Novel Use of Stem-loop DNA in SELEX

Abstract

Aptamers are composed of oligonucleotides and are most recognized for their ability to function much like antibodies. Unlike antibodies, aptamers can be generated in vitro, have a longer shelf life and are potentially faster to synthesize. The current method of aptamer generation is through a cycle known as SELEX, which stands for Systematic Evolution of Ligands through Exponential Enrichment. The SELEX method traditionally uses a library of single-stranded DNA or RNA as a starting material, which is incubated with a target. The nucleotides that bind to the target are separated from the unbound nucleotides, eluted from the target and amplified by PCR. The amplified nucleotides go through several more cycles of SELEX until aptamers with a high binding affinity for the target are produced. Before the double-stranded products from PCR can be used in another cycle they have to be separated into single-strands. The process of regenerating single-stranded DNA from double-stranded DNA is often time consuming and results in a low product yield, decreasing the efficiency of the SELEX process. In order to improve the efficiency of aptamer generation, we explored the use of a novel starting material based on its potential to make SELEX a more automated process. Stem-loop DNA has both double-stranded and single-stranded DNA portions due to its unique secondary structure. It has the ability to retain the single-stranded portion of its special conformation after PCR amplification. After characterization of a specifically designed stem-loop, we incorporated the use of stem-loop DNA as a starting material in SELEX to potentially bypass the regeneration of single-strand DNA in SELEX. The success of this modified approach to SELEX could result in a more efficient method for the generation of aptamers.