Evaluation, synthesis and imaging of therapeutic oligonucleotides

Abstract

A new method for the ³⁴S isotope labelling of phosphorothioate oligonucleotides is reported which allows a practical synthesis of site specifically labeled oligonucleotides. Several applications of this method are reported. It was successfully applied to the quantification of oligonucleotides in tissues and showed superior performance to previously used methods. The application of ³⁴S labeled phosphorothioate oligonucleotides to quantitative Nano Secondary Ion Mass Spectrometry is also shown. Oligonucleotides conjugated to a GLP1 binding peptide were shown to decouple from the peptide quickly in endosomes and escape to the cytoplasm. This process can be influenced by the linker chemistry and helps explain different efficacies in mRNA knockdown when using different conjugation chemistries.

The synthesis and scaleup of 2′-O-AECM nucleotide phosphoramidites is described which includes a new protecting strategy and optimization of reaction conditions. Application of those phosphoramidites in the synthesis of fully 2′-O-AECM modified gapmers is described and they are shown to be active in knocking down MALAT1. 2′-O-AECM modified gapmers are shown to achieve a high level of knockdown if they are conjugated to an endosomal escape enhancing peptide.