Development and Application of Ion Pair Reverse Phase HPLC for the Analysis of DNA Aptamers, dsRNA Biocontrols and mRNA Therapeutics

Nucleic acids are emerging as a powerful new class of therapeutic and play an
increasingly important role in biotechnology. The global mRNA therapeutic market alone
is expected to reach $128.14 billion by 2030. mRNA vaccines significantly reduce vaccine
development time due to their ease of customisation, and DNA/RNA aptamers are
emerging as potential therapeutics and have broader applications in biotechnology.
dsRNA biopesticides are also emerging as novel sustainable pesticides. However, each
of these faces unique challenges; while dsRNA biocontrols such as Ledprona have been
successfully deployed and mRNA vaccines proved vital during the COVID pandemic, they
are subject to strict regulations and quality control. Robust analytical methods are required
to fully characterise these biomolecules to support manufacturing and ensure their safety
and efficacy. ssDNA aptamers are currently developed using in vitro selection (SELEX).
However, high failure rates can impact the development and generation of new aptamers.
This study aimed to develop and utilise ion pair reverse phase HPLC (IP RP HPLC) to
analyse and characterise mRNA therapeutics and dsRNA biocontrols. In addition, it was
proposed that the SELEX procedure be optimised before generating ssDNA aptamers for
the melanoma differentiation-associated gene-9 (MDA-9). IP RP HPLC was used under
denaturing conditions to develop a novel method for the isolation of ssDNA in SELEX. The
novel method enabled the rapid purification of ssDNA with an 80% yield, demonstrating
significant advantages over existing methods. This method was subsequently utilised in
SELEX to obtain ssDNA aptamers to Syntenin-1. Modifications to the SELEX approach
resulted in the successful generation of ssDNA aptamers against Syntenin-1. Binding
affinity studies demonstrated that the ssDNA aptamers had binding affinities in the 100-
600 nM range, with the highest affinity aptamer at 25 nM.
IP RP HPLC was also developed to accurately size dsRNA biocontrols and mRNA
therapeutics. A wide range of ion pair reagents were studied under denaturing and nondenaturing conditions. The results showed that accurate sizing of a range of dsRNA
biocontrols resulted in less than 2.5% error using dibutyl ammonium acetate in the mobile
phase. The IP RP HPLC methods developed in this thesis offer significant advantages
over existing methods for generating ssDNA and accurately sizing dsRNA biocontrols. In
addition, further work building on the ssDNA aptamers against Syntenin-1 could develop
novel therapeutics as inhibitors to a protein important to cancer proliferation.