Fabrizia Nici

Exploitation of a Very Small Peptide Nucleic Acid as a New Inhibitor of miR-509-3p Involved in the Regulation of Cystic Fibrosis Disease-Gene Expression

Computational techniques, and in particular molecular dynamics (MD) simulations, have been successfully used as a complementary technique to predict and analyse the structural behaviour of nucleic acids, including peptide nucleic acid- (PNA-) RNA hybrids. This study shows that a 7-base long PNA complementary to the seed region of miR-509-3p, one of the miRNAs involved in the posttranscriptional regulation of the CFTR disease-gene of Cystic Fibrosis, and bearing suitable functionalization at its N- and C-ends aimed at improving its resistance to nucleases and cellular uptake, is able to revert the expression of the luciferase gene containing the 3′UTR of the gene in A549 human lung cancer cells, in agreement with the MD results that pointed at the formation of a stable RNA/PNA heteroduplex notwithstanding the short sequence of the latter. The here reported results widen the interest towards the use of small PNAs as effective anti-miRNA agents.

Screening Platform toward New Anti-HIV Aptamers Set on Molecular Docking and Fluorescence Quenching Techniques

By using a new rapid screening platform set on molecular docking simulations and fluorescence quenching techniques, three new anti-HIV aptamers targeting the viral surface glycoprotein 120 (gp120) were selected, synthesized, and assayed. The use of the short synthetic fluorescent peptide V35-Fluo mimicking the V3 loop of gp120, as the molecular target for fluorescence-quenching binding affinity studies, allowed one to measure the binding affinities of the new aptamers for the HIV-1 gp120 without the need to obtain and purify the full recombinant gp120 protein. The almost perfect correspondence between the calculated Kd and the experimental EC50 on HIV-infected cells confirmed the reliability of the platform as an alternative to the existing methods for aptamer selection and measuring of aptamer-protein equilibria.

Solid phase synthesis of a thrombin binding aptamer on macroporous silica for label free optical quantification of thrombin

Human α-thrombin (TB) is a serine protease with a crucial role in coagulation and hemostasis. The monitoring of the TB level in blood serum could be of great importance in order to prevent serious damage to human health. In this work, an aptasensor is realized by in situ synthesis of a 17-mer Thrombin Binding Aptamer analogue (TBATT) on silanized macroporous silica (PSi). The interaction between TBATT and TB at different concentrations is monitored by a label-free optical method, spectroscopic reflectometry, and quantified by fast Fourier transform (FFT) analysis. A TBATT-TB affinity constant of 14 ± 8 nM and limit of detection of 1.5 ± 0.3 nM are demonstrated. The selectivity and reversibility of the aptasensor are also proved.

Synthesis and Pharmacological Evaluation of Modified Adenosines Joined to Mono-Functional Platinum Moieties

The synthesis of four novel platinum complexes, bearing N6-(6-amino-hexyl)adenosine or a 1,6-di(adenosin-N6-yl)-hexane respectively, as ligands of mono-functional cisplatin or monochloro(ethylendiamine)platinum(II), is reported. The chemistry exploits the high affinity of the charged platinum centres towards the N7 position of the adenosine base system and a primary amine of an alkyl chain installed on the C6 position of the purine. The cytotoxic behaviour of the synthesized complexes has been studied in A549 adenocarcinomic human alveolar basal epithelial and MCF7 human breast adenocarcinomic cancer cell lines, in order to investigate their effects on cell viability and proliferation.

Self‐Assembly of G‐Rich Oligonucleotides Incorporating a 3′–3′ Inversion of Polarity Site: A New Route Towards G‐Wire DNA Nanostructures

Abstract Obtaining DNA nanostructures with potential applications in drug discovery, diagnostics, and electronics in a simple and affordable way represents one of the hottest topics in nanotechnological and medical sciences. Herein, we report a novel strategy to obtain structurally homogeneous DNA G‐wire nanostructures of known length, starting from the short unmodified G‐rich oligonucleotide d(5′‐CGGT‐3′–3′‐GGC‐5′) (1) incorporating a 3’–3′ inversion of polarity site. The reported approach allowed us to obtain long G‐wire assemblies through 5′–5′ π–π stacking interactions in between the tetramolecular G‐quadruplex building blocks that form when 1 is annealed in the presence of potassium ions. Our results expand the repertoire of synthetic methodologies to obtain new tailored DNA G‐wire nanostructures.

Nanogravimetric and Optical Characterizations of Thrombin Interaction with a Self-Assembled Thiolated Aptamer

Efficient biorecognition of thrombin (TB), a serine protease with crucial role in physiological and pathological blood coagulation, is a hot topic in medical diagnostics. In this work, we investigate the ability of synthetic thrombin aptamer (TBA), immobilized on a gold substrate, to bind thrombin by two different label-free techniques: the quartz crystal microbalance (QCM) and the spectroscopic ellipsometry (SE). By QCM characterization in the range from 20 to 110 nM, we demonstrate high specificity of TBA-TB interaction and determine affinity constant () of nM, system sensitivity of Hz nM−1, and limit of detection (LOD) of pM. The interaction between TBA and TB is also investigated by SE, an all-optical method, by quantifying the thickness increase of the TBA film assembled on gold substrate. AFM characterization of TBA and TB molecules deposited on flat silicon surface is also supplied.

Synthesis of mixed-sequence oligonucleotides on mesoporous silicon: chemical strategies and material stability.

Rapid screening tests in medical diagnostic and environmental analysis are often based on oligonucleotide biochips. In this paper, we studied the stability of functionalized mesoporous silicon supports in the solid-phase synthesis of oligonucleotides, exploiting several chemical procedures. A 19-mer mixed sequence has been successfully synthesized on aminosilane-modified porous silicon photonic structures. The process and the materials have been characterized by optical reflectivity, atomic force microscopy and high-performance liquid chromatography.