Acyclic Identification of Aptamers (AIA) is a streamlined, high-throughput aptamer discovery method which eliminates the lengthy cycles required of in vitro selection, also known as SELEX (Systematic Evolution of Ligands by Exponential Enrichment). Aptamers are functional single-stranded oligonucleotides capable of folding into three-dimensional structures that bind their targets with high specificity. The minimal human alpha-thrombin binding aptamer, d(GGTTGGTGTGGTTGG), has been identified from a minimally flanked DNA library, (pACAC-m15-CACA) in &lt;5 days using AIA. The aptamer has also been identified in a nuclease resistant, modified 2’-OMe RNA/DNA chimera library. Randomized oligonucleotide libraries with an over-representation of all possible sequences eliminate the need for in vitro evolution, reducing the timeline for aptamer discovery from months to days. Amplification free library capture and preparation condenses the timeline further and generates a final product free of amplification bias. Consequently, relative maximum sequence frequencies can be predicted based on the quantity of recovered library, initial degree of over-representation and anticipated high-throughput sequencing data output on Illumina sequencing platforms. Absolute quantification with qPCR CopyCountTM software improves library quantification and distribution of indices (sample “barcodes”), maximizing data output. A novel partitioning method that eliminates the requirement for protein immobilization was also developed. Reversible formaldehyde cross-linking in conjunction with Electrophoretic Mobility Shift Assay was used to successfully identify the thrombin binding aptamer above background in proof of concept experiments using amplification free sample preparation. The ability to perform AIA partitioning in solution provides greater flexibility in target selection, including mixtures of proteins. The method also effectively reduces the aptamer off-rate to zero by covalently linking high and moderate affinity sequences to the protein target during selection, an advantage over protein immobilization for partitioning where loss of some DNA from a reversibly bound complex is inevitable.

Acyclic identification of aptamers (AIA): Rapid identification of DNA and 2-OMe RNA aptamers for human alpha-thrombin

2019 Spring National Meeting

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Acyclic identification of aptamers (AIA): Rapid identification of DNA and 2-OMe RNA aptamers for human alpha-thrombin

Acyclic identification of aptamers (AIA): Rapid identification of DNA and 2-OMe RNA aptamers for human alpha-thrombin