Andre Filipe Rodrigues Miranda
@ubi.pt
PhD Student in Biochemistry
Centro de Investigação em Ciências da Saúde (CICS)
RESEARCH, TEACHING, or OTHER INTERESTS
Biophysics, Biochemistry, Structural Biology, Biotechnology
Scopus Publications
Scholar Citations
Scholar h-index
Scholar i10-index
Scopus Publications
Pedro Lourenço, André Miranda, Maria Paula Cabral Campello, António Paulo, Jean Louis-Mergny, and Carla Cruz
Elsevier BV
Elisa Palma, Cigdem Içhedef, Célia Fernandes, Ana Belchior, Paula Raposinho, Lurdes Gano, André Miranda, David Moreira, Pedro Lourenço, Carla Cruz,et al.
Wiley
AbstractThe main goal of this work was to elucidate the potential relevance of (radio)metal chelates of 99mTc and Re targeting G‐quadruplex structures for the design of new tools for cancer theranostics. 99mTc provides the complexes with the ability to perform single‐photon‐emission computed tomography imaging studies, while the Re complexes should act as anticancer agents upon interaction with specific G4 DNA or RNA structures present in tumor tissues. Towards this goal, we have developed isostructural 99mTc(I) and Re(I) tricarbonyl complexes anchored by a pyrazolyl‐diamine (Pz) chelator carrying a pendant pyridostatin (PDS) fragment as the G4‐binding motif. The interaction of the PDF‐Pz‐Re (8) complex with different G4‐forming oligonucleotides was studied by circular dichroism, fluorescence spectroscopy and FRET‐melting assays. The results showed that the Re complex retained the ability to bind and stabilize G4‐structures from different DNA or RNA sequences, namely those present on the SRC proto‐oncogene and telomeric RNA (TERRA sequence). PDF‐Pz‐Re (8) showed low to moderate cytotoxicity in PC3 and MCF‐7 cancer cell lines, as typically observed for G4‐binders. Biodistribution studies of the congener PDF‐Pz‐99mTc (12) in normal mice showed that the complex undergoes a fast blood clearance with a predominant hepatobiliary excretion, pointing also for a high in vitro stability.
André Miranda, Roi Lopez-Blanco, Jéssica Lopes-Nunes, Ana M. Melo, Maria Paula Cabral Campello, António Paulo, Maria Cristina Oliveira, Jean-Louis Mergny, Paula A. Oliveira, Eduardo Fernandez-Megia,et al.
MDPI AG
Herein, we describe the synthesis of an aptadendrimer by covalent bioconjugation of a gallic acid–triethylene glycol (GATG) dendrimer with the G-quadruplex (G4) AT11 aptamer (a modified version of AS1411) at the surface. We evaluated the loading and interaction of an acridine orange ligand, termed C8, that acts as an anticancer drug and binder/stabilizer of the G4 structure of AT11. Dynamic light scattering experiments demonstrated that the aptadendrimer was approximately 3.1 nm in diameter. Both steady-state and time-resolved fluorescence anisotropy evidenced the interaction between the aptadendrimer and C8. Additionally, we demonstrated that the iodine atom of the C8 ligand acts as an effective intramolecular quencher in solution, while upon complexation with the aptadendrimer, it adopts a more extended conformation. Docking studies support this conclusion. Release experiments show a delivery of C8 after 4 h. The aptadendrimers tend to localize in the cytoplasm of various cell lines studied as demonstrated by confocal microscopy. The internalization of the aptadendrimers is not nucleolin-mediated or by passive diffusion, but via endocytosis. MTT studies with prostate cancer cells and non-malignant cells evidenced high cytotoxicity mainly due to the C8 ligand. The rapid internalization of the aptadendrimers and the fluorescence properties make them attractive for the development of potential nanocarriers.
David Moreira, Daniela Leitão, Jéssica Lopes-Nunes, Tiago Santos, Joana Figueiredo, André Miranda, Daniela Alexandre, Cândida Tomaz, Jean-Louis Mergny, and Carla Cruz
MDPI AG
In this work we explore the structure of a G-rich DNA aptamer termed AT11-L2 (TGGTGGTGGTTGTTGTTGGTGGTGGTGGT; derivative of AT11) by evaluating the formation and stability of G-quadruplex (G4) conformation under different experimental conditions such as KCl concentration, temperature, and upon binding with a variety of G4 ligands (360A, BRACO-19, PDS, PhenDC3, TMPyP4). We also determined whether nucleolin (NCL) can be a target of AT11-L2 G4. Firstly, we assessed by circular dichroism, UV and NMR spectroscopies the formation of G4 by AT11-L2. We observed that, for KCl concentrations of 65 mM or less, AT11-L2 adopts hybrid or multiple topologies. In contrast, a parallel topology predominates for buffer containing 100 mM of KCl. The Tm of AT11-L2 in 100 mM of KCl is 38.9 °C, proving the weak stability of this sequence. We also found that upon titration with two molar equivalents of 360A, BRACO-19 and PhenDC3, the G4 is strongly stabilized and its topology is maintained, while the addition of 3.5 molar equivalents of TMPyP4 promotes the disruption of G4. The KD values between AT11-L2 G4, ligands and NCL were obtained by fluorescence titrations and are in the range of µM for ligand complexes and nM when adding NCL. In silico studies suggest that four ligands bind to the AT11-L2 G4 structure by stacking interactions, while the RBD1,2 domains of NCL interact preferentially with the thymines of AT11-L2 G4. Finally, AT11-L2 G4 co-localized with NCL in NCL-positive tongue squamous cell carcinoma cell line.
Tiago Santos, Jéssica Lopes-Nunes, Daniela Alexandre, André Miranda, Joana Figueiredo, Micael S. Silva, Jean-Louis Mergny, and Carla Cruz
Elsevier BV
Tiago Santos, André Miranda, Lionel Imbert, David Monchaud, Gilmar F. Salgado, Eurico J. Cabrita, and Carla Cruz
Elsevier BV
Tiago Santos, André Miranda, Lionel Imbert, Andreia Jardim, Catarina R.F. Caneira, Virgínia Chu, João P. Conde, Maria Paula Cabral Campello, António Paulo, Gilmar Salgado,et al.
Elsevier BV
Carla Cruz, André Miranda, and Tiago Santos
Elsevier
Josué Carvalho, Jéssica Lopes-Nunes, Joana Figueiredo, Tiago Santos, André Miranda, Micaela Riscado, Fani Sousa, Ana Paula Duarte, Sílvia Socorro, Cândida Teixeira Tomaz,et al.
MDPI AG
The 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.
Joana Figueiredo, Tiago Santos, André Miranda, Daniela Alexandre, Bernardo Teixeira, Pedro Simões, Jéssica Lopes-Nunes, and Carla Cruz
MDPI AG
The non-coding RNAs (ncRNA) are RNA transcripts with different sizes, structures and biological functions that do not encode functional proteins. RNA G-quadruplexes (rG4s) have been found in small and long ncRNAs. The existence of an equilibrium between rG4 and stem−loop structures in ncRNAs and its effect on biological processes remains unexplored. For example, deviation from the stem−loop leads to deregulated mature miRNA levels, demonstrating that miRNA biogenesis can be modulated by ions or small molecules. In light of this, we report several examples of rG4s in certain types of ncRNAs, and the implications of G4 stabilization using small molecules, also known as G4 ligands, in the regulation of gene expression, miRNA biogenesis, and miRNA−mRNA interactions. Until now, different G4 ligands scaffolds were synthesized for these targets. The regulatory role of the above-mentioned rG4s in ncRNAs can be used as novel therapeutic approaches for adjusting miRNA levels.
Joana Figueiredo, André Miranda, Jéssica Lopes-Nunes, Josué Carvalho, Daniela Alexandre, Salete Valente, Jean-Louis Mergny, and Carla Cruz
Elsevier BV
Tiago Santos, André Miranda, Maria P.C. Campello, António Paulo, Gilmar Salgado, Eurico J. Cabrita, and Carla Cruz
Elsevier BV
André Miranda, Tiago Santos, Josué Carvalho, Daniela Alexandre, Andreia Jardim, CatarinaR.F. Caneira, Vírgilio Vaz, Bruno Pereira, Ricardo Godinho, Duarte Brito,et al.
Elsevier BV
André Miranda, Tiago Santos, Eric Largy, and Carla Cruz
MDPI AG
We have designed AS1411-N6, a derivative of the nucleolin (NCL)-binding aptamer AS1411, by adding six nucleotides to the 5′-end that are complementary to nucleotides at the 3′-end forcing it into a stem-loop structure. We evaluated by several biophysical techniques if AS1411-N6 can adopt one or more conformations, one of which allows NCL binding. We found a decrease of polymorphism of G-quadruplex (G4)-forming sequences comparing to AS1411 and the G4 formation in presence of K+ promotes the duplex folding. We also studied the binding properties of ligands TMPyP4, PhenDC3, PDS, 360A, and BRACO-19 in terms of stability, binding, topology maintenance of AS1411-N6, and NCL recognition. The melting experiments revealed promising stabilizer effects of PhenDC3, 360A, and TMPyP4, and the affinity calculations showed that 360A is the most prominent affinity ligand for AS1411-N6 and AS1411. The affinity determined between AS1411-N6 and NCL denoting a strong interaction and complex formation was assessed by PAGE in which the electrophoretic profile of AS1411-N6 showed bands of the dimeric form in the presence of the ligands and NCL.