2026/01/01 by Xuan Han, Tyler Guilbault, Jonathan T. Sczepanski · 1 voice
Biochemistry, Genetics and Molecular Biology · #Advanced biosensing and bioanalysis techniques #DNA and Nucleic Acid Chemistry #RNA and protein synthesis mechanisms
paper · pdf · doi:10.1039/d6cb00057f
openalex publication_date 2026/01/01 · openalex created_date 2026/03/26 · openalex updated_date 2026/07/27
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.