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Funder: Hong Kong PhD Fellowship Scheme ; Funder: Hong Kong Special Administrative Region Government ; G-quadruplexes are non-helical secondary structures that can fold in vivo in both DNA and RNA. In human cells, they can influence replication, transcription and telomere maintenance in DNA, or translation, transcript processing and stability of RNA. We have previously showed that G-quadruplexes are detectable in the DNA of the malaria parasite Plasmodium falciparum, despite a very highly A/T-biased genome with unusually few guanine-rich sequences. Here, we show that RNA G-quadruplexes can also form in P. falciparum RNA, using rG4-seq for transcriptome-wide structure-specific RNA probing. Many of the motifs, detected here via the rG4seeker pipeline, have non-canonical forms and would not be predicted by standard in silico algorithms. However, in vitro biophysical assays verified formation of non-canonical motifs. The G-quadruplexes in the P. falciparum transcriptome are frequently clustered in certain genes and associated with regions encoding low-complexity peptide repeats. They are overrepresented in particular classes of genes, notably those that encode PfEMP1 virulence factors, stress response genes and DNA binding proteins. In vitro translation experiments and in vivo measures of translation efficiency showed that G-quadruplexes can influence the translation of P. falciparum mRNAs. Thus, the G-quadruplex is a novel player in post-transcriptional regulation of gene expression in this major human pathogen. ; UK Medical Research Council [grants MR/K000535/1 and MR/L008823/1] to CJM. Shenzhen Basic Research Project [JCYJ20180507181642811], Research Grants Council of the Hong Kong SAR, China Projects [CityU 11100421, CityU 11101519, CityU 11100218, N_CityU110/17, CityU 21302317], Croucher Foundation [Project No. 9500030, 9509003], State Key Laboratory of Marine Pollution Director Discretionary Fund, City University of Hong Kong [projects 6000711, 7005503, 9667222, 9680261] to CKK. A generous donation from Mr. and Mrs. Sunny Yang, the University Grants Committee Area of Excellence Scheme (AoE/M-403/16), and the Innovation and Technology Commission, Hong Kong Special Administrative Region Government to the State Key Laboratory of Agrobiotechnology (CUHK) to TFC. EYCC was supported by the Hong Kong PhD Fellowship Scheme.

Guanine quadruplexes (G4s) are highly polymorphic four-stranded structures formed within guanine-rich DNA and RNA sequences that play a crucial role in biological processes. The recent discovery of the first G4 structures within mitochondrial DNA has led to a small revolution in the field. In particular, the G-rich conserved sequence block II (CSB II) can form different types of G4s that are thought to play a crucial role in replication. In this study, we decipher the most relevant G4 structures that can be formed within CSB II: RNA G4 at the RNA transcript, DNA G4 within the non-transcribed strand and DNA:RNA hybrid between the RNA transcript and the non-transcribed strand. We show that the more abundant, but unexplored, G6AG7 (37%) and G6AG8 (35%) sequences in CSB II yield more stable G4s than the less profuse G5AG7 sequence. Moreover, the existence of a guanine located 1 bp upstream promotes G4 formation. In all cases, parallel G4s are formed, but their topology changes from a less ordered to a highly ordered G4 when adding small amounts of potassium or sodium cations. Circular dichroism was used due to discriminate different conformations and topologies of nucleic acids and was complemented with gel electrophoresis and fluorescence spectroscopy studies ; This research was funded by the Gobierno de España—Ministerio de Economía y Competitividad, grant number CTQ2014-59020-R, the Xunta de Galicia, grant numbers ED431B 2019/18, ED431G 2019/03 Centro singular de investigación de Galicia accreditation 2019–2022, and Ph.D. fellowship to S.I. and the European Union (European Regional Development Fund—ERDF) ; SI

Non-canonical, four-stranded nucleic acids secondary structures are present within regulatory regions in the human genome and transcriptome. To date, these quadruplex structures include both DNA and RNA G-quadruplexes, formed in guanine-rich sequences, and i-Motifs, found in cytosine-rich sequences, as their counterparts. Quadruplexes have been extensively associated with cancer, playing an important role in telomere maintenance and control of genetic expression of several oncogenes and tumor suppressors. Therefore, quadruplex structures are considered attractive molecular targets for cancer therapeutics with novel mechanisms of action. In this review, we provide a general overview about recent research on the implications of quadruplex structures in cancer, firstly gathering together DNA G-quadruplexes, RNA G-quadruplexes as well as DNA i-Motifs. ; This study was supported by 3TR, IMI2 (H2020-JTI538 IMI2-2018) and by Instituto de Salud Carlos III under the frame of EuroNanoMed III (AC18/00008) to J.A.G.S. The Government of Spain granted with PhD fellowship FPU16/05822 to V.S.M. ; Yes

[EN] Genomes contain rare guanine-rich sequences capable of assembling into four-stranded helical structures, termed G-quadruplexes, with potential roles in gene regulation and chromosome stability. Their mechanical unfolding has only been reported to date by all-atom simulations, which cannot dissect the major physical interactions responsible for their cohesion. Here, we propose a mesoscopic model to describe both the mechanical and thermal stability of DNA G-quadruplexes, where each nucleotide of the structure, as well as each central cation located at the inner channel, is mapped onto a single bead. In this framework we are able to simulate loading rates similar to the experimental ones, which are not reachable in simulations with atomistic resolution. In this regard, we present single-molecule force-induced unfolding experiments by a high-resolution optical tweezers on a DNA telomeric sequence capable of adopting a G-quadruplex conformation. Fitting the parameters of the model to the experiments we find a correct prediction of the rupture-force kinetics and a good agreement with previous near equilibrium measurements. Since G-quadruplex unfolding dynamics is halfway in complexity between secondary nucleic acids and tertiary protein structures, our model entails a nanoscale paradigm for non-equilibrium processes in the cell. ; Work supported by the Spanish Ministry of Economy and Competitiveness (MINECO), grant No. FIS2014-55867, co-financed by FEDER funds. We also thank the support of the Aragon Government and Fondo Social Europeo to FENOL group. Work in J.R.A.-G. laboratory was supported by a grant from MINECO, No. MAT2015-71806-R). ; Bergues-Pupo, A.; Gutiérrez, I.; Arias-Gonzalez, JR.; Falo, F.; Fiasconaro, A. (2017). Mesoscopic model for DNA G-quadruplex unfolding. Scientific Reports. 7:1-13. https://doi.org/10.1038/s41598-017-10849-2 ; S ; 1 ; 13 ; 7 ; Arias-Gonzalez, J. R. Single-molecule portrait of DNA and RNA double helices. Integr. Biol. 6, 904 (2014). ; Burge, S., Parkinson, G. N., Hazel, P., ...

Curaxins and especially the second-generation derivative curaxin CBL0137 have important antitumor activities in multiple cancers such as glioblastoma, melanoma and others. Although most of the authors suggest that their mechanism of action comes from the activation of p53 and inactivation of NF-kB by targeting FACT, there is evidence supporting the involvement of DNA binding in their antitumor activity. In this work, the DNA binding properties of curaxin CBL0137 with model quadruplex DNA oligomers were studied by 1H NMR, CD, fluorescence and molecular modeling. We provided molecular details of the interaction of curaxin with two G-quadruplex structures, the single repeat of human telomere d(TTAGGGT)4 and the c-myc promoter Pu22 sequence. We also performed 1H and 31P NMR experiments were also performed in order to investigate the interaction with duplex DNA models. Our data support the hypothesis that the interaction of curaxin with G-quadruplex may provide a novel insight into the DNA-binding properties of CBL0137, and it will be helpful for the design of novel selective DNA-targeting curaxin analogues. ; This research was funded by PIANO DI SOSTEGNO ALLA RICERCA 2020—Linea 2 azione B (DEFENS) and by the Spanish Ministerio de Ciencia e Innnvacion (PID2019-107158GB-I00) and recognition from the Autonomous Catalan government (2017SGR114). ; Peer reviewed

The conformational equilibria of a guanine-rich sequence found at the promoter region of the human c-kit oncogene are studied by means of circular dichroism spectroscopy (CD) and variable-temperature size exclusion chromatography (SEC). It is shown that the wild sequence ckit21 exists as a mixture of monomeric and multimeric G-quadruplexes. Appropriate mutation of several bases in the wild sequence produces the shift from parallel to antiparallel G-quadruplex, as well as the disappearance of multimeric species. The shift from the antiparallel to the parallel conformation induced by temperature is reflected in both CD and SEC profiles. ; The authors acknowledge funding from the Spanish Government (CTQ2012-38616-C02-02 and CTQ2010-20541-C03-01). ; Peer reviewed

The ability of the guanine-rich strand of the human mitochondrial DNA (mtDNA) to form G-quadruplex structures (G4s) has been recently highlighted, suggesting potential functions in mtDNA replication initiation and mtDNA stability. G4 structures in mtDNA raise the question of their recognition by factors associated with the mitochondrial nucleoid. The mitochondrial transcription factor A (TFAM), a high-mobility group (HMG)-box protein, is the major binding protein of human mtDNA and plays a critical role in its expression and maintenance. HMG-box proteins are pleiotropic sensors of DNA structural alterations. Thus, we investigated and uncovered a surprising ability of TFAM to bind to DNA or RNA G4 with great versatility, showing an affinity similar than to double-stranded DNA. The recognition of G4s by endogenous TFAM was detected in mitochondrial extracts by pull-down experiments using a G4-DNA from the mtDNA conserved sequence block II (CSBII). Biochemical characterization shows that TFAM binding to G4 depends on both the G-quartets core and flanking single-stranded overhangs. Additionally, it shows a structure-specific binding mode that differs from B-DNA, including G4-dependent TFAM multimerization. These TFAM-G4 interactions suggest functional recognition of G4s in the mitochondria. ; We thank Gilles Mirambeau, Dmitry Temiakov and Arka Chakraborty for valuable discussion. The Structural Biology Unit is "Maria de Maeztu" Unit of Excellence awarded by Ministry of Economy and Competitiveness (MINECO) [MDM-2014-0435]. This study was supported by MINECO (BFU2009-07134, BFU2012-33516/ BMC, BFU2015-70645-R), Generalitat de Catalunya (SGR2009-1366, SGR2014-997), the European Union (FP7-HEALTH-2010-261460, FP7-PEOPLE-2011-290246, FP7-HEALTH-2012-306029-2) and the Spanish Research Council for a JAE fellowship to ARC. ; Peer reviewed

Natural phenolic compounds have gained momentum for the prevention and treatment of cancer, but their antitumoral mechanism of action is not yet well understood. In the present study, we screened the antitumoral potential of several phenolic compounds in a cellular model of colorectal cancer (CRC).We selected gallic acid (GA) as a candidate in terms of potency and selectivity and extensively evaluated its biological activity. We report on the role of GA as a ligand of DNA G-quadruplexes (G4s), explaining several of its antitumoral effects, including the transcriptional inhibition of ribosomal and CMYC genes. In addition, GA shared with other established G4 ligands some effects such as cell cycle arrest, nucleolar stress, and induction of DNA damage. We further confirmed the antitumoral and G4-stabilizing properties of GA using a xenograft model of CRC. Finally, we succinctly demonstrate that GA could be explored as a therapeutic agent in a patient cohort with CRC. Our work reveals that GA, a natural bioactive compound present in the diet, affects gene expression by interaction with G4s both in vitro and in vivo and paves the way towards G4s targeting with phenolic compounds. ; Instituto de Salud Carlos III ; European Commission PI21/00497 AC18/00008 ; Next generation EU, Plan de Recuperacion Transformacion y Resiliencia, Agencia Estatal de Investigacion PLEC2021-008094 ; Ministerio de Ciencia e Innovacion from Government of Spain PID2019-104416RB-I00 PID2020-120481RB-I00 ; Ministerio de Universidades from Government of Spain FPU16/05822 FPU17/05413 FPU20/03952 ; University of Almeria FPI-201102

DNA repair inhibitors are one of the latest additions to cancer chemotherapy. In general, chemotherapy produces DNA damage but tumoral cells may become resistant if enzymes involved in DNA repair are overexpressed and are able to reverse DNA damage. One of the most successful drugs based on modulating DNA repair are the poly(ADP-ribose) polymerase 1 (PARP1) inhibitors. Several PARP1 inhibitors have been recently developed and approved for clinical treatments. We envisaged that PARP inhibition could be potentiated by simultaneously modulating the expression of PARP 1 and the enzyme activity, by a two-pronged strategy. A noncanonical G-quadruplex-forming sequence within the PARP1 promoter has been recently identified. In this study, we explored the potential binding of clinically approved PARP1 inhibitors to the G-quadruplex structure found at the gene promoter region. The results obtained by NMR, CD, and fluorescence titration confirmed by molecular modeling demonstrated that two out the four PARP1 inhibitors studied are capable of forming defined complexes with the PARP1 G-quadruplex. These results open the possibility of exploring the development of better G-quadruplex binders that, in turn, may also inhibit the enzyme. ; This research was supported by PIANO DI SOSTEGNO ALLA RICERCA 2020—Linea 2 azione B (DEFENS). ICTS Nanbiosis Oligonucleotide Synthesis Platform (CIBER-BBN). Funding from the Spanish Ministerio de Ciencia e Innovación (PID2019-107158GB-I00) and recognition from the Autonomous Catalan government (2017SGR114) are acknowledged. ; Peer reviewed

G-quadruplex DNAs form four-stranded helical structures and are proposed to play key roles in different cellular processes. Targeting G-quadruplex DNAs for cancer treatment is a very promising prospect. Here, we show that CX-5461 is a G-quadruplex stabilizer, with specific toxicity against BRCA deficiencies in cancer cells and polyclonal patient-derived xenograft models, including tumours resistant to PARP inhibition. Exposure to CX-5461, and its related drug CX-3543, blocks replication forks and induces ssDNA gaps or breaks. The BRCA and NHEJ pathways are required for the repair of CX-5461 and CX-3543-induced DNA damage and failure to do so leads to lethality. These data strengthen the concept of G4 targeting as a therapeutic approach, specifically for targeting HR and NHEJ deficient cancers and other tumours deficient for DNA damage repair. CX-5461 is now in advanced phase I clinical trial for patients with BRCA1/2 deficient tumours (Canadian trial, NCT02719977, opened May 2016). ; This work was supported by the Canadian Breast Cancer Foundation BC/Yukon, BC Cancer Foundation, Stand Up to Cancer Canada (SU2C-AACR-DT-18-15), TFRI Grant 1021, CCSRI Grant 701584, CIHR Grant MOP-126119, Canada Foundation for Innovation and Cancer Research UK. Grant Brown lab is supported by CCSRI Impact Grant 702310 (to G.W.B.) and Ontario Government Scholarship (to B.H.). S.A. is supported by a Canada Research Chair in Molecular Oncology. The Balasubramanian lab is supported by a programme grant (C14303/A17197) and core funding (C14303/A17197) from Cancer Research UK.

Poly ADP-ribose polymerases (PARP) are key proteins involved in DNA repair, maintenance as well as regulation of programmed cell death. For this reason they are important therapeutic targets for cancer treatment. Recent studies have revealed a close interplay between PARP1 recruitment and G-quadruplex stabilization, showing that PARP enzymes are activated upon treatment with a G4 ligand. In this work the DNA binding properties of a PARP-1 inhibitor derived from 7-azaindole-1-carboxamide, (2-[6-(4-pyrrolidin-1-ylmethyl-phenyl)-pyrrolo[2,3-b]pyridin-1-yl]-acetamide, compound 1) with model duplex and quadruplex DNA oligomers were studied by NMR, CD, fluorescence and molecular modelling. We provide evidence that compound 1 is a strong G-quadruplex binder. In addition we provide molecular details of the interaction of compound 1 with two model G-quadruplex structures: the single repeat of human telomeres, d(TTAGGGT)4, and the c-MYC promoter Pu22 sequence. The formation of defined and strong complexes with G-quadruplex models suggests a dual G4 stabilization/PARP inhibition mechanism of action for compound 1 and provides the molecular bases of its therapeutic potential. ; Financial support of the Spanish government is acknowledged (Grant Number PID2019-107158GB-I00). This research was supported by PIANO DI SOSTEGNO ALLA RICERCA 2020—Linea 2 azione B (DEFENS). ; Peer reviewed

AbstractCyclic anthraquinone derivatives (cAQs), which link two side chains of 1,5-disubstituted anthraquinone as a threading DNA intercalator, have been developed as G-quartet (G4) DNA-specific ligands. Among the cAQs, cAQ-mBen linked through the 1,3-position of benzene had the strongest affinity for G4 recognition and stabilization in vitro and was confirmed to bind to the G4 structure in vivo, selectively inhibiting cancer cell proliferation in correlation with telomerase expression levels and triggering cell apoptosis. RNA-sequencing analysis further indicated that differentially expressed genes regulated by cAQ-mBen were profiled with more potential quadruplex-forming sequences. In the treatment of the tumor-bearing mouse model, cAQ-mBen could effectively reduce tumor tissue and had less adverse effects on healthy tissue. These results suggest that cAQ-mBen can be a potential cancer therapeutic agent as a G4 binder.