Evaluation of Fluorescence Resonance Energy Transfer Approaches as a Tool to Quantify the Stability of Antisense Oligodeoxynucleotides

Abstract

Antisense oligodeoxynucleotides (AsODN) are rapidly degraded by nucleases in biological fluids which compromises their efficacy as therapeutic agents. This study evaluated two Fluorescence Resonance Energy Transfer (FRET) approaches, Acceptor Photobleaching and Sensitized Emission, in terms of their suitability for quantification of oligonucleotide stability in various colloidal carrier systems in vitro. The influence of the formulations pH and viscosity on the validity of the two approaches was determined and showed that the donor fluorescence intensity was highly susceptible to pH fluctuations of the medium. Moreover, the Acceptor Photobleaching approach proved to be unsuitable for the proposed studies due to Brownian motion of molecules in liquid formulations, suggesting that this method can only be used for immobilized specimens. The stability of a 30-mer AsODN incorporated into various in situ gelling systems was evaluated using the Sensitized Emission approach. This approach appeared to offer a simple tool to evaluate the stability of AsODN and showed stable molecules over a period of one week. However, a number of criteria, such as photobleaching due to repeated exposure, pH of the surrounding medium and sample preparation need to be carefully considered when performing quantitative FRET measurements. Keywords: Acceptor photobleaching, Brownian motion, Fluorescence resonance energy transfer, In situ gelling systems, Stability, pH, Image analysis, Sensitized emission, Viscosity, High performance liquid chromatography, Electrospray ionisation mass spectrometry