| Popis: |
Chemically modified oligonucleotides find myriad applications in therapeutics and synthetic biology because they can be utilised to modulate biophysical or therapeutic properties, install fluorophores for detection, or to allow orthogonal reactivity through additional functional groups. Whilst oligonucleotides can be principally modified anywhere at their nucleotide subunits, the presented work investigates modifications of the oligonucleotide backbone for therapeutic applications and synthetic biology. Antisense oligonucleotides (ASOs) have become an emerging therapeutic approach to modulate protein expression at the mRNA level to combat hard-to-treat diseases. Stability against nucleolytic degradation, good target hybridisation properties, and efficient delivery into the cell are essential. The chemical modification of the hydrolysable, anionic phosphodiester backbone holds great potential for protection against phosphodiesterases, and to reduce the polyanionic charges for enhanced cellular uptake. In the presented work, two charge-neutral backbone surrogates—the carbamate and amide internucleoside linkages—were investigated for their ability to confer drug-like properties by replacing the natural phosphodiester backbone. Combinations with additional bicyclic sugar modifications and different designs of the ASO segments were explored. The antisense assessment criteria comprised their general hybridisation properties with an RNA target, their stability against nucleolytic degradation, and their potential for enhanced cellular uptake. Backbone-modified oligonucleotides are also versatile constructs for synthetic biology as exemplified by the construction of synthetic genetic polymers, ribozymes, aptamers, or gene editing systems. Amongst those, artificial genetic polymers can be applied for gene synthesis, sequencing, or the production of nanomaterials. A crucial element for backbone modifications in genetic templates is their compatibility to be read by polymerases. In this context, triazole-based internucleoside linkages have been extensively studied as promising replication compatible phosphodiester mimics. This thesis presents a novel triazole backbone structure and its assessment for replication compatibility in terms of continuation, speed, and correctness of the read-through by DNA polymerases. |
