LAUSR

Aptamers composed of mirror‐image L‐(deoxy)ribose nucleic acids, referred to as L‐aptamers, are a promising class of RNA‐binding reagents. Yet, the selectivity of cross‐chiral interactions between L‐aptamers and their RNA targets remain poorly characterized, limiting the potential utility of this approach for applications in biological systems. Herein, we carried out the first comprehensive analysis of cross‐chiral L‐aptamer selectivity using a newly developed “inverse” in vitro selection approach that exploits the genetic nature of the D‐RNA ligand. By employing a library of more than a million target‐derived sequences, we determined the RNA sequence and structural preference of a model L‐aptamer and revealed previously unidentified and potentially broad off‐target RNA binding behaviors. These results provide valuable information for assessing the likelihood and consequences of potential off‐target interactions and reveal strategies to mitigate these effects. Thus, inverse in vitro selection provides several opportunities to advance L‐aptamer technology.