Two perspectives to consider Nucleic Acids

Since their discovery, nucleic acids have been the object of intense and thorough explorations, leading to the understanding of their structure and functions. Their role as genetic information carriers is well known but there are evidences that they are also involved in a series of other less known processes. No longer have nucleic acids to be considered passive structures, but they are dynamic and active macromolecules, able to assume a number of three-dimensional conformations. Precisely, the conformation of nucleic acids, encoded in their primary sequence, is the component that determines the specific and selective interaction with a biological target. This work is essentially divided in two parts reflecting the dual nature of nucleic acids and aimed at developing two different projects, one where nucleic acids represent a valid target for new potential therapeutic agents and the other where nucleic acids, playing an active role in the protein recognition, are developed as diagnostic agents. The first part of my PhD research activity is included in a wider project aimed to identify potential antiviral agents able to interfere with the NC chaperone activity. NC has multiple functions during the HIV-1 replication cycle and, in particular, the chaperone activity of NC is critical in reverse transcription. The recombinant full-length NC protein and its biological activity were deeply characterized: the analysis of the NC structure was completed by circular dichroic spectroscopy (CD). Then, the chaperone activity of NC i.e. its ability to destabilize the secondary structures of the viral nucleic acids TAR and cTAR was evaluated by a Fluorescence Assay and a FRET assay; NC aggregating activity was instead monitored through gel electrophoresis that allowed us to analyze the TAR/cTAR annealing reaction in the presence of the full-length NC protein. The effect of compounds on the chaperone activity of NC was investigated: to achieve this goal, a simple, fast and highly reproducible FRET-based assay for the High Throughput Screening (HTS) of inhibitors of the NC nucleic acids destabilization activity was optimized. A wide range of small molecules with different chemical structures from our in-house chemical library were analyzed, and in this work we focus on molecules with intercalating ability. 2,6-dipeptidyl anthraquinones, known DNA and RNA binders, were analysed as potential NC inhibitors: they could interfere with NC chaperone activity thanks to their attitude of stabilize the secondary structures of RNA and DNA sequences involved in the retrotranscription process. The effect of anthraquinones (series Z, GSF and G) on the chaperone activity of NC was investigated by HTS and the IC50 values on TAR and cTAR were determined for each compound. All tested anthraquinones are active both toward TAR and cTAR: the proposed mechanism of action for anthraquinones was therefore studied in more details by Melting Assays (FRET probes coupled to thermal melting experiments). We analyzed the ability of each compound to bind and stabilize the secondary structure of cTAR, TAR and of the TAR/cTAR hybrid. Melting Assay data supported HTS results, since the most active inhibitors of the chaperone NC activity resulted to be also strong TAR and cTAR stabilizers. Finally, we analyzed by native gel electrophoresis the effect of the most promising anthraquinones on the NC-mediated annealing of TAR with cTAR, to verify the effectiveness of the inhibition of the NC chaperone activity during the step subsequent to TAR and cTAR melting. All the tested anthraquinones resulted to be inhibitors of the TAR/cTAR hybrid formation, according with the NC inhibitory activity analyzed by HTS. Hence, viral nucleic acids involved in reverse transcription represent a new validated target for anti-HIV drugs design and 2,6-dipeptydil anthraquinones were identified as highly active anti-NC compounds which could be further analyzed to be developed as new potential antiviral agents. Moreover, a promising class of anti-HIV agents is represented by compounds which interfere with the Tat-mediated transcription process of the viral replicative cycle. Two classes of compounds were studied as inhibitors of the Tat-TAR complex formation. Quinolones are a promising class of anti-HIV compounds which interfere with the Tat mediated transcription process. To provide new insights into the SAR of antiviral quinolones, WP and HP series were analyzed by Fluorescence Quanching Assay (FQA), a FRET-based platform of screening that allowed the determination of the inhibition constant Ki for each compound. All the tested 6-DFQs of WP series are active as Tat-TAR complex formation inhibitors, with Ki values lower than the control WM5. WP series differs from the control for modifications introduced at the N1 position: these modifications do not result in loss of activity, but rather seem to improve the activity of WP quinolones as inhibitors of the Tat-TAR complex formation. The second class of compounds tested as inhibitors of the Tat-TAR complex formation was constituted by ellagic acid and some synthetic bi- and mono-lactones derivatives. ELs analysis was not possible to be performed by FQA due to a quenching effect observing after the addition of ELs to fluoresceinated Tat. The analysis of the ELs interference on the Tat-TAR complex was therefore performed by EMSA. Compound 1186 can act as Tat-TAR complex formation inhibitor and therefore supports the potential anti-HIV activity of ELs. The second part of my research work is focused on the development, optimization and characterization of a novel sensing system with therapeutic and diagnostic interest employing nucleic acids aptamers for the detection of proteins involved in diverse diseases. Biosensors based on the use of aptamers for specific recognition of an analyte are called "aptasensors". We developed the Sandwich Aptamer Microarray (SAM) with the aim to replace the traditional immunochemical systems used as diagnostics. SAM is a system ELISA-like, using a similar architecture, but totally based on aptamer technology with a "primary aptamer" as selection element and a "secondary aptamer" as a signal transducer. Two DNA aptamers with distinct protein recognition patterns are used in tandem for the simultaneous recognition of the target protein. We developed an aptamer-based microarray for human thrombin detection exploiting two non-overlapping DNA thrombin binding aptamers recognizing different exosites of the target protein. The 15-mer aptamer (TBA1) binds the fibrinogen-binding site, whereas the 29-mer aptamer (TBA2) binds the heparin binding domain. Appropriate chemical modifications were introduced in primary and secondary aptamers to adapt them to the format design and to the detection technology. Extensive analysis on the complex formation between human thrombin and modified aptamers was performed by EMSA, in order to verify in solution whether the chemical modifications introduced would affect aptamers/protein recognition. The validated system was then applied to the aptamer microarray, using the solid phase system devised by the solution studies. Our SAM system based on aptamer recognition of an analyte resulted to be efficient and specific, and different detection aptamers and methods can be utilized. The use of the indirect method (TBA2- biotin plus Cy3-streptavidin) allows lower LOD and LOQ when compared to TBA2-Cy5 (direct method) even in the presence of biological fluids. SAM exhibits a limit of detection comparable to other systems described in the literature employing the same aptamers and different technologies but has the advantage of a simple set up. In view of future applications of SAM in multiplex microarray systems, we enlarged the SAM system analysis on the VEGF165 detection using two anti-VEGF165 aptamers that recognize different sites of the protein. Following the same approach adopted for thrombin, a deep analysis and optimization of binary and ternary complexes formation between the modified anti-VEGF165 aptamers and VEGF165 in solution was performed prior to verify the sandwich aptamer formation in solid phase. The sandwich aptamer formation was verified also for VEGF165. This pioneering work suggests that SAM, developed for thrombin and VEGF165 detection, could represent a useful tool to monitor protein concentration in blood or plasma and represents a potential application of aptamers in diagnostic.