Modification of DNA aptamers for sensing applications

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Allabush, Francia (2019). Modification of DNA aptamers for sensing applications. University of Birmingham. Ph.D.

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Abstract

Current clinical assays employ a number of different molecular recognition elements, with the most prevalent using antibodies as a consequence of their high selectivity towards target analytes. However, antibody based assays carry a number of limitations. In recent years, aptamers have been highlighted as suitable alternatives to antibodies. Aptamers are single stranded oligonucleotides which fold to bind to non-oligonucleotide based analytes. However, aptamers are often degraded by nuclease enzymes and are thus not ideal for analysing clinical samples. Fortunately, their instability to nuclease digestion can be rectified by the use of modified aptamers. This thesis aims to contribute to the growing field of modified aptamer development by introducing two vastly different but innovative modifications for incorporating aptamers into biosensing applications.
MIPs: This approach incorporates aptamers into molecularly imprinted polymers, or MIPs. Often noted as a more stable substitute to both antibodies and aptamers, MIPs are formed when the analyte of interest is imprinted into a polymer. The synthesis of the acrylamide modified thymidine molecule is described, and the feasibility of the molecule to polymerise and be incorporated into DNA via solid phase oligonucleotide synthesis is reported. Modified aptamer sequences with various placements of acrylamide groups are then synthesised, and their ability to polymerise and bind to thrombin/complementary DNA targets is studied. These studies demonstrate that placement of the modification needs to be carefully considered in order to maximise aptamer-target interactions.
Liposomes: This approach uses aptamers as target recognition elements in liposomes for biosensing applications. Aptamer based sensor components anchored in fluidic membranes can be engineered to respond to external triggers outside the liposome, leading to a chemical signal within the liposome. These components are designed to mimic the properties of transmembrane proteins in nature. This thesis describes the synthesis of these components for vesicle incorporation. DNA-bischolesterol and DNA-bischolesterol-dye conjugates were successfully synthesised, purified by reversed phase HPLC, and characterized by mass spectrometry. The constructs afforded have the potential to bind to target molecules and ultimately be adopted into biosensing systems with liposomes.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):