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Title: Proximity dependent ligation selection : a new approach to generating DNA aptamers
Author: Chumphukam, Orada
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2013
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Nucleic acid aptamers are a group of molecules which emerge from large random sequence pools through in vitro selection, a process called Systematic Evolution of Ligands by EXponential enrichment (SELEX). These recognition molecules are a potential alternative to antibodies and offer a greater thermal stability, robustness and chemical versatility. The first part of this thesis describes the selection of an aptamer against acetylcholinesterase (AChE) using conventional in vitro selection process. The pool enrichment was observed during 15 rounds of selection and a chosen aptamer, R15/19, was characterized. The R15/19 binds with dissociation constant of 55±8pM but does not inhibit the enzyme. This aptamer can be used as a part of immobilization mediator. However, this manual in vitro selection procedure is time consuming as it needs multiple rounds of selection. Therefore, a novel aptamer selection method has been developed that is aimed at shortening selection times. Taking an advantage of multiple interactions, it is possible to generate a single sequence that contains two binding sites through a single selection process. The approach is adopted from proximity dependent DNA ligation assay (PLA) and called 'proximity ligation selection' (PLS). Instead of using one random pool for selection, with PLS, we use two. These two starting pools were designed to contain different additional sequences, apart from the primer and random regions, at either the 5' or the 3' end. The basis of this method is only in the presence of target protein molecules, is there a close proximity of pairs of bound sequences. And only if those pairs are in an appropriate orientation in which the 3' hydroxyl terminal of one sequence is sufficiently close to the 5'-phosphate terminal of the other sequence, are the extended free ends of each member of the proximity oligonucleotide pairs were brought closely by base-pairing to subsequently added connector oligonucleotide. The hybridized complexes are then joined using a DNA ligase, allowing the formation of a ligated product that contains a dual binding site on the target. Thrombin and lysozyme were used as model targets. For thrombin, none of the PLS products showed a G-quadruplex signature in CD spectroscopy. Using the SPR, the Kd of the aptamer dimer 2/19-5 (from F and R pool) and 4/19-7 (from TBA29plus and R pool) are 1.6μM and 1.2μM, respectively. The ELONA test was used to measure a Kd for each monomer and there was no binding observed from each monomer of the aptamer against thrombin. In the case of lysozyme, the SPR and ELONA revealed the Kd of the aptamer dimer (from two starting pools) of 8.7μM and 9nM, respectively. The Kd of each constitute of anti-lysozyme aptamer dimer using ELONA showed the Kd of 35.5 and 51.5nM. The Kd obtained from SPR was high, suggesting a low binding affinity. We assume that the aptamer molecule can freely fold into a favored structure once it meets the target and thus can perform binding better than in the SPR. Although we did not succeed in generating aptamer dimers that had higher affinity than monomers, there are several steps in the procedure that need to be further improved.
Supervisor: Cass, Tony Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available