/trends , Colorimetry , Electrochemical Techniques , Fluorescent Dyes , Humans , Pandemics , SARS-CoV-2
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