Simultaneously Isolating Multiple Biosensors for Multiple Targets from a Single SELEX Procedure

Abstract

SELEX is a selective amplification technique based on the assumption that functional nucleic acids (FNAs) can be found in a pool of chemically synthesized random nucleic acid sequences. These FNAs can either bind to a target of interest (aptamers) or catalyze a chemical reaction (DNAzymes and ribozymes) or both (aptazyme). The aptazymes discussed herein, are RNA-cleaving DNAzymes that become catalytically active upon target recognition at the aptamer domain. In chapter 2, two SELEX experiments were performed in parallel to determine whether it was possible to obtain multiple aptazymes from a single SELEX procedure. Two different crude intercellular mixtures (CIM) each containing 35 unique overexpressed ASKA clone gene products were used. After 10 rounds of SELEX, the sequence pool was analyzed using Illumina Genome Analyzer. The sequences tested appeared to bind to a protein other than the 35 gene products of interest. To redirect these SELEX experiments towards the gene products of interest, 12 gene products from each parallel selection was purified and incubated with sequences from round 6 of the CIM SELEX. A total of 13 rounds of selection were performed using these purified proteins. The sequences were analyzed and found to either not cleave at all or self-cleave. In chapter 3 a similar SELEX approach was explored using 36 purified proteins, from the CIM SELEX to isolate protein-binding DNA aptamers. This SELEX approach served as another means to explore isolating multiple aptamers against multiple targets. However both the RNA-cleaving aptazyme and protein-binding DNA aptamer SELEX experiments both experienced challenges in non-specific binding. Although negative selection steps were taken in order to avoid non-specific binding species, such sequences were still isolated. In order for these approaches to be successful, negative selection steps are required to remove any self-cleaving and non-specific FNAs. Although these studies did not conclusively give rise to the desired FNAs, it has produced some insight to the potential setbacks associated with developing a screen for multiple targets.