Development of analytical and sensitive methods to detect and quantify therapeutic oligonucleotides

Abstract

Therapeutic antisense oligonucleotides (ASOs) had a breakthrough in 1998 when fomivirsen, an inhibitor of human cytomegalovirus, was approved for medical use. Scientists discovered the medical potential of therapeutic oligonucleotides in treatments of various conditions and diseases, leading to the research field gaining more popularity. A challenge within this research field is the detection and evaluation of different oligonucleotide sequences, as no established quantitative and sensitive methodologies are present for these. The aim of this thesis is to develop analytical and sensitive methods for detection and quantification of therapeutic oligonucleotides. Particularly antisense oligonucleotides (ASOs), for application in in vitro experiments associated with fields such as cancer research. The ASOs included in this project are EBER1-5 and EBER1-6. The two-tailed RT-qPCR method, used for detection of miRNA, was adapted and modified in this project. The method was changed so that instead of a reverse transciption, a PCR was performed to anneal and elongate the two-tailed primer with the ASO. The PCR was then followed by a qPCR analysis. Different deviations to the initial protocol as well as to the designs of the primers and oligonucleotides, were performed during the project. The results showed that the method is promising as detection was possible to approximately 105 molecules, which corresponds to a concentration of 83 fM. Still, improvements need to be done for the binding and the elongation in the two-tailed part of the method to be able to reliably detect lower amount of molecules.