We report the structural effect of 2'‐deoxy‐2',2'‐difluorocytidine (dFdC) insertions ... more We report the structural effect of 2'‐deoxy‐2',2'‐difluorocytidine (dFdC) insertions in the DNA strand of a DNA : RNA hybrid duplex and in a self‐complementary DNA : DNA duplex. In both cases, the modification slightly destabilizes the duplex and provokes minor local distortions that are more pronounced in the case of the DNA : RNA hybrid. Analysis of the solution structures determined by NMR methods show that dFdC is an adaptable derivative that adopts North type sugar conformation when inserted in pure DNA, or a South sugar conformation in the context of DNA : RNA hybrids. In this latter context, South sugar pucker favors the formation of a 2'F⋅⋅H8 attractive interaction with a neighboring purine, which compensates the destabilizing effect of base pair distortions. These interactions share some features with pseudohydrogen bonds described previously in other nucleic acids structures with fluorine modified sugars.
An entry from the Cambridge Structural Database, the world's repository for small molecule cr... more An entry from the Cambridge Structural Database, the world's repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
Conjugation of small molecules such as lipids or receptor ligands to anti-cancer drugs has been u... more Conjugation of small molecules such as lipids or receptor ligands to anti-cancer drugs has been used to improve their pharmacological properties. In this work, we studied the biological effects of several small-molecule enhancers into a short oligonucleotide made of five floxuridine units. Specifically, we studied adding cholesterol, palmitic acid, polyethyleneglycol (PEG 1000), folic acid and triantennary N-acetylgalactosamine (GalNAc) as potential enhancers of cellular uptake. As expected, all these molecules increased the internalization efficiency with different degrees depending on the cell line. The conjugates showed antiproliferative activity due to their metabolic activation by nuclease degradation generating floxuridine monophosphate. The cytotoxicity and apoptosis assays showed an increase in the anti-cancer activity of the conjugates related to the floxuridine oligomer, but this effect did not correlate with the internalization results. Palmitic and folic acid conjugates ...
Some lung and ovarian tumors are connected to the loss of expression of SMARCA4 gene. In its prom... more Some lung and ovarian tumors are connected to the loss of expression of SMARCA4 gene. In its promoter region, a 44-nucleotides long guanine sequence prone to form G-quadruplex structures has been studied by means of spectroscopic techniques (circular dichroism, molecular absorption and nuclear magnetic resonance), size exclusion chromatography and multivariate analysis. The results have shown that the central 21-nucleotides long sequence comprising four guanine tracts of disparate length is able to fold into a pH-dependent ensemble of G-quadruplex structures. Based on acid-base titrations and melting experiments of wild and mutated sequences, the formation of a C·C+ base pair between cytosine bases present at the two lateral loops is shown to promote a reduction in conformational heterogeneity, as well as an increase in thermal stability. The formation of this base pair is characterized by a pKa value of 7.1 ± 0.2 at 20 °C and 150 mM KCl. This value, higher than those usually found ...
Most of the applications involving oligonucleotide probes are based on hybridization with complem... more Most of the applications involving oligonucleotide probes are based on hybridization with complementary targets to form a duplex. When the target sequence is a polypurine-polypyrimidine track it is possible to use special triplex forming oligonucleotides (TFO). Parallel clamps and polypurine reversed hairpins (PPRH) are special cases of TFOs in which the polypurine Watson-Crick strand is linked to either a polypyrimidine Hoogsteen strand or a polypurine reversed Hoogsteen strand. The high stability of the triplex formed upon binding of these TFOs with the polypyrimidine target allows targeting of single and double stranded nucleic acid sequences as these TFOs induce strand displacement. This targeting technology covers from gene silencing of genomic DNA and mRNA to detection of nucleic acids of biological interest.
The development of new strategies for detecting miRNAs has become a crucial step in the diagnosti... more The development of new strategies for detecting miRNAs has become a crucial step in the diagnostic field. miRNA profiles depend greatly on the sample and the analytical platform employed, leading sometimes to contradictory results. In this work we study the use of modified parallel tail-clamps to detect a miRNA sequence involved in tumor suppression by triplex formation. Thermal denaturing curves and CD measurements have been performed to confirm that parallel clamps carrying 8-aminoguanine form the most stable triplex structures with their target miRNA. The modified tail-clamps have been tested as bioreceptors in a SPR biosensor for the detection of miRNA-145. The detection limit was improved 2.4 times demonstrating that a stable triplex structure is formed between target miRNA and 8-aminoguanine tail-clamp bioreceptor. This new approach is an essential step toward the label-free and reliable detection of miRNA signatures for diagnostic purposes.
A comparative study of the cleavage efficiency of succinyl, phthaloyl, oxalyl, 2-(2-nitrophenyl)e... more A comparative study of the cleavage efficiency of succinyl, phthaloyl, oxalyl, 2-(2-nitrophenyl)ethyl, 9-fluorenylmethyl, and 2nitrophenyl supports in 0.5M DBU solutions is described. A decrease in cleavage efficiency is observed when small oligonucleotides containing thymidine are linked to the supports. In these conditions oxalyl supports gave the best yields followed by 2-(2-nitrophenyl)ethyl and 9fluorenylmethyl supports.
An aptamer-capped mesoporous material for the selective and sensitive detection of α-thrombin in ... more An aptamer-capped mesoporous material for the selective and sensitive detection of α-thrombin in human plasma and serum has been prepared and characterised.
Pneumocystis jirovecii is a fungus responsible for human Pneumocystis pneumonia, one of the most ... more Pneumocystis jirovecii is a fungus responsible for human Pneumocystis pneumonia, one of the most severe infections encountered in immunodepressed individuals. The diagnosis of Pneumocystis pneumonia continues to be challenging due to the absence of specific symptoms in infected patients. Moreover, the standard diagnostic method employed for its diagnosis involves mainly PCR-based techniques, which besides being highly specific and sensitive, require specialized personnel and equipment and are time-consuming. Our aim is to demonstrate an optical biosensor methodology based on surface plasmon resonance to perform such diagnostics in an efficient and decentralized scheme. The biosensor methodology employs poly-purine reverse-Hoogsteen hairpin probes for the detection of the mitochondrial large subunit ribosomal RNA (mtLSU rRNA) gene, related to P. jirovecii detection. The biosensor device performs a real-time and label-free identification of the mtLSU rRNA gene with excellent selectivi...
Curaxins and especially the second-generation derivative curaxin CBL0137 have important antitumor... more 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 D...
A full understanding of biomolecular function requires an analysis of both the dynamic properties... more A full understanding of biomolecular function requires an analysis of both the dynamic properties of the system of interest and the identification of those dynamics that are required for function. We describe NMR methods based on metabolically directed specific isotope labeling for the identification of molecular disorder and/or conformational transitions on the RNA backbone ribose groups. These analyses are complemented by the use of synthetic covalently modified nucleotides constrained to a single sugar pucker, which allow functional assessment of dynamics by selectively removing a minor conformer identified by NMR from the structural ensemble.
We report the structural effect of 2'‐deoxy‐2',2'‐difluorocytidine (dFdC) insertions ... more We report the structural effect of 2'‐deoxy‐2',2'‐difluorocytidine (dFdC) insertions in the DNA strand of a DNA : RNA hybrid duplex and in a self‐complementary DNA : DNA duplex. In both cases, the modification slightly destabilizes the duplex and provokes minor local distortions that are more pronounced in the case of the DNA : RNA hybrid. Analysis of the solution structures determined by NMR methods show that dFdC is an adaptable derivative that adopts North type sugar conformation when inserted in pure DNA, or a South sugar conformation in the context of DNA : RNA hybrids. In this latter context, South sugar pucker favors the formation of a 2'F⋅⋅H8 attractive interaction with a neighboring purine, which compensates the destabilizing effect of base pair distortions. These interactions share some features with pseudohydrogen bonds described previously in other nucleic acids structures with fluorine modified sugars.
An entry from the Cambridge Structural Database, the world's repository for small molecule cr... more An entry from the Cambridge Structural Database, the world's repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
Conjugation of small molecules such as lipids or receptor ligands to anti-cancer drugs has been u... more Conjugation of small molecules such as lipids or receptor ligands to anti-cancer drugs has been used to improve their pharmacological properties. In this work, we studied the biological effects of several small-molecule enhancers into a short oligonucleotide made of five floxuridine units. Specifically, we studied adding cholesterol, palmitic acid, polyethyleneglycol (PEG 1000), folic acid and triantennary N-acetylgalactosamine (GalNAc) as potential enhancers of cellular uptake. As expected, all these molecules increased the internalization efficiency with different degrees depending on the cell line. The conjugates showed antiproliferative activity due to their metabolic activation by nuclease degradation generating floxuridine monophosphate. The cytotoxicity and apoptosis assays showed an increase in the anti-cancer activity of the conjugates related to the floxuridine oligomer, but this effect did not correlate with the internalization results. Palmitic and folic acid conjugates ...
Some lung and ovarian tumors are connected to the loss of expression of SMARCA4 gene. In its prom... more Some lung and ovarian tumors are connected to the loss of expression of SMARCA4 gene. In its promoter region, a 44-nucleotides long guanine sequence prone to form G-quadruplex structures has been studied by means of spectroscopic techniques (circular dichroism, molecular absorption and nuclear magnetic resonance), size exclusion chromatography and multivariate analysis. The results have shown that the central 21-nucleotides long sequence comprising four guanine tracts of disparate length is able to fold into a pH-dependent ensemble of G-quadruplex structures. Based on acid-base titrations and melting experiments of wild and mutated sequences, the formation of a C·C+ base pair between cytosine bases present at the two lateral loops is shown to promote a reduction in conformational heterogeneity, as well as an increase in thermal stability. The formation of this base pair is characterized by a pKa value of 7.1 ± 0.2 at 20 °C and 150 mM KCl. This value, higher than those usually found ...
Most of the applications involving oligonucleotide probes are based on hybridization with complem... more Most of the applications involving oligonucleotide probes are based on hybridization with complementary targets to form a duplex. When the target sequence is a polypurine-polypyrimidine track it is possible to use special triplex forming oligonucleotides (TFO). Parallel clamps and polypurine reversed hairpins (PPRH) are special cases of TFOs in which the polypurine Watson-Crick strand is linked to either a polypyrimidine Hoogsteen strand or a polypurine reversed Hoogsteen strand. The high stability of the triplex formed upon binding of these TFOs with the polypyrimidine target allows targeting of single and double stranded nucleic acid sequences as these TFOs induce strand displacement. This targeting technology covers from gene silencing of genomic DNA and mRNA to detection of nucleic acids of biological interest.
The development of new strategies for detecting miRNAs has become a crucial step in the diagnosti... more The development of new strategies for detecting miRNAs has become a crucial step in the diagnostic field. miRNA profiles depend greatly on the sample and the analytical platform employed, leading sometimes to contradictory results. In this work we study the use of modified parallel tail-clamps to detect a miRNA sequence involved in tumor suppression by triplex formation. Thermal denaturing curves and CD measurements have been performed to confirm that parallel clamps carrying 8-aminoguanine form the most stable triplex structures with their target miRNA. The modified tail-clamps have been tested as bioreceptors in a SPR biosensor for the detection of miRNA-145. The detection limit was improved 2.4 times demonstrating that a stable triplex structure is formed between target miRNA and 8-aminoguanine tail-clamp bioreceptor. This new approach is an essential step toward the label-free and reliable detection of miRNA signatures for diagnostic purposes.
A comparative study of the cleavage efficiency of succinyl, phthaloyl, oxalyl, 2-(2-nitrophenyl)e... more A comparative study of the cleavage efficiency of succinyl, phthaloyl, oxalyl, 2-(2-nitrophenyl)ethyl, 9-fluorenylmethyl, and 2nitrophenyl supports in 0.5M DBU solutions is described. A decrease in cleavage efficiency is observed when small oligonucleotides containing thymidine are linked to the supports. In these conditions oxalyl supports gave the best yields followed by 2-(2-nitrophenyl)ethyl and 9fluorenylmethyl supports.
An aptamer-capped mesoporous material for the selective and sensitive detection of α-thrombin in ... more An aptamer-capped mesoporous material for the selective and sensitive detection of α-thrombin in human plasma and serum has been prepared and characterised.
Pneumocystis jirovecii is a fungus responsible for human Pneumocystis pneumonia, one of the most ... more Pneumocystis jirovecii is a fungus responsible for human Pneumocystis pneumonia, one of the most severe infections encountered in immunodepressed individuals. The diagnosis of Pneumocystis pneumonia continues to be challenging due to the absence of specific symptoms in infected patients. Moreover, the standard diagnostic method employed for its diagnosis involves mainly PCR-based techniques, which besides being highly specific and sensitive, require specialized personnel and equipment and are time-consuming. Our aim is to demonstrate an optical biosensor methodology based on surface plasmon resonance to perform such diagnostics in an efficient and decentralized scheme. The biosensor methodology employs poly-purine reverse-Hoogsteen hairpin probes for the detection of the mitochondrial large subunit ribosomal RNA (mtLSU rRNA) gene, related to P. jirovecii detection. The biosensor device performs a real-time and label-free identification of the mtLSU rRNA gene with excellent selectivi...
Curaxins and especially the second-generation derivative curaxin CBL0137 have important antitumor... more 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 D...
A full understanding of biomolecular function requires an analysis of both the dynamic properties... more A full understanding of biomolecular function requires an analysis of both the dynamic properties of the system of interest and the identification of those dynamics that are required for function. We describe NMR methods based on metabolically directed specific isotope labeling for the identification of molecular disorder and/or conformational transitions on the RNA backbone ribose groups. These analyses are complemented by the use of synthetic covalently modified nucleotides constrained to a single sugar pucker, which allow functional assessment of dynamics by selectively removing a minor conformer identified by NMR from the structural ensemble.
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Papers by Anna Aviñó