Directed molecular evolution of novel DNA aptamers raised against an antibiotic resistant escherichia coli.

  • Robert Michael Nicholas Gowland

Student thesis: Doctoral Thesis

Abstract

Antimicrobial resistance presents a serious, worldwide threat to public health. New tools are required to combat the evolving problem. Here we report the systematic evolution of nucleic acid ligands to live Escherichia coli cells. Two SELEX methods were used to generate aptamers to the HB101: pAT153 strain of antibiotic resistant bacterial cells. These different Cell-SELEX methods were compared to analyse how DNA adapts to selective evolutionary pressure. The first method was performed using asymmetric PCR as the mechanism of single strand regeneration, with target cells taken from bacterial colonies. The second method used a novel strand protection exonuclease method for single strand regeneration and target cells taken from live cultures.

Sequences evolved during both methods were analysed and stored in plasmid libraries.These methods produced a combined total of fifty-eight putative DNA ligands (aptamers). These sequences were analysed using alignment and structural prediction software and six candidate molecules were taken for further analysis. Evolved sequences were synthesised and target cell binding assays were performed using fluorescence laser-scanning confocal microscopy to visualise binding. A number of control sequences were also analysed. These included scrambled versions of each aptamer sequence, complementary oligonucleotides to deform binding structures and positive control sequences of published cell binding aptamers. Five of the six novel sequences exhibited cell specific localisation. Two of these sequences, named Oligo 3.1and Oligo 4.4 (one from each SELEX Method) demonstrated apparent ligand binding affinities in the low-micromolar range (KDapp Oligo 3.1= 3.35 μM. KDapp Oligo 4.4 = 6.69 μM).
Date of AwardJun 2015
Original languageEnglish
Awarding Institution
  • University of Portsmouth
SupervisorDarren Gowers (Supervisor), Garry Scarlett (Supervisor) & Andrew Pickford (Supervisor)

Cite this

'