Untitled

Funder

Organizational Unit

Publications

Naphthalene amine support for G-quadruplex isolation

Publication . Ferreira, João; Santos, Tiago; Pereira, Patrícia; Corvo, Marta C.; Queiroz, João; Sousa, Fani; Cruz, Carla

G-quadruplex (G4) is involved in many biological processes, such as telomere function, gene expression and DNA replication. The selective isolation of G4 using affinity ligands that bind tightly and selectively is a valuable strategy for discovering new G4 binders for the separation of G4 from duplexes or the discrimination of G4 structures. In this work, one affinity chromatographic support was prepared using a naphthalene amine as a G4 binder. The ligand was immobilized on epoxy-activated Sepharose CL-6B using a long spacer arm and was characterized by HR-MAS spectroscopy. The supercoiled (sc) isoform of pVAX1-LacZ and pVAX1-G4 was isolated from a native sample. Also, the recovery and isolation of the plasmid isoforms from Escherichia coli lysate samples were achieved using an ionic gradient with different concentrations of NaCl in 10 mM Tris-HCl (pH 7.4). The retention times of different DNA/single strand sequences that can form G4, such as, c-MYC, c-kit1, c-kit2, tetrameric, telomeric (23AG), thrombin aptamer (TBA) and 58Sγ3 in this support were evaluated. Our experimental results suggest that the support exhibits selectivity for parallel c-MYC and c-kit1 G4s. In vitro transcription was performed using purified sc pVAX1-G4 and pPH600 to induce G4 formation and circular dichroism (CD) analysis confirmed that both transcripts adopt a parallel G4 topology.

2017-08-07Journal article

Restricted access

Plasmid purification by using a new naphthalene tripodal support

Publication . Santos, Tiago; Proença, Z.; Queiroz, João; Tomaz, C. T.; Cruz, Carla

The aim of this work was to employ a new naphthalene tripodal support for the isolation of supercoiled (sc) isoform of plasmid (pDNA) from a native sample. This support is for the first time synthesized and used in pDNA purification. The naphthalene tripodal ligand was synthesized and characterized to assess its purity and subsequently immobilized onto an epoxy-activated Sepharose CL-6B, using mild conditions and resulting in a ligand density of 0.32 mmol naphthalene tripodal/g derivatized Sepharose CL-6B. The complete characterization of naphthalene tripodal Sepharose CL-6B support was performed by High Resolution Magic Angle Spinning (HR-MAS) NMR spectroscopy, scanning electron microscopy (SEM) and elemental analysis. The affinity was measured by SPR biosensor between naphthalene tripodal ligand immobilized on the surface and sc pVAX1-LacZ and the KD was 8.65 10 8 ± 1.0 10 8 M in 10 mM Tris-HCl pH 8.0, at T = 25 C, indicating a high affinity. For comparison reasons, the affinity ligand 3,8-diamino-6-phenylphe nanthridine (DAPP) was also immobilized on the chip surface and the KD for sc pVAX1-LacZ is lower than with naphthalene tripodal. Saturation transfer difference-nuclear magnetic resonance spectroscopy (STD-NMR) experiments showed that the interactions between the naphthalene tripodal–Sepharose CL-6B and DAPP-Sepharose supports and the 50-mononucleotides are mainly hydrophobic and p-p stacking. The isolation of sc pDNA isoform was achieved with low salt concentrations, using 95 mM NaCl in binding step and 550 mM NaCl in elution step at T = 4 C and pH 8, thus reducing the economic and environmental impact.

2017-06-30Journal article

Open access

Current progress on microRNAs-based therapeutics in neurodegenerative diseases

Publication . Pereira, Patrícia; Queiroz, João; Figueiras, Ana; Sousa, Fani

MicroRNAs (miRNAs)-based therapy has recently emerged as a promising strategy in the treatments of neurodegenerative diseases. Thus, in this review, the most recent and important challenges and advances on the development of miRNA therapeutics for brain targeting are discussed. In particular, this review highlights current knowledge and progress in the field of manufacturing, recovery, isolation, purification, and analysis of these therapeutic oligonucleotides. Finally, the available miRNA delivery systems are reviewed and an analysis is presented in what concerns to the current challenges that have to be addressed to ensure their specificity and efficacy. Overall, it is intended to provide a perspective on the future of miRNA-based therapeutics, focusing the biotechnological approach to obtain miRNAs. WIREs RNA 2017, 8:e1409. doi: 10.1002/wrna.1409 For further resources related to this article, please visit the WIREs website.

2017Journal article

Restricted access