This thesis is divided into seven projects exploring the chemical engineering of peptide nucleic acids (PNAs) and DNA hybridization networks. Six projects focus on chemically modifying PNAs through artificial nucleobases or backbone modifications to improve their physicochemical and hybridization properties, enabling applications ranging from oligonucleotide sensing and signal amplification to cellular delivery. The remaining project investigates DNA hybridization networks for logic-gated intracellular drug delivery.
The first chapter describes the development of a novel pseudocomplementary G:C base pair based on N-7-methyl guanine and G-clamp. This pair combines self-avoiding properties with efficient recognition of native G:C base pairs, enabling DNA:PNA invasion complexes under physiological salt conditions. These assemblies were applied to develop a rapid lateral flow assay for SARS-CoV-2 RNA detection, which could additionally discriminate between Omicron and Delta variants through mismatch recognition.
The second chapter addresses a major limitation in PNA chemistry: the laborious synthesis of different backbone-modified monomers. A universal γ-propargyl PNA monomer compatible with Fmoc-based solid-phase peptide synthesis was developed, providing a versatile platform for introducing diverse side-chain modifications through copper-catalysed azide–alkyne cycloaddition (CuAAC) click chemistry. The resulting modified PNAs were applied to improve PNA-based hybridization chain reactions (HCRs).
Building on this platform, the third chapter systematically investigates how differently charged side chains influence DNA:PNA and PNA:PNA duplex formation. A parallel melting-temperature screening method based on a fluorophore–quencher system was established for use with standard qPCR instruments, while surface plasmon resonance (SPR) measurements provided detailed kinetic insights. The results revealed beneficial and detrimental effects of positive and negative charges on hybridization and showed that the helical preorganization induced by γ-modifications remains substantially lower than that of DNA.
The fourth chapter explores logic-gated intracellular drug delivery using DNA recognition motifs, including affibodies and aptamers, to trigger HCRs only upon dual biomarker recognition. Systematic investigation of drug-loading strategies identified triply modified hydrophobic moieties as essential for efficient cellular delivery. This led to the development of a non-toxic bystander hydrophobic peptide capable of acting as a carrier for different caged drugs, creating a plug-and-play platform for conditional drug delivery with potential applications in oncology.
The fifth chapter builds on the previous findings by introducing a novel constrained pyrrolidine PNA backbone designed to enhance preorganization while maintaining compatibility with aqueous environments. Monomers compatible with both Mtt- and Fmoc-based solid-phase synthesis were developed and shown to support late-stage functionalization. Remarkably, the resulting constrained PNAs displayed hybridization kinetics approaching those of DNA.
Finally, the sixth chapter uses the γ-propargyl PNA platform to investigate chemical modifications that enhance cellular uptake. A broad range of modifications, including thiol-mediated uptake carriers and hydrophobic masked groups, were evaluated directly in cells using the chloroalkane penetration assay.
