Synthesis, structure and application of backbone modified nucleic acids

Modified backbones are versatile modifications which can improve biological half-lives, have good structural biocompatibility and can alter the biophysical properties of oligonucleotides. This work presents the synthesis and biophysical properties of oligonucleotides modified with carbamate-locked nucleic acids (CBM-LNA) for antisense applications, the crystallisation and structural analysis of amide-locked nucleic acids (LNA-amides) and explores peptide translation using modified triazole backbones in circular mRNAs (Tz-circRNA). From these studies it is shown that CBM-LNAs present viable templates for potential antisense applications, that LNA-amides are good structural mimics as phosphodiesters and that Tz-circRNAs are viable templates for protein expression.
Carbamate backbones are four-atom, charge neutral linkages that were previously found to be duplex destabilising and unsuitable for antisense applications. In this work, we combine the enzymatic stability of the CBM backbone with the thermodynamic stability of locked nucleic acids (LNA). CBM-LNA phosphoramidite dimers were synthesised and incorporated into oligonucleotide sequences. Biophysical characterisation using UV melting, showed that LNA-CBM1-LNA modifications were able to restore duplex stability against RNA targets and that LNA-CBMs have enhanced enzymatic resistance. Compared to previous CBM modifications, LNA-CBMs now present suitable modifications for potential antisense applications.
LNA-amides have good thermodynamic properties which makes them suitable candidates for therapeutic applications. The aim of our work is to show that LNA-amides are good structural mimics as phosphodiesters and suggest the features that provide LNA-amides with good biophysical properties. XRD crystallography was used to obtain three different LNA-amide modified oligonucleotides between 2.5-2.8 Å resolution. Analysis of these structures showed that LNA-amides are structurally alike to phosphodiesters and that they cause minimal duplex disruption. The amide and LNA modifications were found to work harmoniously together. To provide good duplex stability, modified backbones should have good flexibility towards their 5ˈ-end and should consist of four-atoms in length if they contain a carbonyl functionality.
Triazole backbones have good biocompatibility for replication and transcription mechanisms. In this work we aim to complete the central dogma and translate peptides using triazole modified circular mRNA templates. This will allow for the chemical synthesis of circular mRNAs > 100 nt without the need for enzymes, plasmid preparation or bacterial cell culture. Linear precursor RNAs were modified with 5ˈ-azide and 3ˈ-alkyne functional groups. These were then subjected to the CuAAC reaction to form a triazole linkage contained within the coding region of a circular mRNA. Encoded with FLAG epitopes, the triazole circular RNAs were used in cell free prokaryotic protein translation and analysed by immunoblotting techniques. From these results it is shown that triazoles can be successfully translated by the prokaryotic ribosome and yield up to 50% as much peptide as an unmodified circular RNA.

Keywords: Antisense
Oligonucleotides
Nucleic Acids
DNA
Carbamates
Modified backbones
RNA
circRNA
Advisor: Brown, T
El-Sagheer, A