Mutagenesis-based evolution enables rapid retargeting of l-aptamers for structured RNA recognition

Mirror-image l-(deoxy)ribose nucleic acid aptamers (l-aptamers) enable high-affinity, selective recognition of native structured RNAs via shape-based, cross-chiral interactions rather than Watson-Crick base pairing. However, achieving this level of specificity has typically required de novo selection from fully randomized libraries for each new target. Here, we report the first application of mutagenesis-based evolution to an existing cross-chiral l-aptamer to rapidly reprogram target specificity. Starting from a parental aptamer recognizing the HIV-1 TAR RNA hairpin, we generated two evolved aptamers that, together with the parent, form a set of orthogonal binders capable of discriminating RNA hairpins differing by a single nucleotide. Deep sequencing revealed that retargeting requires extensive remodeling of the aptamer fold rather than incremental sequence changes, reflecting the dependence of cross-chiral interactions on global RNA architecture rather than local sequence complementarity. Finally, RNA pulldown experiments demonstrate that these orthogonal l-aptamers retain strict selectivity in a competitive and structurally complex context, including extended flanking sequences and multiple RNA targets. Taken together, this work establishes mutagenesis-based evolution as a versatile strategy for rapidly retargeting cross-chiral l-aptamers and reinforces the unique advantages of structure-based RNA recognition, providing a framework for developing adaptable and multiplexable RNA-targeting strategies.