Chemical Synthesis of New Modified Nucleotides Towards Improved Antisense Oligonucleotide Drugs

Research output: ThesisPh.D. thesis


The use of nucleic acids in therapeutics and biotechnology keeps increasing and a continued interest has called for the development of novel nucleotides with augmented properties and new features. The antisense approach aims to introduce oligonucleotides that have a therapeutic effect on the host but can deliver such an effect in a reversible manner, i.e., once the oligonucleotide stop being present at the targeted location, so does the therapeutic effect. This is a substantially different approach compared to changing the very genome, which can lead to irreversible ramifications on the host.

The thesis is divided into two areas; the first part (chapter 4) includes the use of modified nucleotides in the gap region of gapmer ASOs and the second part (chapters 5 and 6) presents
nucleotide building blocks and oligonucleotides that have been functionalized with cationic moieties.

In the first part, a new class of 2’-deoxy-2’-fluoro-3’-C-hydroxymethyl-β-D-lyxose configured nucleotides is presented. The oligonucleotides modified with these modifications have been evaluated for their duplex stability, nuclease resistance, RNase H recruitment, RNase H cleavage control and antisense activity in HeLa cells. After incorporation into oligonucleotide this new class of nucleotides do not disrupt the formed duplex. Additionally, a slight improvement in nuclease resistance can be observed relative to the standard DNA nucleotides. The important property of this new class of building blocks is the ability to act as good RNase H substrates and to influence the RNase H cleavage
in the targeted RNA to a high degree. Ultimately, terminating RNase H activity for an RNA strand that has one single nucleobase mutation. Finally, when incorporated twice in the gap region of a
gapmer antisense oligonucleotide, this new class of nucleotides improved the antisense activity in vitro.

The second part has been split into two chapters (5 and 6). In chapter 5, three 2’-amino-LNAT monomers and their ability to improve the duplex and triplex stability, together with nuclease
resistance for the modified oligonucleotides, is presented. High duplex stability is observed for all modified oligonucleotides, together with improved nuclease resistance. Interestingly, the linker length between the terminal amino-groups of the bis-amino linker and the attachment site on the sugar scaffold played an important role in the triplex stability.

Chapter 6 focuses on cationic nucleotide derivatives capacity to improve the antisense activity of antibacterial peptide-oligonucleotide conjugates. Notably, the 2’-amino-LNA-T derivatives improves the duplex and triplex stability to a significant degree, while offering a versatile method of
modifying the oligonucleotides through copper-catalysed click reactions. Interestingly, inserting cationic nucleotide derivatives in the peptide-oligonucleotide conjugates have demonstrated to be a successful method of improving the antibacterial activity of such systems that works through the antisense approach. Additionally, these systems seem to be selective towards certain bacterial families.
Original languageEnglish
Awarding Institution
  • University of Southern Denmark
  • Wengel, Jesper Thagaard, Principal supervisor
Date of defence7. Apr 2022
Publication statusPublished - 29. Mar 2022


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