Author: Kerrilee Evelyn Allan
Allan, Kerrilee Evelyn, 2012 Free Solution Capillary Electrophoresis for Purity and Identification of Synthetic Oligonucleotides on Polymer Modified Capillary Surfaces, Flinders University, School of Chemical and Physical Sciences
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This thesis describes a capillary electrophoretic method to determine the identity and purity of oligonucleotides (ODNs) and phosphorothioate antisense oligonucleotides (PS-ODNs) using cationic copolymers as a dynamic coating, based on monomers of ethylpyrrolidine methacrylate (EPyM) and methyl methacrylate (MMA). ODNs are short fragments of single-stranded (ss) or double-stranded (ds) deoxyribonucleic acid (DNA) and their uses range from biological systems to drug delivery and pharmaceutical analysis. In particular, antisense oligonucleotides (AS-ODNs) have a role as therapeutic molecules which inhibit expression of target genes by binding to a specific RNA sequence, blocking the translation process. AS-ODNs exist in various modified forms of which PS-ODNs are the most common. It is therefore necessary to have methods in place to determine the identity and purity of these modified AS-ODNs. Capillary electrophoresis (CE) has been extensively used for separation of DNA fragments focusing on large strands. Consequently, there is a significant gap in the literature regarding the analysis of short strands of DNA (such as ODNs). DNA strands below the DNA persistence length (pDNA) are rod-like and non-free draining, and exhibit different migration patterns in free solution compared to larger strands which cannot be separated in this way. Operating in free solution has the advantage of only requiring surface confined capillary modification for electroosmotic flow (EOF) suppression allowing for charge-based separation in which larger, more negatively strands exhibit a greater mobility. In this thesis, homopolymers and copolymers based on EPyM and MMA were prepared by conventional free radical (CFR) and reversible addition-fragmentation chain transfer (RAFT) polymerisation. This work reports for the first time the use of a RAFT block copolymer for the separation of ODNs. Polymer solutions were prepared for surface-confined capillary modification via physical adsorption and the adsorptive properties (onto silicon (Si) wafers) were investigated via atomic force microscopy (AFM). It was observed that the effectiveness of these polymers for capillary surface modification via adsorption was highly dependent on the polymer concentration, tacticity and hence, stereochemistry. A dilute polymer solution of CFR poly(ethylpyrrolidine methacrylate-co-methyl methacrylate) (PEPyM-co-PMMA) random copolymer (1 mg mL-1) provided a homogenous surface coating owing to the polymer chains being hydrodynamically separated allowing for adsorption as individual globules. Whereas the RAFT poly(ethylpyrrolidine methacrylate-block-methyl methacrylate) (PEPyM-b-PMMA) block copolymer provided an uneven and incomplete surface coverage owing to the rigidity of the polymer chains. Enhanced CE separation of ODNs (ds and ss) ranging from 16 - 20 base pairs (bp) (containing the same 16 base (b) sequence) was achieved in free solution on a capillary dynamically coated with CFR PEPyM-co-PMMA random copolymer (21/79). Fast and efficient surface modification was achieved on bare fused-silica capillaries activated with hydrochloric acid (HCl), H2O, sodium hydroxide (NaOH) and Tris(hydroxymethyl)aminomethane (Tris)-borate (100 mM)/urea (7M) buffer, by flushing with polymer solution and H2O followed by a final treatment with running buffer. 1 bp resolution (Rs) and was seen for long (30 cm) and short (8 cm) capillaries with partial Rs of the 16 bp and 17 bp mixture. Co-migration of some strands was attributed to ODN-ODN interactions during migration. These interactions were confirmed by the 16 bp peak migration time (tm) not being additive within each mixture. The 1 bp Rs achieved for the complementary sequence strands was improved by up to 37 % for separation of dsODNs containing non-complementary sequences. Interestingly, separation of a dsODN mixture containing two 16 bp strands of different sequences resulted in partial Rs (0.52) suggesting that the free solution mobility of dsODNs was sequence dependent. Differential mobilities for ssODN fragments of the same length were also observed. The method described herein was capable of resolving mixtures of PS-ODNs and ODNs, (including Rs of ss impurities from ds fragments) indicating that this method is suitable for determining ODN and PS-ODN purity. Both ODN and PS-ODN mobility was found to be proportional to an increase in bp length, suggesting the separation mechanism is based on the free solution mobility and ion-pairing between the surface and the analyte. The developed method has the advantage of fast and simple preparation, easy regeneration, extended capillary life-time, unrestricted polymer solubility, and enhanced stability under harsh conditions (high voltage and temperature, and extreme pH).
Keywords: oligonucleotides,capillary electrophoresis,polymer surfaces,DNA,antisense oligonucleotides
Subject: Chemistry thesis
Thesis type: Doctor of Philosophy
School: School of Chemical and Physical Sciences
Supervisor: Associate Professor Amanda Ellis