# Biophysical and biological properties of splice-switching oligonucleotides and click conjugates containing LNA-phosphothiotriester linkages

## TL;DR

This paper explores new types of oligonucleotides with improved stability and biological activity for potential disease treatments.

## Contribution

A straightforward synthesis method for oligonucleotides with LNA-phosphothiotriester linkages and functional groups is introduced.

## Key findings

- Oligonucleotides with LNA-phosphothiotriester linkages showed excellent duplex stability with DNA and RNA.
- Amino acid conjugates demonstrated significantly higher splice-switching activity compared to carbohydrate conjugates.
- Over 60 oligonucleotides were synthesized and evaluated for biophysical and biological properties.

## Abstract

Antisense oligonucleotides hold great promise in the treatment of disease, but their efficacy is limited by modest bioavailability and toxicity. Charge-neutral phosphorus-based backbones can potentially improve biological properties, but oligonucleotides with such modifications are challenging to synthesize. Here, we report the straightforward synthesis of a range of oligonucleotides containing multiple LNA alkyl phosphothiotriester nucleotides and evaluate their biophysical and biological properties. Several functional groups were incorporated into the triester linkages, including 2-butyl, 2-hexyl, 3-octyl, 4-trifluoromethyl cyclohex-1-yl, hexadecyl, and 4-pentyn-2-yl. The alkyne triesters were functionalized with carbohydrates, amino acids, heptaethylene glycol, spermine, and thiazole orange through CuAAC click chemistry. Analysis of over 60 oligonucleotides showed that almost all displayed excellent duplex stability with both complementary DNA and RNA and good splice-switching activity in an in vitro reporter assay. Amino acid conjugates showed significantly higher activity than carbohydrate conjugates via gymnosis.

Graphical Abstract