Structure of a DNA G-quadruplex that Modulates SP1 Binding Sites Architecture in HIV-1 Promoter.

Nucleic acid sequences containing guanine tracts are able to form non-canonical DNA or RNA structures known as G-quadruplexes (or G4s). These structures, based on the stacking of G-tetrads, are involved in various biological processes such as gene expression regulation. Here, we investigated a G4 forming sequence, HIVpro2, derived from the HIV-1 promoter. This motif is located 60 nucleotides upstream of the proviral Transcription Starting Site (TSS) and overlaps with two SP1 transcription factor binding sites. Using NMR spectroscopy, we determined that HIVpro2 forms a hybrid type G4 structure with a core that is interrupted by a single nucleotide bulge. An additional reverse-Hoogsteen AT base pair is stacked on top of the tetrad. SP1 transcription factor is known to regulate transcription activity of many genes through the recognition of Guanine-rich duplex motifs. Here, the formation of HIVpro2 G4 may modulate SP1 binding sites architecture by competing with the formation of the canonical duplex structure. Such DNA structural switch potentially participates to the regulation of viral transcription and may also interfere with HIV-1 reactivation or viral latency.

Structural Perspective into the Interaction of an Oncogenesis-Relevant pre-miRNA G-Quadruplex Ligand Carrier with the Protein Nucleolin.

The structural determinants of the interaction of the G-quadruplex (G4) motif found in precursor miRNA 149 (rG4) with the acridine orange derivative C8 , a G4 ligand stabilizer possessing anticancer activity, and the protein nucleolin (overexpressed in cancer cells) were investigated by Nuclear Magnetic Resonance (NMR) spectroscopy. For the rG4/C8 complex, the results revealed a strong stabilizing interaction between the aromatic core and the iodinated ring of the C8 ligand with the rG4 structure. The NMR study revealed also different interaction patterns between nucleolin and rG4 and nucleolin and rG4/C8 complex. In the absence of the ligand, rG4 establishes interactions with polar residues of the protein while for the rG4/C8 complex, these contacts are mainly established with amino acids that have hydrophobic side chains. However, nucleolin chemical shift perturbation studies in the presence of rG4 or rG4/C8 reveal the same location between domains 1 and 2 of the protein, which suggests that the rG4 and rG4/C8 complex bind in this region. This puzzling structural study opens a new framework to study rG4/ligand/nucleolin complexes that might impact the biogenesis of miRNA 149.

Guidelines for G-quadruplexes: I. In vitro characterization.

Besides the well-known DNA double-helix, non-canonical nucleic acid structures regulate crucial biological activities. Among these oddities, guanine-rich DNA sequences can form unusual four-stranded secondary structures called G-quadruplexes (G4s). G4-prone sequences have been found in the genomes of most species, and G4s play important roles in essential processes such as transcription, replication, genome integrity and epigenetic regulation. Here, we present a short overview of G-quadruplexes followed by a detailed description of the biophysical and biochemical methods used to characterize G4s in vitro. The principles, experimental details and possible shortcomings of each method are discussed to provide a comprehensive view of the techniques used to study these structures. We aim to provide a set of guidelines for standardizing research on G-quadruplexes; these guidelines are not meant to be a dogmatic set of rules, but should rather provide useful information on the methods currently used to study these fascinating motifs.

Deciphering RNA G-quadruplex function during the early steps of HIV-1 infection.

G-quadruplexes (G4s) are four-stranded nucleic acid structures formed by the stacking of G-tetrads. Here we investigated their formation and function during HIV-1 infection. Using bioinformatics and biophysics analyses we first searched for evolutionary conserved G4-forming sequences in HIV-1 genome. We identified 10 G4s with conservation rates higher than those of HIV-1 regulatory sequences such as RRE and TAR. We then used porphyrin-based G4-binders to probe the formation of the G4s during infection of human cells by native HIV-1. The G4-binders efficiently inhibited HIV-1 infectivity, which is attributed to the formation of G4 structures during HIV-1 replication. Using a qRT-PCR approach, we showed that the formation of viral G4s occurs during the first 2 h post-infection and their stabilization by the G4-binders prevents initiation of reverse transcription. We also used a G4-RNA pull-down approach, based on a G4-specific biotinylated probe, to allow the direct detection and identification of viral G4-RNA in infected cells. Most of the detected G4-RNAs contain crucial regulatory elements such as the PPT and cPPT sequences as well as the U3 region. Hence, these G4s would function in the early stages of infection when the viral RNA genome is being processed for the reverse transcription step.

RNA G-quadruplex forming regions from SARS-2, SARS-1 and MERS coronoviruses.

COVID-19 (Corona Virus Disease 2019), SARS (Severe Acute Respiratory Syndrome) and MERS (Middle East Respiratory Syndrome) are infectious diseases each caused by coronavirus outbreaks. Small molecules and other therapeutics are rapidly being developed to treat these diseases, but the threat of new variants and outbreaks argue for the identification of additional viral targets. Here we identify regions in each of the three coronavirus genomes that are able to form G-quadruplex (G4) structures. G4s are structures formed by DNA or RNA with a core of two or more stacked planes of guanosine tetrads. In recent years, numerous DNA and RNA G4s have emerged as promising pharmacological targets for the treatment of cancer and viral infection. We use a combination of bioinformatics and biophysical approaches to identify conserved RNA G4 regions from the ORF1A and S sequences of SARS-CoV, SARS-CoV-2 and MERS-CoV. Although a general depletion of G4-forming regions is observed in coronaviridae, the preservation of these selected G4 sequences support a significance in viral replication. Targeting these RNA structures may represent a new antiviral strategy against these viruses distinct from current approaches that target viral proteins.

G-quadruplex structure of the C. elegans telomeric repeat: a two tetrads basket type conformation stabilized by a non-canonical C-T base-pair.

The Caenorhabditis elegans model has greatly contributed to the understanding of the role of G-quadruplexes in genomic instability. The GGCTTA repeats of the C. elegans telomeres resemble the GGGTTA repeats of the human telomeres. However, the comparison of telomeric sequences (Homo sapiens, Tetrahymena, Oxytricha, Bombyx mori and Giardia) revealed that small changes in these repeats can drastically change the topology of the folded G-quadruplex. In the present work we determined the structure adopted by the C. elegans telomeric sequence d[GG(CTTAGG)3]. The investigated C. elegans telomeric sequence is shown to fold into an intramolecular two G-tetrads basket type G-quadruplex structure that includes a C-T base pair in the diagonal loop. This work sheds light on the telomeric structure of the widely used C. elegans animal model.

Gold(III) porphyrins: Synthesis and interaction with G-quadruplex DNA.

G-quadruplex nucleic acids (G4s) are RNA and DNA secondary structures involved in the regulation of multiple key biological processes. They can be found in telomeres, oncogene promoters, RNAs, but also in viral genomes. Due to their unique structural features, very distinct from the canonical duplexes or single-strands, G4s represent promising pharmacological targets for small molecules, namely G4-ligands. Gold(III) penta-cationic porphyrins, as specific G4 ligands, are able to inhibit HIV-1 infectivity and their antiviral activity correlates with their affinity for G4s. Up to now, one of the best antiviral compounds is meso-5,10,15,20-tetrakis[4-(N-methyl-pyridinium-2-yl)phenyl]porphyrinato gold(III) (1). Starting from this compound, we report a structure/affinity relationship study of gold(III) cationic porphyrins to find out the best porphyrin candidate for functionalization, in order to study the antiviral mechanism of action of these gold(III) porphyrins.

The beginning and the end: flanking nucleotides induce a parallel G-quadruplex topology.

Genomic sequences susceptible to form G-quadruplexes (G4s) are always flanked by other nucleotides, but G4 formation in vitro is generally studied with short synthetic DNA or RNA oligonucleotides, for which bases adjacent to the G4 core are often omitted. Herein, we systematically studied the effects of flanking nucleotides on structural polymorphism of 371 different oligodeoxynucleotides that adopt intramolecular G4 structures. We found out that the addition of nucleotides favors the formation of a parallel fold, defined as the 'flanking effect' in this work. This 'flanking effect' was more pronounced when nucleotides were added at the 5'-end, and depended on loop arrangement. NMR experiments and molecular dynamics simulations revealed that flanking sequences at the 5'-end abolish a strong syn-specific hydrogen bond commonly found in non-parallel conformations, thus favoring a parallel topology. These analyses pave a new way for more accurate prediction of DNA G4 folding in a physiological context.

Thermal and pH Stabilities of i-DNA: Confronting in vitro Experiments with Models and In-Cell NMR Data.

Recent studies indicate that i-DNA, a four-stranded cytosine-rich DNA also known as the i-motif, is actually formed in vivo; however, a systematic study on sequence effects on stability has been missing. Herein, an unprecedented number of different sequences (271) bearing four runs of 3-6 cytosines with different spacer lengths has been tested. While i-DNA stability is nearly independent on total spacer length, the central spacer plays a special role on stability. Stability also depends on the length of the C-tracts at both acidic and neutral pHs. This study provides a global picture on i-DNA stability thanks to the large size of the introduced data set; it reveals unexpected features and allows to conclude that determinants of i-DNA stability do not mirror those of G-quadruplexes. Our results illustrate the structural roles of loops and C-tracts on i-DNA stability, confirm its formation in cells, and allow establishing rules to predict its stability.

Symmetric and dissymmetric carbohydrate-phenyl ditriazole derivatives as DNA G-quadruplex ligands: Synthesis, biophysical studies and antiproliferative activity.

Here we present a novel G4-binding family of compounds based on a central core of phenyl ditriazole (PDTZ) modified with carbohydrates and phenyl pyrrolidinyl side-chains. Their synthesis was achieved using controlled click chemistry conditions to obtain both, symmetric and dissymmetric carb-PDTZ derivatives without any intermediate protecting steps through an optimized methodology. Binding of the new carb-PDTZ to a variety of G-quadruplex motifs was examined using different biophysical techniques. The symmetric carb-PDTZ derivatives were not able to stabilize G4, but the dissymmetric ones (containing one sugar and one phenyl pyrrolidinyl side-chain) did. Interestingly, the dissymmetric carb-PDTZ derivatives showed much higher G4 vs duplex DNA selectivity than the control compound PDTZ 1, which contains two phenyl pyrrodilinyl side-chains and no carbohydrates. Their potential antitumoral activity was also investigated by in vitro cytotoxicity measurements on different cancerous cell lines. All carb-PDTZ derivatives showed higher IC50 values than the control PDTZ 1, probably due to the lack of compound stability of some derivatives and to lower cellular uptake.

Author Correction: RNA synthesis is modulated by G-quadruplex formation in Hepatitis C virus negative RNA strand.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

G-Quadruplex binding optimization by gold(iii) insertion into the center of a porphyrin.

Porphyrins represent a valuable class of ligands for G-quadruplex nucleic acids. Herein, we evaluate the binding of cationic porphyrins metallated with gold(iii) to G-quadruplex DNA and we compare it with other porphyrin derivatives. The G-quadruplex stabilization capacity and the selectivity of the various porphyrins were evaluated by biophysical and biochemical assays. The porphyrins were also tested as inhibitors of telomerase. It clearly appeared that the insertion of gold(iii) ion in the center of the porphyrin increases the binding affinity of the porphyrin for the G-quadruplex target. Together with modelling studies, it is possible to propose that the insertion of the square planar gold(iii) ion adds an extra positive charge on the complex and decreases the electron density in the porphyrin aromatic macrocycle, both properties being in favour of stronger electrostatic and π-staking interactions.

Multicarbazole scaffolds for selective G-quadruplex binding.

Herein we report a new class of G-quadruplex stabilising ligands, multicarbazoles, which display high G-quadruplex DNA selectivity in the presence of 250 times excess duplex DNA. We report the synthesis of these compounds in moderate to high yields. Ligands in the series with optimal G-quadruplex selectivity contain an N-propylamino chain length where the amino functionalities are either pyrrolidine or piperidine.

RNA synthesis is modulated by G-quadruplex formation in Hepatitis C virus negative RNA strand.

DNA and RNA guanine-rich oligonucleotides can form non-canonical structures called G-quadruplexes or "G4" that are based on the stacking of G-quartets. The role of DNA and RNA G4 is documented in eukaryotic cells and in pathogens such as viruses. Yet, G4 have been identified only in a few RNA viruses, including the Flaviviridae family. In this study, we analysed the last 157 nucleotides at the 3'end of the HCV (-) strand. This sequence is known to be the minimal sequence required for an efficient RNA replication. Using bioinformatics and biophysics, we identified a highly conserved G4-prone sequence located in the stem-loop IIy' of the negative strand. We also showed that the formation of this G-quadruplex inhibits the in vitro RNA synthesis by the RdRp. Furthermore, Phen-DC3, a specific G-quadruplex binder, is able to inhibit HCV viral replication in cells in conditions where no cytotoxicity was measured. Considering that this domain of the negative RNA strand is well conserved among HCV genotypes, G4 ligands could be of interest for new antiviral therapies.

Unexpected Position-Dependent Effects of Ribose G-Quartets in G-Quadruplexes.

To understand the role of ribose G-quartets and how they affect the properties of G-quadruplex structures, we studied three systems in which one, two, three, or four deoxyribose G-quartets were substituted with ribose G-quartets. These systems were a parallel DNA intramolecular G-quadruplex, d(TTGGGTGGGTTGGGTGGGTT), and two tetramolecular G-quadruplexes, d(TGGGT) and d(TGGGGT). Thermal denaturation experiments revealed that ribose G-quartets have position-dependent and cumulative effects on G-quadruplex stability. An unexpected destabilization was observed when rG quartets were presented at the 5'-end of the G stack. This observation challenges the general belief that RNA residues stabilize G-quadruplexes. Furthermore, in contrast to past proposals, hydration is not the main factor determining the stability of our RNA/DNA chimeric G-quadruplexes. Interestingly, the presence of rG residues in a central G-quartet facilitated the formation of additional tetramolecular G-quadruplex topologies showing positive circular dichroism signals at 295 nm. 2D NMR analysis of the tetramolecular TGgGGT (lowercase letter indicates ribose) indicates that Gs in the 5'-most G-quartet adopt the syn conformation. These analyses highlight several new aspects of the role of ribose G-quartets on G-quadruplex structure and stability, and demonstrate that the positions of ribose residues are critical for tuning G-quadruplex properties.

Quadruplex Turncoats: Cation-Dependent Folding and Stability of Quadruplex-DNA Double Switches.

Quadruplex (G4) nucleic acids, a family of secondary structures formed by guanine-rich sequences, exhibit an important structural polymorphism. We demonstrate here that G-rich DNA sequences may function as a double switch based on different triggers, provided that their quadruplex structures and stability display a high dependence on cation nature and concentration. A first switch is based on a remarkable antiparallel-to-parallel conversion, taking place in a few seconds at room temperature by addition of low KCl amounts to a sodium-rich sample. The second switch involves the conversion of alternative antiparallel quadruplex structures binding only one cation, formed in the presence of sub-millimolar potassium or strontium concentrations, to parallel structures by increasing the cation concentration. Incidentally, extremely low K(+) or Sr(2+) concentrations (≤5 equiv) are sufficient to induce G4 formation in a buffer devoid of other G4-promoting cations, and we suggest that the alternative structures observed contain only two tetrads. Such DNA systems are biological relevant targets, can be used in nanotechnology applications, and are valuable methodological tools for understanding DNA quadruplex folding, notably at low cation concentrations. We demonstrate that this behavior is not restricted to a narrow set of sequences but can also be found for other G-quadruplex-forming motifs, arguing for widespread applications.

The exception that confirms the rule: a higher-order telomeric G-quadruplex structure more stable in sodium than in potassium.

DNA and RNA guanine-quadruplexes (G4s) are stabilized by several cations, in particular by potassium and sodium ions. Generally, potassium stabilizes guanine-quartet assemblies to a larger extent than sodium; in this article we report about a higher-order G4 structure more stable in sodium than in potassium. Repeats of the DNA GGGTTA telomeric motif fold into contiguous G4 units. Using three independent approaches (thermal denaturation experiments, isothermal molecular-beacon and protein-binding assays), we show that the (GGGTTA)7GGG sequence, folding into two contiguous G4 units, exhibits an unusual feature among G4 motifs: despite a lower thermal stability, its sodium conformation is more stable than its potassium counterpart at physiological temperature. Using differential scanning calorimetry and mutated sequences, we show that this switch in the relative stability of the sodium and potassium conformations (occurring around 45 °C in 100 mM cation concentration) is the result of a more favorable enthalpy change upon folding in sodium, generated by stabilizing interactions between the two G4 units in the sodium conformation. Our work demonstrates that interactions between G4 structural domains can make a higher-order structure more stable in sodium than in potassium, even though its G4 structural domains are individually more stable in potassium than in sodium.

Anticancer molecule AS1411 exhibits low nanomolar antiviral activity against HIV-1.

During clinical trials, a number of fully characterized molecules are dropped along the way because they do not provide enough benefit for the patient. Some of them show limited side effects and might be of great use for other applications. AS1411 is a nucleolin-targeting aptamer that underwent phase II clinical trials as anticancer agent. Here, we show that AS1411 exhibits extremely potent antiviral activity and is therefore an attractive new lead as anti-HIV agent.

G-quadruplexes in viruses: function and potential therapeutic applications.

G-rich nucleic acids can form non-canonical G-quadruplex structures (G4s) in which four guanines fold in a planar arrangement through Hoogsteen hydrogen bonds. Although many biochemical and structural studies have focused on DNA sequences containing successive, adjacent guanines that spontaneously fold into G4s, evidence for their in vivo relevance has recently begun to accumulate. Complete sequencing of the human genome highlighted the presence of ∼300,000 sequences that can potentially form G4s. Likewise, the presence of putative G4-sequences has been reported in various viruses genomes [e.g., Human immunodeficiency virus (HIV-1), Epstein-Barr virus (EBV), papillomavirus (HPV)]. Many studies have focused on telomeric G4s and how their dynamics are regulated to enable telomere synthesis. Moreover, a role for G4s has been proposed in cellular and viral replication, recombination and gene expression control. In parallel, DNA aptamers that form G4s have been described as inhibitors and diagnostic tools to detect viruses [e.g., hepatitis A virus (HAV), EBV, cauliflower mosaic virus (CaMV), severe acute respiratory syndrome virus (SARS), simian virus 40 (SV40)]. Here, special emphasis will be given to the possible role of these structures in a virus life cycle as well as the use of G4-forming oligonucleotides as potential antiviral agents and innovative tools.

Backbone-independent nucleic acid binding by splicing factor SUP-12 reveals key aspects of molecular recognition.

Cellular differentiation is frequently accompanied by alternative splicing, enabled by the expression of tissue-specific factors which bind to pre-mRNAs and regulate exon choice. During Caenorhabditis elegans development, muscle-specific expression of the splicing factor SUP-12, together with a member of the Fox-1 family of splicing proteins, generates a functionally distinct isoform of the fibroblast growth factor receptor EGL-15. Using a combination of NMR spectroscopy and isothermal titration calorimetry, we determined the mode of nucleic acid binding by the RNA recognition motif domain of SUP-12. The calculated structures provide the first atomic details of RNA and DNA binding by the family of proteins that include SUP-12, RBM24, RBM38/RNPC1, SEB-4 and XSeb4R. This information was further used to design strategic mutations to probe the interaction with ASD-1 and to quantitatively perturb splicing in vivo.