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The i-motif structure is formed in cytosine-rich sequences, its building block being the cytosine·cytosine + base pair. This structure is particularly stable at pH values below physiological (∼7.4) and, because of that, it has not attracted as much biological interest as other non-canonical structures such as the G-quadruplex. Nowadays, the proposal of potential roles in vivo, as well as nanotechnological applications, has produced an increasing interest in its study. In this context, the present work provides an overall picture of the i-motif structure. Those aspects related to formation and stability, such as chemical modifications or the interaction with ligands, are discussed. Special attention has been made to the i-motif structures that could have a hypothetical role in vivo, such as those present near the promoter region of several oncogenes. ; We acknowledge funding from the Spanish government (CTQ2012-38616-C02-02 and CTQ-2010-20541-C03). Sanae Benabou thanks the Spanish Ministerio de Economía y Competitividad for a PhD grant. ; Peer reviewed

The influence of pH, temperature and the cationic porphyrin TMPyP4 on the conformational equilibria of the cytosine-rich strand of the human telomeric sequence 5′-(C3TA2)4-3′, as well as those of the sequence 5′-(C3TT2)4-3′, was studied. The presence of adenine bases at the loops causes the formation of two different intramolecular i-motif structures with a pH-transition midpoint around 4.6, which stability is lower than the i-motif formed by the sequence 5′-(C3TT2)4-3′. The stoichiometries of the complexes formed by these i-motif structures with TMPyP4 are also influenced by the presence of adenine at the loops. ; We acknowledge funding from both the Spanish government (CTQ2009-11572 and CTQ2010-20541-C03-01) and Catalan government(2009 SGR 45 and 2009 SGR 238). ; Peer reviewed

Silver nanoclusters (AgNCs) prepared by the reduction of silver ions in the presence of DNA oligonucleotides have attracted great interest as potential diagnostic tools for their tunable and high fluorescent properties. In this work, three DNA sequences that consist of a 12-nucleotide long probe sequence at the 5′-end linked to the complementary sequence to three miRNAs are studied. First, the interaction of these sequences with Ag(I) was characterized by means of circular dichroism spectroscopy. By applying multivariate methods to the analysis of spectroscopic data, two complexes with different Ag(I) : DNA ratios were resolved. Secondly, the impact of several experimental variables, such as temperature, borohydride concentration and reaction time, on the formation of AgNCs templated by these three sequences was studied. Finally, the fluorescence properties of the duplexes formed by DNA probes with complementary DNA or miRNA sequences were studied. The results presented here highlight the role of the secondary structure adopted by the DNA probe on the fluorescence properties of DNA-stabilized AgNCs which, in turn, affect the development of methods for miRNA detection. ; We thank the contribution of Gerard Pastor and Sara Guardia (University of Barcelona). Funding from the Spanish Ministerio de Ciencia e Innovación (PID2019-107158GB-I00) and recognition from the Autonomous Catalan government (2017SGR114) are acknowledged. ; Peer reviewed

Recently, the presence of i-motif structures at C-rich sequences in human cells and their regulatory functions have been demonstrated. Despite numerous steady-state studies on i-motif at neutral and slightly acidic pH, the number and nature of conformation of this biological structure are still controversial. In this work, the fluorescence lifetime of labelled molecular beacon i-motif-forming DNA sequences at different pH values is studied. The influence of the nature of bases at the lateral loops and the presence of a Watson–Crick-stabilized hairpin are studied by means of time-correlated single-photon counting technique. This allows characterizing the existence of several conformers for which the fluorophore has lifetimes ranging from picosecond to nanosecond. The information on the existence of different i-motif structures at different pH values has been obtained by the combination of classical global decay fitting of fluorescence traces, which provides lifetimes associated with the events defined by the decay of each sequence and multivariate analysis, such as principal component analysis or multivariate curve resolution based on alternating least squares. Multivariate analysis, which is seldom used for this kind of data, was crucial to explore similarities and differences of behaviour amongst the different DNA sequences and to model the presence and identity of the conformations involved in the pH range of interest. The results point that, for i-motif, the intrachain contact formation and its dissociation show lifetimes ten times faster than for the open form of DNA sequences. They also highlight that the presence of more than one i-motif species for certain DNA sequences according to the length of the sequence and the composition of the bases in the lateral loop. ; Funding from Spanish government [CTQ2014-61758-EXP, CTQ2014-52588-R, CTQ2015-66254-C2-2-P]; Autonomous Catalan government [2017SGR114]. Funding for open access charge: University of Bracelona [CTQ2015-66254-C2-2-P] and Université de Lille, LASIR CNRS. ; Peer reviewed

The i-motif is a DNA structure formed by cytosine-rich sequences, very relevant from a biochemical point of view and potentially useful in nanotechnology as pH-sensitive nanodevices or nanomotors. To provide a different view on the structural changes and dynamics of direct excitation processes involving i-motif structures, the use of rapid-scan FTIR spectroscopy is proposed. Hybrid hard- and soft-modelling based on the Multivariate Curve Resolution by Alternating Least Squares (MCR-ALS) algorithm has been used for the resolution of rapid-scan FTIR spectra and the interpretation of the photochemically induced time-dependent conformational changes of i-motif structures. The hybrid hard- and soft-modelling version of MCR-ALS (HS-MCR), which allows the introduction of kinetic models to describe process behavior, provides also rate constants associated with the transitions modeled. The results show that UV irradiation does not produce degradation of the studied sequences but induces the formation of photodimers. The presence of these affect much more the stability of i-motif structures formed by short sequences than that of those formed by longer sequences containing additional structural stabilizing elements, such as hairpins. ; Sanae Benabou thanks the LAboratoire de Spectrochimie Infrarouge et Raman (LASIR) for all the support, especially, Dr. Isabelle Waele for her help in the acquisition of rapid-scan FTIR measurements. Funding from Spanish government (CTQ2017-84415-R and CTQ2015-66254-C2-2-P) and recognition from the Autonomous Catalan government (2017SGR753 and 2017SGR114) are acknowledged. ; Peer reviewed

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

Cytosine-rich oligonucleotides are capable of forming complex structures known as i-motif with increasingly studied biological properties. The study of sequences prone to form i-motifs located near the promoter region of genes may be difficult because these sequences not only contain repeats of cytosine tracts of disparate length but also these may be separated by loops of varied nature and length. In this work, the formation of intramolecular i-motif structures by a long sequence located upstream of the promoter region of the SMARCA4 gene has been demonstrated. Nuclear Magnetic Resonance, Circular Dichroism, Gel Electrophoresis, Size-Exclusion Chromatography, and multivariate analysis have been used. Not only the wild sequence (5'-TC3T2GCTATC3TGTC2TGC2TCGC3T2G2TCATGA2C4-3') has been studied but also several other truncated and mutated sequences. Despite the apparent complex sequence, the results showed that the wild sequence may form a relatively stable and homogeneous unimolecular i-motif structure, both in terms of pH or temperature. The model ligand TMPyP4 destabilizes the structure, whereas the presence of 20% (w/v) PEG200 stabilized it slightly. This finding opens the door to the study of the interaction of these kind of i-motif structures with stabilizing ligands or proteins. © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM) ; S.L. thanks Maria Solà (IBMB-CSIC, Barcelona, Spain) for support. Xavier Subirats (University of Barcelona, Spain) is thanked for helpful discussions. Funding from Spanish government (CTQ2014-61758-EXP, CTQ2014-52588-R and CTQ2015-66254-C2-2-P) and recognition from the Autonomous Catalan government (2014SGR1106) are acknowledged. ; Peer reviewed

[Background] Cytosine- and guanine-rich regions of DNA are capable of forming complex structures named i-motifs and G-quadruplexes, respectively. In the present study the solution equilibria at nearly physiological conditions of a 34-base long cytosine-rich sequence and its complementary guanine-rich strand corresponding to the first intron of the n-myc gene were studied. Both sequences, not yet studied, contain a 12-base tract capable of forming stable hairpins inside the i-motif and G-quadruplex structures, respectively. ; [Methods] Spectroscopic, mass spectrometry and separation techniques, as well as multivariate data analysis methods, were used to unravel the species and conformations present. ; [Results] The cytosine-rich sequence forms two i-motifs that differ in the protonation of bases located in the loops. A stable Watson-Crick hairpin is formed by the bases in the first loop, stabilizing the i-motif structure. The guanine-rich sequence adopts a parallel G-quadruplex structure that is stable throughout the pH range 3-7, despite the protonation of cytosine and adenine bases at lower pH values. The presence of G-quadruplex aggregates was confirmed using separation techniques. When mixed, G-quadruplex and i-motif coexist with the Watson-Crick duplex across a pH range from approximately 3.0 to 6.5. ; [Conclusions] Two cytosine- and guanine-rich sequences in n-myc gene may form stable i-motif and G-quadruplex structures even in the presence of long loops. pH modulates the equilibria involving the intramolecular structures and the intermolecular Watson-Crick duplex. ; [General significance] Watson-Crick hairpins located in the intramolecular G-quadruplexes and i-motifs in the promoter regions of oncogenes could play a role in stabilizing these structures. © 2013 Elsevier B.V. ; We acknowledge funding from the Spanish government (CTQ2012-38616-C02-02 and CTQ2010-20541-C03-01) ; Peer Reviewed

The excellent self-assembling properties of DNA and the excellent specificity of the antibodies to detect analytes of small molecular weight under competitive conditions have been combined in this study. Three oligonucleotide sequences (N1up, N2up, and N3up) have been covalently attached to three steroidal haptens (8, hG, and 13) of three anabolic-androgenic steroids (AAS), stanozolol (ST), tetrahydrogestrinone (THG), and boldenone (B), respectively. The synthesis of steroid-oligonucleotide conjugates has been performed by the reaction of oligonucleotides carrying amino groups with carboxyl acid derivatives of steroidal haptens. Due to the chemical nature of the steroid derivatives, two methods for coupling the haptens and the ssDNA have been studied: a solid-phase coupling strategy and a solution-phase coupling strategy. Specific antibodies against ST, THG, and B have been used in this study to asses the possibility of using the self-assembling properties of the DNA to prepare biofunctional SPR gold chips based on the immobilization of haptens, by hybridization with the complementary oligonucleotide strands possessing SH groups previously immobilized. The capture of the steroid-oligonucleotide conjugates and subsequent binding of the specific antibodies can be monitored on the sensogram due to variations produced on the refractive index on top of the gold chip. The resulting steroid-oligonucleotide conjugates retain the hybridization and specific binding properties of oligonucleotides and haptens as demonstrated by thermal denaturation experiments and surface plasmon resonance (SPR). ; This work was supported by grants from the Ministry of Science and Innovation, MICINN (MAT2011-29335-C03-01 and CTQ2010-20541-C03-01), CCEE (FUNMOL, FP7-NMP-213382-2), Generalitat de Catalunya (2009/SGR/1343, 2009/SGR/208), and CIBER-BBN. CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. Núria Tort has a FI_B fellowship from the AGAUR (Agència de Gestió d'Ajuts Universitaris i de Recerca) of the (Generalitat de Catalunya) Government of Catalonia. ; Peer reviewed

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

[EN] The development of new strategies to detect microRNAs (miRNAS) has become an important challenge in thebiomedical ¿eld. We report herein the use of oligonucleotide-gated silica nanoparticles for the detection ofmiRNA-145. In the proposed design, mesoporous silica nanoparticles (MSNs) are loaded with a ¿uorescentreporter (rhodamine B) and pores are blocked by speci¿c DNA oligonucleotides. The opening of the gated systemand dye delivery is selectively controlled by DNA-miRNA recognition. Moreover, the use of DNA capture probesable to form duplex or triplex structures between target miRNA and the complementary oligonucleotides hasbeen studied. By this simple procedure a limit of detection as low as 0.25 pM was found for miRNA. The methodwas successfully applied to detect miRNA-145 in serum samples, which demonstrates the high potential of thesecapped materials to detect miRNA for diagnostic purposes ; We thank the Spanish Government (projects MAT2015-64139-C4-1-R and CTQ2014-52588-R (MINECO/FEDER)), the Generalitat Valenciana (project PROMETEOII/2014/047) and the Generalitat de Catalunya (2014/SGR/624) for support. A.R. thanks UPV for her predoctoral fellowship. S.S. thanks the Instituto de Salud Carlos III and the European Social Fund for the financial support "Sara Borrell" (CD16/000237). V.C.-N. thanks Ministerio de Educacion, Cultura y Deporte for his predoctoral grant. The authors also thank the Electron Microscopy Service at the UPV for support. ; Ribes, À.; Santiago Felipe, S.; Aviñó, A.; Candela-Noguera, V.; Eritja, R.; Sancenón Galarza, F.; Martínez-Máñez, R. (2018). Design of oligonucleotide-capped mesoporous silica nanoparticles for the detection of miRNA-145 by duplex and triplex formation. Sensors and Actuators B Chemical. 277:598-603. https://doi.org/10.1016/j.snb.2018.09.026 ; S ; 598 ; 603 ; 277

2,2,7-trimethylguanosine (TMG) binding proteins from human cells were purified through TMG-affinity columns. TMG synthesis was improved and the TMG obtained was shown to be similar to the TMG in the 5' cap of the UsnRNAs. The eluates obtained with TMG-affinity chromatographies were very different from those isolated with m7G-affinity columns, thus suggesting that specific TMG- binding proteins were obtained. The fraction may be enriched with factors associated with import and/or hypermethylation of UsnRNPs. ; This work was supported by PGC grant no PB92-0004, and partially by a grant from the Marató TV3 and Asociación Española Contra el Cáncer. We also thank the Alexander von Humboldt Foundation for providing initial help with several equipment. We thank J.M. Barrios. X. Jimenez and R. Perez for technical assistance, and all members of the M. Bach-Elias group for comments about the manuscript. C. Codony was the recipient of a fellowship from PGC; R.B. Cicarelli was the recipient from a fellowship from CAPES and Spanish MEC; A. Khaouja was the recipient of a fellowship from the Moroccan government and Diana Bahia was recipient of a CNPq fellow. We also thank Marti Cullell for revising the manuscript. ; Peer Reviewed

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

[EN] Pneumocystis pneumonia (PcP) is a disease produced by the opportunistic infection of the fungus Pneumocystis jirovecii. As delayed or unsuitable treatments increase the risk of mortality, the development of rapid and accurate diagnostic tools for PcP are of great importance. Unfortunately, current standard methods present severe limitations and are far from adequate. In this work, a time-competitive, sensitive and selective biosensor based on DNA-gated nanomaterials for the identification of P. jirovecii is presented. The biosensor consists of a nanoporous anodic alumina (NAA) scaffold which pores are filled with a dye reporter and capped with specific DNA oligonucleotides. In the presence of P. jirovecii genomic DNA, the gated biosensor is open, and the cargo is delivered to the solution where it is monitored through fluorescence spectroscopy. The use of capping oligonucleotides able to form duplex or triplex with P. jirovecii DNA is studied. The final diagnostic tool shows a limit of detection (LOD) of 1 nM of target complementary DNA and does not require previous amplification steps. The method was applied to identify DNA from P. jirovecii in unmodified bronchoalveolar lavage, nasopharyngeal aspirates, and sputum samples in 60 min. This is a promising alternative method for the routinely diagnosis of Pneumocystis pneumonia. ; This research was funded by the Spanish Government, (projects RTI2018-100910-B-C41 (MCUI/AEI/FEDER, UE) and CTQ2017-84415-R), the Generalitat Valenciana (project PROMETEO/2018/024) and CIBER-BBN (projects NANOPATH and CANDI-EYE). ; Pla, L.; Aviñó, A.; Eritja, R.; Ruiz-Gaitán, A.; Pemán, J.; Friaza, V.; Calderón, EJ. (2020). Triplex hybridization-based nanosystem for the rapid screening of pneumocystis pneumonia in clinical samples. Journal of Fungi. 6(4):1-14. https://doi.org/10.3390/jof6040292 ; S ; 1 ; 14 ; 6 ; 4 ; Fauchier, T., Hasseine, L., Gari-Toussaint, M., Casanova, V., Marty, P. M., & Pomares, C. (2016). Detection of Pneumocystis jirovecii by Quantitative PCR To ...

5480 5482 49 48 ; S ; An aptamer-capped mesoporous material for the selective and sensitive detection of &-thrombin in human plasma and serum has been prepared and characterised. The authors thank the Spanish Government (project MAT2012-38429-C04-01, CTQ2010-20541) the Generalitat Valenciana (project PROMETEO/2009/016) and the CIBER-BBN for their support. C. C. thanks the Universitat Politecnica de Valencia (UPV PAID-06-11) for support. M.O. is grateful to the Universitat Politecnica de Valencia for her grant. Oroval Cucarella, MDM.; Climent Terol, E.; Coll Merino, MC.; Eritja, R.; Aviñó, A.; Marcos Martínez, MD.; Sancenón Galarza, F. (2013). An aptamer-gated silica mesoporous material for thrombin detection. Chemical Communications. 49(48):5480-5482. https://doi.org/10.1039/c3cc42157k Patel, K., Angelos, S., Dichtel, W. R., Coskun, A., Yang, Y.-W., Zink, J. I., & Stoddart, J. F. (2008). Enzyme-Responsive Snap-Top Covered Silica Nanocontainers. Journal of the American Chemical Society, 130(8), 2382-2383. doi:10.1021/ja0772086 Schlossbauer, A., Kecht, J., & Bein, T. (2009). Biotin-Avidin as a Protease-Responsive Cap System for Controlled Guest Release from Colloidal Mesoporous Silica. Angewandte Chemie International Edition, 48(17), 3092-3095. doi:10.1002/anie.200805818 Bernardos, A., Aznar, E., Marcos, M. D., Martínez-Máñez, R., Sancenón, F., Soto, J., … Amorós, P. (2009). Enzyme-Responsive Controlled Release Using Mesoporous Silica Supports Capped with Lactose. Angewandte Chemie International Edition, 48(32), 5884-5887. doi:10.1002/anie.200900880 Park, C., Kim, H., Kim, S., & Kim, C. (2009). Enzyme Responsive Nanocontainers with Cyclodextrin Gatekeepers and Synergistic Effects in Release of Guests. Journal of the American Chemical Society, 131(46), 16614-16615. doi:10.1021/ja9061085 Thornton, P. D., & Heise, A. (2010). Highly Specific Dual Enzyme-Mediated Payload Release from Peptide-Coated Silica Particles. Journal of the American Chemical Society, 132(6), 2024-2028. doi:10.1021/ja9094439 ...