Optimization of RNA Aptamer SELEX Methods: Improved Aptamer Transcript 3′-End Homogeneity, PAGE Purification Yield, and Target-Bound Aptamer RNA Recovery

Since its inception in the early 1990s, SELEX remains the gold standard for discovering RNA aptamers specific for proteins and small molecules. The SELEX process has undergone countless modifications and now encompasses a breadth of innovative selection schemes to pare an aptamer library toward target-specific aptamers. Common to all these RNA aptamer SELEX processes are the steps for the preparation of DNA template and in vitro transcription of aptamer RNA. These steps have remained mostly unchanged over the past three decades and would benefit from optimization. We focused on three key areas: improving the homogeneity of in vitro transcribed aptamer RNA, increasing the efficiency of in vitro transcribed aptamer RNA purification by PAGE, and improving the quality of target-bound aptamer RNA recovered during SELEX. Together, these optimizations contribute toward a more efficient SELEX process and are applicable to both protein-based and cell-based RNA aptamer selections.