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- G-Quadruplex Aptamer Beacon for Detection of Prostate Cancer BiomarkerPublication . Miranda, André Filipe Rodrigues; Cruz, Carla Patrícia Alves Freire Madeira; Conde, João Pedro Estrela Rodrigues; Chu, VirgíniaThe prostate is the major male reproductive gland involved in male fertility and plays an important role in triggering of molecular pathways relevant to fertility success. Unfortunately, in Portugal prostate cancer is the most common cancer type among men, being asymptomatic in earlier stages. Thus, is important early detection of disease. NCL is a multifunctional protein involved in multiple biological processes under both physiological and pathological processes and can have several cellular localizations. Cell surface protein overexpression was found restricted to cancer cells, namely in prostate cancer cells. Thus, we can consider NCL as a potential biomarker for cancer diagnosis and a target for cancer treatment. The AS1411 is an aptamer capable to recognise and binds specifically NCL and have a therapeutic effect on cancer cells through of induction of antiproliferative activity. Beyond its therapeutic use, AS1411 can be used in imaging and diagnostic, particularly on aptasensors development. One of the most relevant characteristics of this aptamer is the ability to fold in a G4 conformation, a secondary structure of nucleic acids. G4 structure confers stabilization to sequence and availability to bind NCL. Thus, in this work is presented the first approach of use AS1411 aptamer to prostate cancer diagnosis, namely through the design of molecular beacon (MB) designated by AS1411N5. Initially, biophysical characterization of AS1411-N5 was done by circular dichroism, nuclear magnetic resonance or fluorometric spectroscopies. Additionally, it was performed microfluidic experiments, to detect NCL using AS1411-N5 in biological samples. The results demonstrated that the proposed AS1411-N5 adopt a G4 structure and it is capable to bind with specificity and selectivity NCL, even in plasma of human patients with prostate cancer.
- Molecular Beacon Assay Development for Severe Acute Respiratory Syndrome Coronavirus 2 DetectionPublication . Carvalho, Josué; Nunes, J. Lopes; Figueiredo, Joana; Santos, Tiago; Miranda, André; Riscado, Micaela; Sousa, Fani; Duarte, A. P.; Socorro, Sílvia; Tomaz, Cândida; Felgueiras, Mafalda; Teixeira, Rui; Faria, Conceição; Cruz, CarlaThe fast spread of SARS-CoV-2 has led to a global pandemic, calling for fast and accurate assays to allow infection diagnosis and prevention of transmission. We aimed to develop a molecular beacon (MB)-based detection assay for SARS-CoV-2, designed to detect the ORF1ab and S genes, proposing a two-stage COVID-19 testing strategy. The novelty of this work lies in the design and optimization of two MBs for detection of SARS-CoV-2, namely, concentration, fluorescence plateaus of hybridization, reaction temperature and real-time results. We also identify putative G-quadruplex (G4) regions in the genome of SARS-CoV-2. A total of 458 nasopharyngeal and throat swab samples (426 positive and 32 negative) were tested with the MB assay and the fluorescence levels compared with the cycle threshold (Ct) values obtained from a commercial RT-PCR test in terms of test duration, sensitivity, and specificity. Our results show that the samples with higher fluorescence levels correspond to those with low Ct values, suggesting a correlation between viral load and increased MB fluorescence. The proposed assay represents a fast (total duration of 2 h 20 min including amplification and fluorescence reading stages) and simple way of detecting SARS-CoV-2 in clinical samples from the upper respiratory tract.
- Non-canonical DNA secondary structures as a therapeutic strategy for lung cancerPublication . Miranda, André Filipe Rodrigues ; Cruz, Carla Patrícia Alves Freire Madeira; Mergny, Jean-Louis; Oliveira, Paula Alexandra Martins deNucleic acids store and control genetic information and, beyond the canonical B-form DNA double helix, can adopt a variety of non-canonical architectures, including G-quadruplex (G4s), i-motifs (iMs), triplexes, R-loops, and Z-DNA/Z-RNA. These structures are widespread across genomes and transcriptomes and play important biological roles (transcription, replication, translation, and genome stability). They are recognized as regulatory hotspots enriched at promoters, enhancers, telomeres, untranslated regions, and viral genomes and their involvement in cancer, viral infection, neurodegeneration, and inflammatory disorders. Their in vivo functions have also opened therapeutic avenues both as drug targets and therapeutic tools. As drug targets, these non-canonical structures can be modulated to alter cellular behavior, while as therapeutic agents, nucleic acids can be engineered to adopt non-canonical folds that bind key cellular partners and exert biological effects. This dual role is unusual for other approaches and positions non-canonical structures at the forefront of modern drug discovery. These structures are emerging as important tools in oncological research and therapeutic development. Proto-oncogenes encode proteins essential to normal physiology; however, when mutated or amplified become oncogenes, they drive unchecked growth and survival. The transcription factors (TFs) are a particularly important class of proto-oncogenes that bind to DNA and recruit co-regulators, orchestrating broad gene-expression programs. Their sweeping control of transcription makes them powerful cancer drivers but also challenging drug targets. Unlike enzymes with well-defined catalytic pockets, TFs often rely on flexible, intrinsically disordered regions for interactions, contributing to their reputation as “undruggable.” Numerous TFs have been reported as dysregulated in cancer and are frequently correlated with poor prognosis and resistance to chemotherapy. Other TF are gaining relevance, such as B-MYB (MYBL2), a transcription factor from the MYB family required for normal cell-cycle progression, which acts as an oncogene when overexpressed or deregulated. Functionally, the B-MYB protein serves as a master cell-cycle regulator by integrating into multiprotein assemblies, most notably the DREAM and MMB complexes, that coordinate cell-cycle gene expression. Beyond these physiological functions, B-MYB is upregulated in multiple cancers, such as breast and lung, and its overexpression is correlated with aggressive disease, treatment resistance, and unfavorable prognosis. The molecular mechanisms linking B-MYB to tumorigenesis include gene amplification, cell cycle deregulation, genomic instability, apoptosis suppression, post-transcriptional and post-translational modifications, or can contribute to epithelial-to-mesenchymal transition. Thus, B-MYB TF can be seen as a relevant clinical biomarker and one of the main orchestrators of carcinogenesis; however, until now, no pharmacological therapy against B-MYB has been developed, also related to its “undruggable” profile. Also, it’s known, by bioinformatic analysis of the human genome, that oncogene promoter regions are G/C rich around transcription start sites (TSS), which allows the formation of structures called G4 or iM. The G4s are formed by the self-association of four guanine bases in a quasi-planar arrangement via Hoogsteen bonds and are described as having a gene regulatory function, namely at the transcriptional level, while the iM is formed by Hoogsteen bonds between protonated and deprotonated cytosines. Motivated by these observations, the central objective of this thesis was to explore alternative routes to target the B-MYB oncogene using non-canonical nucleic-acid structures: first as drug targets and later as therapeutics. The thesis started by the identification of G-rich sequences at the B-MYB promoter capable of forming G4 structures. The identification of G-rich sequences was performed using the G4Hunter algorithm, and their conservation across mammalian species was also verified. The experimental validation of their formation was performed by combining 10 biophysical and biochemical methods. Later, the in-cell relevance of G4 structures was evaluated employing the G4access method, which reveals that from the predicted sequences, only the most stable one (B-MYB 43R) was shown to be significantly formed in cells, evidencing a potential impact on the transcription of this gene to its location closest to the TSS. Next, the ability of C-rich sequences to form iM structure was evaluated. Again, the iM-forming sequences were predicted using the G4Hunter algorithm and the experimental validation started by circular dichroism (CD) and nuclear magnetic resonance (NMR) to determine if the sequences fold into an iM structure. Then, stability parameters such as pHT (pH transitional midpoint) were determined by acquiring spectra at different pH values (between pH=5 and pH=8; 0.25-unit increments). The thermal stability and thermodynamic parameters were also calculated using the denaturation and renaturation melting curves. Then, the in-cell formation was assessed using iM-CUT&Tag experiments in HEK293T, which revealed the formation of the iB-MYB 43 that was characterized to have the highest Tm and the highest pHT among the studied sequences. After this characterization, the interaction of small molecules with iB-MYB structures was assessed. As a general tendency, the ligands did not affect the CD spectral shape; however, some of them evidenced changes in secondary structure or thermal destabilization. After evaluating the capacity to form G4 or iM, we continued to validate the G4 formation within a cellular environment using a G4-triggered fluorogenic hybridization probe. This strategy circumvents the limitations of the antibodies and small-molecule probes, which show a lack of structural selectivity labeling diverse DNA and RNA G4s indiscriminately, and cannot specifically target the desired G4. The molecular probe was composed of G4-recognizing light-up ligand (acridine derivative) with an antisense oligonucleotide that hybridizes adjacent region of B-MYB G4. Before probe synthesis using a click chemistry approach, the ligands were photophysically characterized, biophysically evaluated against G4, and further validated spectroscopically and in cells. Cellular studies confirmed the co-localization between the molecular probe and B-MYB G4 in-cell, offering promise for future applications in cancer research in terms of targeted therapies and monitoring of G4. Although G4s are highly dynamic and topologically diverse, promoter G4s are commonly parallel, whereas antiparallel forms are underreported and less characterized. In earlier work, we identified a B-MYB promoter sequence (B-MYB 26RA), that forms an antiparallel G4. Thus, a biophysical pipeline, starting in solution NMR spectroscopy alongside in-cell NMR studies, was made to predict a three-dimensional model (antiparallel quadruplex-duplex junction) and to assess the formation in a live complex environment of B-MYB 26RA. Interestingly, B-MYB 26RA evidenced an ionic sensitivity to K+ and Na+, increasing their topological dynamics, and demonstrated that, when associated with PhenDC3 ligand, a conformational shift from hybrid to antiparallel topology happens. Then, we moved to B-MYB 43R G4 to discuss the therapeutic relevance and the capacity to be targeted by small molecules. Thus, using spectroscopic methods, the G4 formation and its interaction with a panel of G4-stabilizing ligands were confirmed. From the tested ligands, PhenDC3 and TMPyP4 demonstrated the highest binding affinity and stabilization and revealed that both ligands inhibited proliferation and migration in two lung cancer cell lines (A549 and H1299), with PhenDC3 showing potent cytotoxic and cytostatic effects. Furthermore, PhenDC3 upregulated B-MYB expression despite its strong phenotypic effects, highlighting the complexity of G4-targeting mechanisms and supporting the growing evidence that ligands do not always downregulate their target genes. Finally, we explored the use of Polypurine Reverse-Hoogsteen (PPRH) hairpins as an alternative therapeutic approach to target the B-MYB promoter. PPRH are DNA oligonucleotides that fold into intramolecular hairpins and bind complementary polypyrimidine, forming stable DNA triplexes, impeding transcription, and displacing the G-rich strand, inducing G4 formation, enabling dual-level regulation of oncogene expression. Thus, using a bioinformatic tool (TFO Searching Tool), a PPRH was designed against the G4-forming region at the B-MYB promoter (B-MYB 43R), and then experimentally assessed the triplex formation using biophysical methods. The biological effects of designed PPRH, evaluated in lung cellular models (A549, H1299 and MRC-5), demonstrated a low cell viability and clonogenic capacity accompanied by mRNA expression reduction and proteomic profile alterations. Thus, the combination of triplex-based approaches with G4 biology could be a future venue for therapy. Overall, this thesis establishes non-canonical nucleic-acid structures as both actionable targets and therapeutic agents. As a target, the formation of G4 and iM structures at the promoter region of the B-MYB oncogene; was provided new insights about structure and topological dynamics according to the surrounding environment; was validated their formation in-cell, as well as explored the interaction with small molecules. On the other side, the therapeutic potential was explored using PPRH, which revealed a selective and powerful tool to target B-MYB. Together, these findings provide fundamental insights and open new avenues for translational research on the B-MYB oncogene.