Targeting double-stranded DNA with locked nucleic acid-based (LNA) oligonucleotides (ON) and DNA-binding peptide conjugates

Abstract: In this thesis we have studied two different types of molecules that bind to the major groove of the DNA: (i) oligonucleotides (ONs) containing locked nucleic acid (LNA) and in combination with intercalating agents (Twisted Intercalating Nucleic Acid, TINA, and Benzoquinoquinoxaline, BQQ) and (ii) peptides mimicking the leucine zipper transcription factor (GCN4 bZIP TF). Both ONs and peptides bind to the DNA double helix aiming to serve as transcription regulators of disease related genes. In paper I, we developed a clamp type LNA ON (bisLNA) having the capacity to invade and bind double-stranded supercoiled DNA, as demonstrated by chemical probing. BisLNA has a dual mode of binding to duplex DNA, by Hoogsteen interactions forming a triplex structure and by double strand invasion (DSI) forming new Watson-Crick hydrogen bonds. Optimization of the bisLNA construct was carried out in particular regarding the number and location of LNA nucleotides and the length of the triplex-forming and the strand-invading arms, and we achieved 30% DSI using plasmid DNA. In paper II, by combining EMSA and molecular dynamics (MD) simulations we evaluated the structural features of modified ONs in stabilizing both duplex and triplex structures; we found that reduction in the LNA content at the 3’-end of a triplex forming oligonucleotide (TFO) destabilized the triplex. Moreover, we demonstrated that positioning TINA at the 3’-end of a TFO had an advantageous effect on triplex stability, and BQQ was able to stabilize the pyrimidine motif triplex containing LNA ON and TINA. MD simulation showed that LNA-substitution in the pyrimidine strand of a duplex alters the double helix structure, changing x-displacement, slide and twist allowing triplex formation through enhanced TFO major groove accommodation. Finally, we also elucidated the mechanism of bisLNA binding to a dsDNA, which basically involves a two-step process where the triplex is formed first followed by double strand invasion. Finally, in paper III, four models of the GCN4 bZIP TF were evaluated, but only one achieved high sequence specific dsDNA binding as was demonstrated by electrophoretic mobility shift assay (EMSA) and the obtained dissociation constant. The same peptide showed a better uptake when evaluated in macrophages, demonstrating the potential of peptide-steroid based TF mimics. In summary, the results provide a basis for further development of DNA binding chemical compounds, which could potentially have future applications in medicine and biotechnology in the future.