The i‐motif DNA, also known as i‐DNA, is a non‐canonical DNA secondary structure formed by cytosi... more The i‐motif DNA, also known as i‐DNA, is a non‐canonical DNA secondary structure formed by cytosine‐rich sequences, consisting of two intercalated parallel‐stranded duplexes held together by hemi‐protonated cytosine–cytosine+ (C:C+) base pairs. The growing interest in the i‐DNA structure as a target in anticancer therapy increases the need for tools for a rapid and meaningful interpretation of the spectroscopic data of i‐DNA samples. Herein, we analyzed the circular dichroism (CD) and thermal difference UV‐absorbance spectra (TDS) of 255 DNA sequences by means of multivariate data analysis, aiming at unveiling peculiar spectral regions that could be used as diagnostic features during the analysis of i‐DNA‐forming sequences.
Starting from a chemoproteomic-driven approach, novel human telomeric G-quadruplex binding protei... more Starting from a chemoproteomic-driven approach, novel human telomeric G-quadruplex binding proteins were identified that directly bind the DNA structure in vitro and colocalize with such structures in cells.
A proof of principle study on the use of nanoDSF as a screening tool for G-quadruplex targeting c... more A proof of principle study on the use of nanoDSF as a screening tool for G-quadruplex targeting compounds.
Differential Scanning Calorimetry (DSC) is a straightforward methodology to characterize the ener... more Differential Scanning Calorimetry (DSC) is a straightforward methodology to characterize the energetics of thermally-induced transitions of DNA and other biological macromolecules. Therefore, DSC has been used to study the thermodynamic stability of several nucleic acids structures. G-quadruplexes are among the most important non-canonical nucleic acid architectures that are receiving great consideration. This article reports examples on the contribution of DSC to the knowledge of G-quadruplex structures. The selected case studies show the potential of this method in investigating the structure stability of G-quadruplex forming nucleic acids, and in providing information on their structural complexity. Indeed, DSC can determine thermodynamic parameters of G-quadruplex folding/unfolding processes, but it can also be useful to reveal the formation of multiple conformations or the presence of intermediate states along the unfolding pathway, and to evaluate the impact of chemical modifications on their structural stability. This article aims to show that DSC is an important complementary methodology to structural techniques, such as NMR and X-ray crystallography, in the study of G-quadruplex forming nucleic acids.
The complex between distamycin A and the parallel DNA quadruplex [d(TGGGGT)]4 has been studied by... more The complex between distamycin A and the parallel DNA quadruplex [d(TGGGGT)]4 has been studied by 1 H NMR spectroscopy and isothermal titration calorimetry (ITC). To unambiguously assert that distamycin A interacts with the grooves of the quadruplex [d(TGGGGT)]4, we have analyzed the NMR titration profile of a modified quadruplex, namely [d(TGG Me GGT)]4, and we have applied the recently developed differential frequency-saturation transfer difference (DF-STD) method, for assessing the ligand-DNA binding mode. The three-dimensional structure of the 4:1 distamycin A/[d(TGGGGT)]4 complex has been determined by an in-depth NMR study followed by dynamics and mechanics calculations. All results unequivocally indicate that distamycin molecules interact with [d(TGGGGT)] 4 in a 4:1 binding mode, with two antiparallel distamycin dimers that bind simultaneously two opposite grooves of the quadruplex. The affinity between distamycin A and [d(TGGGGT)] 4 enhances (∼10-fold) when the ratio of distamycin A to the quadruplex is increased. In this paper we report the first three-dimensional structure of a groovebinder molecule complexed to a DNA quadruplex structure.
PNA-DNA chimeras present the interesting properties of PNA, such as the high binding affinity to ... more PNA-DNA chimeras present the interesting properties of PNA, such as the high binding affinity to complementary single-strand (DNA or RNA), and the resistance to nuclease and protease degradation. At the same time, the limitations of an oligomer containing all PNA residues, such as low water solubility, self-aggregation, and low cellular uptake, are effectively overcome. Further, PNA-DNA chimeras possess interesting biological properties as antisense agents. We have explored the ability of PNA-DNA chimeric strands to assemble in quadruplex structures. The rate constant for association of the quadruplexes and their thermodynamic properties have been determined by CD spectroscopy and differential scanning calorimetry (DSC). Thermal denaturation experiments indicated higher thermal and thermodynamic stabilities for chimeric quadruplexes in comparison with the corresponding unmodified DNA quadruplex. Singular value decomposition analysis (SVD) suggests the presence of kinetically stable intermediate species in the quadruplex formation process. The experimental results have been discussed on the basis of molecular dynamic simulations. The ability of PNA-DNA chimeras to form stable quadruplex structures expands their potential utility as therapeutic agents.
Intracellular environment is crowded with biomolecules that occupy a significant fraction (up to ... more Intracellular environment is crowded with biomolecules that occupy a significant fraction (up to 40%) of the cellular volume, with a total concentration in the range 300-400mg/ml. Recently, the effect of crowding/dehydrating agents on the DNA G-quadruplexes has become a subject of an increasing interest. Crowding and/or dehydrating agents have been used to simulate how G-quadruplexes behave under cell-mimicking conditions characterized by a large excluded volume and a lower water activity. Indeed, the presence of both steric crowding and a lower water activity can affect G-quadruplex stability, their folding/unfolding kinetics, as well as their binding processes with proteins or small ligands. Many of these effects can be explored experimentally by measuring the dependence of the conformational stability, isomerisation kinetics and equilibria on the concentration of cosolutes which do not interact with the molecules (G-quadruplexes) under investigation. Spectroscopic methodologies, ...
. Quantitative Descriptive Analysis results. fruity leaf grassy bitter pungent sweet almond artic... more . Quantitative Descriptive Analysis results. fruity leaf grassy bitter pungent sweet almond artichoke apple tomato rosemary *R 2 (cum) indicate how well the variation of the variable is explained. **Q 2 (cum) indicate how well the variable can be predicted.
Immortality of tumour cells is strictly correlated to telomerase activity. Telomerase is overexpr... more Immortality of tumour cells is strictly correlated to telomerase activity. Telomerase is overexpressed in about 85% of tumour cells and maintains telomere length contributing to cell immortalisation, whereas in somatic cells telomeres progressively shorten until cell death occurs by apoptosis. Different drugs can promote telomeric G-rich overhangs which fold into quadruplex structures that inhibit telomerase activity. Detailed studies on drug-quadruplex complexes are essential to understand quadruplex recognition and address drug design. This review will discuss the energetic aspects of quadruplex-drug interactions with a particular attention to physico-chemical methodologies.
Here we report the X-ray crystal structure of nemorosone, a polyprenylated benzophenone derivativ... more Here we report the X-ray crystal structure of nemorosone, a polyprenylated benzophenone derivative that presents biological activity and potential use as antimicrobial, cytotoxic and antioxidant drug. On the basis of crystallographic data, we carried out a theoretical investigation based on state-of-the-art density functional approaches. A remarkable agreement has been found between experimental findings and quantum mechanical results. Furthermore, the theoretical calculations allowed us to dissect the structural and energetic features of the nemorosone molecule, thus paving the route toward the design of new synthetic drugs.
Aptamer-based drugs represent an attractive approach in pharmacological therapy. The most studied... more Aptamer-based drugs represent an attractive approach in pharmacological therapy. The most studied aptamer, thrombin binding aptamer (TBA), folds into a well-defined quadruplex structure and binds to its target with good specificity and affinity. Modified aptamers with improved biophysical properties could constitute a new class of therapeutic aptamers. In this study we show that the modified thrombin binding aptamer (mTBA), 39 GGT 59 -59 TGGTGTGGTTGG 39 , which also folds into a quadruplex structure, is more stable than its unmodified counterpart and shows a higher thrombin affinity. The stability of the modified aptamer was investigated using differential scanning calorimetry, and the energetics of mTBA and TBA binding to thrombin was characterized by means of isothermal titration calorimetry (ITC). ITC data revealed that TBA/thrombin and mTBA/ thrombin binding stoichiometry is 1:2 for both interactions. Structural models of the two complexes of thrombin with TBA and with mTBA were also obtained and subjected to molecular dynamics simulations in explicit water. Analysis of the models led to an improvement of the understanding of the aptamer-thrombin recognition at a molecular level.
Activation of the multicomponent enzyme NADPH oxidase requires the interaction between the tandem... more Activation of the multicomponent enzyme NADPH oxidase requires the interaction between the tandem SH3 domain of the cytosolic subunit p47 phox and the cytoplasmic tail of membrane-bound p22 phox . In the resting state, p47 phox exists in an autoinhibited conformation stabilized by intramolecular contacts between the SH3 domains and an adjacent polybasic region. Phosphorylation of three serine residues, Ser 303 , Ser 304 , and Ser 328 within this polybasic region has been shown to be sufficient for the disruption of the intramolecular interactions thereby inducing an active state of p47 phox . This active conformation is accessible to the cytoplasmic tail of p22 phox and initiates the formation of the membrane-bound functional enzyme complex. Molecular dynamics simulations reveal insights in the mechanism of activation of the autoinhibited form of p47 phox by in silico phosphorylation, of the three serine residues, Ser 303 , Ser 304 , and Ser 328 . The simulations highlight the major collective coordinates generating the opening and the closing of the two SH3 domains and the residues that cause the unmasking of the p22 phox binding site.
A series of trisubstituted naphthalimides have been synthesized and evaluated as telomeric G-quad... more A series of trisubstituted naphthalimides have been synthesized and evaluated as telomeric G-quadruplex ligands by biophysical methods. Affinity for telomeric G-quadruplex AGGG(TTAGGG) 3 binding was first screened by fluorescence titrations. Subsequently, the interaction of the telomeric G-quadruplex with compounds showing the best affinity has been studied by isothermal titration calorimetry and UVmelting experiments. The two best compounds of the series tightly bind the telomeric quadruplex with a 2:1 drug/DNA stoichiometry. These derivatives have been further evaluated for their ability to inhibit telomerase by a TRAP assay and their pharmacological properties by treating melanoma (M14) and human lung cancer (A549) cell lines with increasing drug concentrations. A dose-dependent inhibition of cell proliferation was observed for all cellular lines during short-term treatment.
A novel fluorescent thrombin binding aptamer (TBA), conjugated with the environmentally sensitive... more A novel fluorescent thrombin binding aptamer (TBA), conjugated with the environmentally sensitive dansyl probe at the 3′-end and a β-cyclodextrin residue at the 5′-end, has been efficiently synthesized exploiting Cu(I)-catalyzed azide−alkyne cycloaddition procedures. Its conformation and stability in solution have been studied by an integrated approach, combining in-depth NMR, CD, fluorescence, and DSC studies. ITC measurements have allowed us to analyze in detail its interaction with human thrombin. All the collected data show that this bis-conjugated aptamer fully retains its Gquadruplex formation ability and thrombin recognition properties, with the terminal appendages only marginally interfering with the conformational behavior of TBA. Folding of this modified aptamer into the chairlike, antiparallel G-quadruplex structure, promoted by K + and/or thrombin binding, typical of TBA, is associated with a net fluorescence enhancement, due to encapsulation of dansyl, attached at the 3′-end, into the apolar cavity of the β-cyclodextrin at the 5′-end. Overall, the structural characterization of this novel, bis-conjugated TBA fully demonstrates its potential as a diagnostic tool for thrombin recognition, also providing a useful basis for the design of suitable aptamer-based devices for theranostic applications, allowing simultaneously both detection and inhibition or modulation of the thrombin activity.
The cKit87up sequence d( 5 0 AGGGAGGGCGC-TGGGAGGAGGG 3 0 ) can form a unique G-quadruplex structu... more The cKit87up sequence d( 5 0 AGGGAGGGCGC-TGGGAGGAGGG 3 0 ) can form a unique G-quadruplex structure in the promoter region of the human c-kit protooncogene. It provides a peculiar platform for the design of selective quadruplex-binding agents, which could potentially repress the protooncogene transcription. In this study, we examined the binding of a small library of PNA probes (P1ÀP5) targeting cKit87up quadruplex in either K þ -or NH 4 þ -containing solutions by using a combination of UV, CD, PAGE, ITC, and ESI-MS methodologies. Our results showed that (1) P1ÀP4 interact with the cKit87up quadruplex, and (2) the binding mode depends on the quadruplex stability. In K þ buffer, P1ÀP4 bind the ckit87up quadruplex structure as "quadruplex-binding agents". The same holds for P1 in NH 4 þ solution. On the contrary, in NH 4 þ solution, P2ÀP4 overcome the quadruplex structure by forming PNA/DNA hybrid complexes, thus acting as "quadruplex openers".
The use of small molecules that bind and stabilize G-quadruplex structures is emerging as a promi... more The use of small molecules that bind and stabilize G-quadruplex structures is emerging as a promising way to inhibit telomerase activity in tumor cells. In this paper, isothermal titration calorimetry (ITC) and 1 H NMR studies have been conducted to examine the binding of distamycin A and its two carbamoyl derivatives (compounds 1 and 2) to the target [d(TGGGGT)] 4 and d[AG 3 (T 2 AG 3 ) 3 ] quadruplexes from the Tetrahymena and human telomeres, respectively. The interactions were examined using two different buffered solutions containing either K þ or Na þ at a fixed ionic strength, to evaluate any influence of the ions present in solution on the binding behaviour. Experiments reveal that distamycin A and compound 1 bind the investigated quadruplexes in both solution conditions; conversely, compound 2 appears to have a poor affinity in any case. Moreover, these studies indicate that the presence of different cations in solution affects the stoichiometry and thermodynamics of the interactions.
To assess the potential of mutations from the L1 loop of the tumour suppressor p53 as second-site... more To assess the potential of mutations from the L1 loop of the tumour suppressor p53 as second-site suppressors, the effect of H115N and S116M on the p53 'hot spot' mutations has been investigated using the double-mutant approach. The effects of these two mutants on the p53 hot spots in terms of thermal stability and DNA binding were evaluated. The results show that: (i) the p53 mutants H115N and S116M are thermally more stable than wild-type p53; (ii) H115N but not S116M is capable of rescuing the DNA binding of one of the most frequent p53 mutants in cancer, R248Q, as shown by binding of R248Q/H115N to gadd45 (the promoter of a gene involved in cell-cycle arrest); (iii) the double mutant R248Q/H115N is more stable than wild-type p53;
A simple, cheap, and highly reproducible affinity chromatography-based method has been developed ... more A simple, cheap, and highly reproducible affinity chromatography-based method has been developed for the screening of G-quadruplex binders. The tested compounds were flowed through a polystyrene resin functionalized with an oligonucleotide able to form, in proper conditions, a G-quadruplex structure. Upon cation-induced control of the folding/ unfolding processes of the immobilized G-quadruplex-forming sequence, small molecules specifically interacting with the oligonucleotide structure were first captured and then released depending on the used working solution. This protocol, first optimized for different kinds of known Gquadruplex ligands and then applied to a set of putative ligands, has allowed one to fully reuse the same functionalized resin batch, recycled for several tens of experiments without loss in efficiency and reproducibility.
The i‐motif DNA, also known as i‐DNA, is a non‐canonical DNA secondary structure formed by cytosi... more The i‐motif DNA, also known as i‐DNA, is a non‐canonical DNA secondary structure formed by cytosine‐rich sequences, consisting of two intercalated parallel‐stranded duplexes held together by hemi‐protonated cytosine–cytosine+ (C:C+) base pairs. The growing interest in the i‐DNA structure as a target in anticancer therapy increases the need for tools for a rapid and meaningful interpretation of the spectroscopic data of i‐DNA samples. Herein, we analyzed the circular dichroism (CD) and thermal difference UV‐absorbance spectra (TDS) of 255 DNA sequences by means of multivariate data analysis, aiming at unveiling peculiar spectral regions that could be used as diagnostic features during the analysis of i‐DNA‐forming sequences.
Starting from a chemoproteomic-driven approach, novel human telomeric G-quadruplex binding protei... more Starting from a chemoproteomic-driven approach, novel human telomeric G-quadruplex binding proteins were identified that directly bind the DNA structure in vitro and colocalize with such structures in cells.
A proof of principle study on the use of nanoDSF as a screening tool for G-quadruplex targeting c... more A proof of principle study on the use of nanoDSF as a screening tool for G-quadruplex targeting compounds.
Differential Scanning Calorimetry (DSC) is a straightforward methodology to characterize the ener... more Differential Scanning Calorimetry (DSC) is a straightforward methodology to characterize the energetics of thermally-induced transitions of DNA and other biological macromolecules. Therefore, DSC has been used to study the thermodynamic stability of several nucleic acids structures. G-quadruplexes are among the most important non-canonical nucleic acid architectures that are receiving great consideration. This article reports examples on the contribution of DSC to the knowledge of G-quadruplex structures. The selected case studies show the potential of this method in investigating the structure stability of G-quadruplex forming nucleic acids, and in providing information on their structural complexity. Indeed, DSC can determine thermodynamic parameters of G-quadruplex folding/unfolding processes, but it can also be useful to reveal the formation of multiple conformations or the presence of intermediate states along the unfolding pathway, and to evaluate the impact of chemical modifications on their structural stability. This article aims to show that DSC is an important complementary methodology to structural techniques, such as NMR and X-ray crystallography, in the study of G-quadruplex forming nucleic acids.
The complex between distamycin A and the parallel DNA quadruplex [d(TGGGGT)]4 has been studied by... more The complex between distamycin A and the parallel DNA quadruplex [d(TGGGGT)]4 has been studied by 1 H NMR spectroscopy and isothermal titration calorimetry (ITC). To unambiguously assert that distamycin A interacts with the grooves of the quadruplex [d(TGGGGT)]4, we have analyzed the NMR titration profile of a modified quadruplex, namely [d(TGG Me GGT)]4, and we have applied the recently developed differential frequency-saturation transfer difference (DF-STD) method, for assessing the ligand-DNA binding mode. The three-dimensional structure of the 4:1 distamycin A/[d(TGGGGT)]4 complex has been determined by an in-depth NMR study followed by dynamics and mechanics calculations. All results unequivocally indicate that distamycin molecules interact with [d(TGGGGT)] 4 in a 4:1 binding mode, with two antiparallel distamycin dimers that bind simultaneously two opposite grooves of the quadruplex. The affinity between distamycin A and [d(TGGGGT)] 4 enhances (∼10-fold) when the ratio of distamycin A to the quadruplex is increased. In this paper we report the first three-dimensional structure of a groovebinder molecule complexed to a DNA quadruplex structure.
PNA-DNA chimeras present the interesting properties of PNA, such as the high binding affinity to ... more PNA-DNA chimeras present the interesting properties of PNA, such as the high binding affinity to complementary single-strand (DNA or RNA), and the resistance to nuclease and protease degradation. At the same time, the limitations of an oligomer containing all PNA residues, such as low water solubility, self-aggregation, and low cellular uptake, are effectively overcome. Further, PNA-DNA chimeras possess interesting biological properties as antisense agents. We have explored the ability of PNA-DNA chimeric strands to assemble in quadruplex structures. The rate constant for association of the quadruplexes and their thermodynamic properties have been determined by CD spectroscopy and differential scanning calorimetry (DSC). Thermal denaturation experiments indicated higher thermal and thermodynamic stabilities for chimeric quadruplexes in comparison with the corresponding unmodified DNA quadruplex. Singular value decomposition analysis (SVD) suggests the presence of kinetically stable intermediate species in the quadruplex formation process. The experimental results have been discussed on the basis of molecular dynamic simulations. The ability of PNA-DNA chimeras to form stable quadruplex structures expands their potential utility as therapeutic agents.
Intracellular environment is crowded with biomolecules that occupy a significant fraction (up to ... more Intracellular environment is crowded with biomolecules that occupy a significant fraction (up to 40%) of the cellular volume, with a total concentration in the range 300-400mg/ml. Recently, the effect of crowding/dehydrating agents on the DNA G-quadruplexes has become a subject of an increasing interest. Crowding and/or dehydrating agents have been used to simulate how G-quadruplexes behave under cell-mimicking conditions characterized by a large excluded volume and a lower water activity. Indeed, the presence of both steric crowding and a lower water activity can affect G-quadruplex stability, their folding/unfolding kinetics, as well as their binding processes with proteins or small ligands. Many of these effects can be explored experimentally by measuring the dependence of the conformational stability, isomerisation kinetics and equilibria on the concentration of cosolutes which do not interact with the molecules (G-quadruplexes) under investigation. Spectroscopic methodologies, ...
. Quantitative Descriptive Analysis results. fruity leaf grassy bitter pungent sweet almond artic... more . Quantitative Descriptive Analysis results. fruity leaf grassy bitter pungent sweet almond artichoke apple tomato rosemary *R 2 (cum) indicate how well the variation of the variable is explained. **Q 2 (cum) indicate how well the variable can be predicted.
Immortality of tumour cells is strictly correlated to telomerase activity. Telomerase is overexpr... more Immortality of tumour cells is strictly correlated to telomerase activity. Telomerase is overexpressed in about 85% of tumour cells and maintains telomere length contributing to cell immortalisation, whereas in somatic cells telomeres progressively shorten until cell death occurs by apoptosis. Different drugs can promote telomeric G-rich overhangs which fold into quadruplex structures that inhibit telomerase activity. Detailed studies on drug-quadruplex complexes are essential to understand quadruplex recognition and address drug design. This review will discuss the energetic aspects of quadruplex-drug interactions with a particular attention to physico-chemical methodologies.
Here we report the X-ray crystal structure of nemorosone, a polyprenylated benzophenone derivativ... more Here we report the X-ray crystal structure of nemorosone, a polyprenylated benzophenone derivative that presents biological activity and potential use as antimicrobial, cytotoxic and antioxidant drug. On the basis of crystallographic data, we carried out a theoretical investigation based on state-of-the-art density functional approaches. A remarkable agreement has been found between experimental findings and quantum mechanical results. Furthermore, the theoretical calculations allowed us to dissect the structural and energetic features of the nemorosone molecule, thus paving the route toward the design of new synthetic drugs.
Aptamer-based drugs represent an attractive approach in pharmacological therapy. The most studied... more Aptamer-based drugs represent an attractive approach in pharmacological therapy. The most studied aptamer, thrombin binding aptamer (TBA), folds into a well-defined quadruplex structure and binds to its target with good specificity and affinity. Modified aptamers with improved biophysical properties could constitute a new class of therapeutic aptamers. In this study we show that the modified thrombin binding aptamer (mTBA), 39 GGT 59 -59 TGGTGTGGTTGG 39 , which also folds into a quadruplex structure, is more stable than its unmodified counterpart and shows a higher thrombin affinity. The stability of the modified aptamer was investigated using differential scanning calorimetry, and the energetics of mTBA and TBA binding to thrombin was characterized by means of isothermal titration calorimetry (ITC). ITC data revealed that TBA/thrombin and mTBA/ thrombin binding stoichiometry is 1:2 for both interactions. Structural models of the two complexes of thrombin with TBA and with mTBA were also obtained and subjected to molecular dynamics simulations in explicit water. Analysis of the models led to an improvement of the understanding of the aptamer-thrombin recognition at a molecular level.
Activation of the multicomponent enzyme NADPH oxidase requires the interaction between the tandem... more Activation of the multicomponent enzyme NADPH oxidase requires the interaction between the tandem SH3 domain of the cytosolic subunit p47 phox and the cytoplasmic tail of membrane-bound p22 phox . In the resting state, p47 phox exists in an autoinhibited conformation stabilized by intramolecular contacts between the SH3 domains and an adjacent polybasic region. Phosphorylation of three serine residues, Ser 303 , Ser 304 , and Ser 328 within this polybasic region has been shown to be sufficient for the disruption of the intramolecular interactions thereby inducing an active state of p47 phox . This active conformation is accessible to the cytoplasmic tail of p22 phox and initiates the formation of the membrane-bound functional enzyme complex. Molecular dynamics simulations reveal insights in the mechanism of activation of the autoinhibited form of p47 phox by in silico phosphorylation, of the three serine residues, Ser 303 , Ser 304 , and Ser 328 . The simulations highlight the major collective coordinates generating the opening and the closing of the two SH3 domains and the residues that cause the unmasking of the p22 phox binding site.
A series of trisubstituted naphthalimides have been synthesized and evaluated as telomeric G-quad... more A series of trisubstituted naphthalimides have been synthesized and evaluated as telomeric G-quadruplex ligands by biophysical methods. Affinity for telomeric G-quadruplex AGGG(TTAGGG) 3 binding was first screened by fluorescence titrations. Subsequently, the interaction of the telomeric G-quadruplex with compounds showing the best affinity has been studied by isothermal titration calorimetry and UVmelting experiments. The two best compounds of the series tightly bind the telomeric quadruplex with a 2:1 drug/DNA stoichiometry. These derivatives have been further evaluated for their ability to inhibit telomerase by a TRAP assay and their pharmacological properties by treating melanoma (M14) and human lung cancer (A549) cell lines with increasing drug concentrations. A dose-dependent inhibition of cell proliferation was observed for all cellular lines during short-term treatment.
A novel fluorescent thrombin binding aptamer (TBA), conjugated with the environmentally sensitive... more A novel fluorescent thrombin binding aptamer (TBA), conjugated with the environmentally sensitive dansyl probe at the 3′-end and a β-cyclodextrin residue at the 5′-end, has been efficiently synthesized exploiting Cu(I)-catalyzed azide−alkyne cycloaddition procedures. Its conformation and stability in solution have been studied by an integrated approach, combining in-depth NMR, CD, fluorescence, and DSC studies. ITC measurements have allowed us to analyze in detail its interaction with human thrombin. All the collected data show that this bis-conjugated aptamer fully retains its Gquadruplex formation ability and thrombin recognition properties, with the terminal appendages only marginally interfering with the conformational behavior of TBA. Folding of this modified aptamer into the chairlike, antiparallel G-quadruplex structure, promoted by K + and/or thrombin binding, typical of TBA, is associated with a net fluorescence enhancement, due to encapsulation of dansyl, attached at the 3′-end, into the apolar cavity of the β-cyclodextrin at the 5′-end. Overall, the structural characterization of this novel, bis-conjugated TBA fully demonstrates its potential as a diagnostic tool for thrombin recognition, also providing a useful basis for the design of suitable aptamer-based devices for theranostic applications, allowing simultaneously both detection and inhibition or modulation of the thrombin activity.
The cKit87up sequence d( 5 0 AGGGAGGGCGC-TGGGAGGAGGG 3 0 ) can form a unique G-quadruplex structu... more The cKit87up sequence d( 5 0 AGGGAGGGCGC-TGGGAGGAGGG 3 0 ) can form a unique G-quadruplex structure in the promoter region of the human c-kit protooncogene. It provides a peculiar platform for the design of selective quadruplex-binding agents, which could potentially repress the protooncogene transcription. In this study, we examined the binding of a small library of PNA probes (P1ÀP5) targeting cKit87up quadruplex in either K þ -or NH 4 þ -containing solutions by using a combination of UV, CD, PAGE, ITC, and ESI-MS methodologies. Our results showed that (1) P1ÀP4 interact with the cKit87up quadruplex, and (2) the binding mode depends on the quadruplex stability. In K þ buffer, P1ÀP4 bind the ckit87up quadruplex structure as "quadruplex-binding agents". The same holds for P1 in NH 4 þ solution. On the contrary, in NH 4 þ solution, P2ÀP4 overcome the quadruplex structure by forming PNA/DNA hybrid complexes, thus acting as "quadruplex openers".
The use of small molecules that bind and stabilize G-quadruplex structures is emerging as a promi... more The use of small molecules that bind and stabilize G-quadruplex structures is emerging as a promising way to inhibit telomerase activity in tumor cells. In this paper, isothermal titration calorimetry (ITC) and 1 H NMR studies have been conducted to examine the binding of distamycin A and its two carbamoyl derivatives (compounds 1 and 2) to the target [d(TGGGGT)] 4 and d[AG 3 (T 2 AG 3 ) 3 ] quadruplexes from the Tetrahymena and human telomeres, respectively. The interactions were examined using two different buffered solutions containing either K þ or Na þ at a fixed ionic strength, to evaluate any influence of the ions present in solution on the binding behaviour. Experiments reveal that distamycin A and compound 1 bind the investigated quadruplexes in both solution conditions; conversely, compound 2 appears to have a poor affinity in any case. Moreover, these studies indicate that the presence of different cations in solution affects the stoichiometry and thermodynamics of the interactions.
To assess the potential of mutations from the L1 loop of the tumour suppressor p53 as second-site... more To assess the potential of mutations from the L1 loop of the tumour suppressor p53 as second-site suppressors, the effect of H115N and S116M on the p53 'hot spot' mutations has been investigated using the double-mutant approach. The effects of these two mutants on the p53 hot spots in terms of thermal stability and DNA binding were evaluated. The results show that: (i) the p53 mutants H115N and S116M are thermally more stable than wild-type p53; (ii) H115N but not S116M is capable of rescuing the DNA binding of one of the most frequent p53 mutants in cancer, R248Q, as shown by binding of R248Q/H115N to gadd45 (the promoter of a gene involved in cell-cycle arrest); (iii) the double mutant R248Q/H115N is more stable than wild-type p53;
A simple, cheap, and highly reproducible affinity chromatography-based method has been developed ... more A simple, cheap, and highly reproducible affinity chromatography-based method has been developed for the screening of G-quadruplex binders. The tested compounds were flowed through a polystyrene resin functionalized with an oligonucleotide able to form, in proper conditions, a G-quadruplex structure. Upon cation-induced control of the folding/ unfolding processes of the immobilized G-quadruplex-forming sequence, small molecules specifically interacting with the oligonucleotide structure were first captured and then released depending on the used working solution. This protocol, first optimized for different kinds of known Gquadruplex ligands and then applied to a set of putative ligands, has allowed one to fully reuse the same functionalized resin batch, recycled for several tens of experiments without loss in efficiency and reproducibility.
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Papers by Bruno Pagano