| Description |
The selection of functional aptamers using the Systematic Evolution of Ligands via Exponential enrichment, or SELEX, has been a powerful tool for the generation of nucleic acid affinity reagents. Many elegant methods have been devised to tailor the selection process to the creation of aptamers with specific functionality. Each SELEX strategy must be specifically designed to achieve a particular outcome such as catalytic activity or increased nuclease stability. As nucleic acid aptamers grow in popularity, the need for methods to customize the selection process to generate specific functionality increases. We have developed a novel SELEX method for the generation of RNA aptamers with catalytic activity. This method utilized an immunoprecipitation step to specifically pull down RNA sequences capable of self-alkylation. Using this strategy, we have generated functional ribozymes capable of self-labeling with a reactive fluorescein dye. We have also explored the use of nucleic acids with a modified backbone. There are many benefits to modified nucleic acids including their superior nuclease resistance. Threose Nucleic Acids show great promise as alternatives to native DNA or RNA. This is, in part, because much work has been done to develop enzymes that can recognize them. This allowed us to propose and test a selection method for the generation of high affinity TNA aptamers. We have also worked to expand the functionality of TNA by synthesizing and testing TNA with additional modifications such as β, γ -modified-l-tTTP mimics. Another method we have used to push the boundaries of current aptamer selection methodology utilizes denaturing-reducing conditions to select for aptamers that function in conditions that denature proteins such as antibodies. We have developed a SELEX protocol that allows us to directly select for DNA aptamers that bind with high affinity under these conditions. We have shown successful selection of an affinity reagent under these conditions and continue to optimize the selection process. |
