This study explores the potential of DNA triplexes for gene regulation and antigene therapy, focusing on enhancing their stability for in vivo applications. While DNA triplexes play important biological roles, a stable triplex structure requires an uninterrupted polypurine strand. In biological contexts, their stability is reduced due to sequence composition, where pyrimidine interruptions are often found in the potential binding area, limiting their therapeutic potential. The goal of this work is to develop synthetic backbones and base pair DNA modifications to improve triplex formation, using native mass spectrometry to assess stability and binding affinity. The study also investigates cell-penetrating peptides (CPPs) and nanoparticle-based delivery methods for the efficient bacterial delivery of oligonucleotides to combat antibiotic-resistant bacteria.
Using electrospray ionization mass spectrometry (ESI-MS), UV-vis spectroscopy, and isothermal titration calorimetry (ITC), we examined triplex formation in model and bacterial triplex target sequences with pyrimidine base interruptions. Oligonucleotides were modified through oxidative amination and thiol alkylation using 4-thioldioxyuracil to incorporate alkyne and azide handles for click chemistry. Initial tests focused on simple base pair modifications with minimal steric hindrance to optimize triplex formation, with plans to implement more complex modifications to enhance hydrogen bonding and stability.
Preliminary results show that locked nucleic acid (LNA)-modified triplex-forming oligonucleotides (TFOs) have a higher propensity for triplex formation than DNA-only and dSpacer-modified TFOs. When analysing biologically relevant sequences, LNA-modified TFOs were the only ones to form triplexes with Pseudomonas aeruginosa gene targets, demonstrating their therapeutic potential. CPP-conjugated oligonucleotides and nanoparticle-based systems will be explored to compare their effectiveness in delivery of triplex-forming oligonucleotides.
Overall, this work aims to improve the stability and delivery of triplex-forming oligonucleotides for targeted gene therapies in bacterial cells, with the potential to lead to new antimicrobial treatments targeting specific bacterial genes.