Masters Thesis

Selection of DNA Aptamers Targeting Microbial Virulence Factors Using SELEX

Nucleic acid Aptamers are short single-stranded oligonucleotides that bind to target molecules with high affinity and specificity. Although they function like antibodies, there are several properties that aptamers possess that make them superior for certain applications. Despite an initial monetary and time investment in the selection of specific aptamers, once the sequences have been identified, the aptamers can be inexpensively synthesized, without the need to involve animals, and conveniently modified or labeled. DNA aptamers are of particular interest due to their stability at ambient temperature compared to antibodies, which makes them very suitable for portable assays. This study is aimed at selecting single-stranded DNA (ssDNA) aptamers targeting two virulence factors from two different microorganisms, i.e. Potato Virus Y (PVY) and Shiga toxin type 2 (STX2) produced by Escherichia coli O157:H7. The goal of the study is to determine (1) if recombinant viral proteins could be used to select aptamers that efficiently target live viruses, (2) if our DNA aptamers are capable of replacing antibodies in immunoassays for detecting STX2, and (3) if STX2-targeting aptamers can neutralize STX2. Aptamer selection utilizes a process called Systematic Evolution of Ligands by EXponential enrichment (SELEX). Each cycle of SELEX involves binding a pool of randomized oligonucleotides to immobilized targets, eluting the bound aptamers, amplifying the aptamers by PCR, and regenerating the ssDNA pool. Recombinant PVY coat protein purified from bacteria and commercial STX2 subunit were used as targets for SELEX. After about 8-12 cycles of SELEX, a pool of ssDNA containing enriched target-binding aptamers was obtained. The ssDNAs were subjected to next generation sequencing and the data was analyzed to identify target-binding candidates. The top candidate aptamers were synthesized and subject to verification by a modified ELISA protocol, named Enzyme-Linked Aptamer-Sorbent Assay (ELASA), which was developed using commercial Shiga toxin type 1 (STX1) protein with a known aptamer targeting STX1. Unfortunately, the top candidates of PVY and STX2-binding aptamers were verified to be non-specific. However, by using the STX1 aptamers synthesized based on published data, we successfully developed an ELASA that could detect STX1 B subunit effectively. This assay could differentiate between STX1 and STX2 despite of a high sequence similarity between these two toxins, making the assay an effective diagnostic tool that could replace antibodies in immunoassays.

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