Dissertação para obtenção do Grau de Mestre em
Genética Molecular e BiomedicinaThe main objective of the work presented in this thesis was to develop a gene silencing system by taking advantage of the nanovectorization capability and optical properties of gold nanoparticles. The idea is based on the construction of a DNA structure containing a therapeutic oligonucleotide with the ability to form Hoogsteen hydrogen bonds with double-stranded DNA, producing a DNA triple helix, besides silencing the gene of interest. Hoogsteen bonds, more unstable than the conventional Watson-Crick bonds, permit the achievement of lower melting temperatures. This attribute, coupled with the ability to generate heat by laser irradiation of the gold nanoparticles used, will allow the release of the therapeutic oligonucleotide and subsequent gene silencing without significant increase in the medium’s temperature. Thus, the thesis comprises three major sections: structure design and formation, vectorization, and gene expression silencing; the tasks involved in each of these sections were conducted in parallel.
The design of the obtained structure took into account the desired melting temperature, stability at physiological conditions of the sequence-forming nucleotides, the number of Hoogsteen bonds and ionic conditions. To evaluate the formation of this structure, spectroscopic techniques were mainly used: FRET analysis and ultraviolet melting curves. Both approaches allowed the identification of interactions in the presence of therapeutic oligonucleotide compared with its absence, which may indicate structure formation. In addition, melting curves allowed the determination of the temperature of release of this oligonucleotide – 40ºC. The double-stranded DNA functionalization to gold nanoparticles has been achieved, but there was no difference in electrophoretic migration when the three oligonucleotides were present. However, the therapeutic oligonucleotide was able to efficiently inhibit gene expression in in vitro transcription and translation assays with efficiency up to 95% and 60% respectively
