| Name | Concetta |
| Surname | Giancola |
| Institution | University of Naples – Federico II |
| concetta.giancola@unina.it | |
| Address | Department of Pharmacy, University of Napoli Federico II, Via D. Montesano 49, 80131, Napoli, Italy; InterUniversity Center for Research in Neurosciences, University of Campania "Luigi Vanvitelli", Napoli, Italy |
Dominant diseases are single gene disorders occurring in the heterozygous state. The mutated allele exerts a dominant effect because it produces an abnormal polypeptide that interferes with the function of the normal allele product. Peptide Nucleic Acids (PNAs) offer a route for a potential therapy for dominant diseases by selectively silencing the allele carrying the dominant mutation. Here, we have synthesized and studied the properties of a 15-mer PNA fully complementary to the site of the c.5272-38T>A sequence variation, which identifies a recurrent mutant COL7A1 allele causing dominant dystrophic epidermolysis bullosa (DDEB), a mendelian disease characterized by skin blistering. The PNA was conjugated with four lysine residues at the C-terminus and a fluorescent probe at the N-terminus. Physico-chemical results proved the formation of a stable, selective PNA/mutant-DNA heteroduplex in vitro. Intriguingly, when transfected into normal human fibroblasts, the PNA correctly localized in the cell nucleus. Our results open new therapeutic possibilities for patients with DDEB.
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 G-quadruplex 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.
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.
G-quadruplex unfolding within a sequence of two quadruplex units was characterized by gel electrophoresis, calorimetry and spectroscopy. The obtained results suggest that the kinetics and thermodynamics of the individual quadruplex unfolding are affected by its interaction with other DNA secondary structural elements.
Prion protein (PrP) is involved in lethal neurodegenerative diseases, and many issues remain unclear about its physio-pathological role. Quadruplex-forming nucleic acids (NAs) have been found to specifically bind to both PrP cellular and pathological isoforms. To clarify the relevance of these interactions, thermodynamic, kinetic and structural studies have been performed, using isothermal titration calorimetry, surface plasmon resonance and circular dichroism methodologies. Three quadruplex-forming sequences, d(TGGGGT), r(GGAGGAGGAGGA), d(GGAGGAGGAGGA), and various forms of PrP were selected for this study. Our results showed that these quadruplexes exhibit a high affinity and specificity toward PrP, with K(D) values within the range 62÷630 nM, and a weaker affinity toward a PrP-β oligomer, which mimics the pathological isoform. We demonstrated that the NA quadruplex architecture is the structural determinant for the recognition by both PrP isoforms. Furthermore, we spotted both PrP N-terminal and C-terminal domains as the binding regions involved in the interaction with DNA/RNAs, using several PrP truncated forms. Interestingly, a reciprocally induced structure loss was observed upon PrP-NA interaction. Our results allowed to surmise a quadruplex unwinding-activity of PrP, that may have a feedback in vivo.
G-quadruplex ligands are potential anticancer agents as telomerase inhibitors and potential transcriptional regulators of oncogenes. The search for best-in-class drugs is addressed to identify small molecules able to promote and stabilize G-quadruplex structures. What features should the G-quadruplex ligands possess? They should have selective antiproliferative effects on cancer cells and induce telomerase inhibition or oncogene suppression. One of the main challenges in their design and synthesis is to make the ligands selective for G-quadruplex DNA. These features should be amplified by careful analyses of physico-chemical aspects of G-quadruplex-drug interactions. In particular, the study of the energetics of G-quadruplex-drug interactions can enhance drug design by providing thermodynamic parameters that give quantitative information on the biomolecular interactions important for binding. The main methodologies used to gain information on energetics of binding are based on spectroscopic or calorimetric principles. Spectroscopic techniques such as fluorescence and circular dichroism are rapid and cheap methods, but are not sufficient to characterize completely the thermodynamics of interaction. Calorimetric techniques such as isothermal titration calorimetry offer a direct measure of binding enthalpy, in addition to the stoichiometry and affinity constants. With the complete thermodynamic signature of drug-target interaction, dissecting the enthalpic and entropic components of binding is possible, which can be a useful aid to decision-making during drug optimization.
The abasic sites represent one of the most frequent lesions of DNA and most of the events able to generate such modifications involve guanine bases. G-rich sequences are able to form quadruplex structures that have been proved to be involved in several important biological processes.
Targeting of DNA secondary structures, such as G-quadruplexes, is now considered an appealing opportunity for drug intervention in anticancer therapy. So far, efforts made in the discovery of chemotypes able to target G-quadruplexes mainly succeeded in the identification of a number of polyaromatic compounds featuring end-stacking binding properties. Against this general trend, we were persuaded that the G-quadruplex grooves can recognize molecular entities with better drug-like and selectivity properties. From this idea, a set of small molecules was identified and the structural features responsible for G-quadruplex recognition were delineated. These compounds were demonstrated to have enhanced affinity and selectivity for the G-quadruplex over the duplex structure. Their ability to induce selective DNA damage at telomeric level and to induction of apoptosis and senescence on tumor cells is herein experimentally proven.
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, like circular dichroism, UV and fluorescence, have been widely employed to study G-quadruplexes in dilute solution. Here we focus on some aspects that need to be taken into account when employing such techniques in the presence of large amount of a cosolute. Additionally, we discuss possible problems/artifacts that arise in setting experiments in presence of these commonly employed cosolutes and in interpreting the results.
G-quadruplex structures are an attractive target for the development of anticancer drugs, as their formation in human telomere induces a DNA damage response followed by apoptosis in cancer cells. However, the development of new anticancer drugs by means of structural-based drug design is hampered by a lack of accurate information on the exact G-quadruplex conformation adopted by the human telomeric DNA under physiological conditions. Several groups reported that, in a molecular crowded, cell-like environment, simulated by polyethylene glycol (PEG), the human telomeric DNA adopts the parallel G-quadruplex conformation. These studies have suggested that 40% (w/v) PEG concentration induces complete structural conversion from the other known human telomeric G-quadruplex conformations to the parallel G-quadruplex, thus simplifying the high structural polymorphism existing in the absence of PEG. In this study, we demonstrate that the structural conversion to the parallel G-quadruplex is not a complete reaction at physiological temperature. We report a complete kinetic and thermodynamic characterization of the conformational transitions involving the (TTAGGG)(4)TT and (TTAGGG)(8)TT human telomeric DNA sequences in K(+) solution containing PEG. Our data show that the hybrid-type and parallel conformations coexist at equilibrium in the presence of PEG at physiological temperature and the degree of the quadruplex interconversion depends on the PEG molecular weight. Further, we find that telomeric DNA folds in the parallel quadruplex in the seconds time scale, a much larger time scale than the one reported for the hybrid quadruplex folding (~ms). The whole of our data allow us to predict the relative amount of each G-quadruplex conformation as a function of temperature and time. The effect of other crowding agents like Ficoll 400 and glycerol on the quadruplex interconversion has been also explored.