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

Area of Science:

• Biochemistry and Molecular Biology
• RNA Therapeutics
• Synthetic Biology

Background:

• Mirror-image l-ribonucleic acid aptamers (l-aptamers) offer high-affinity, shape-based recognition of structured RNAs.
• Current methods for generating specific l-aptamers typically require *de novo* selection for each target.
• Reprogramming existing l-aptamers for new targets remains a significant challenge.

Purpose of the Study:

• To investigate the application of mutagenesis-based evolution for reprogramming cross-chiral l-aptamers.
• To develop a set of orthogonal l-aptamers with enhanced specificity for RNA hairpins.
• To establish a versatile strategy for adaptable and multiplexable RNA-targeting.

Main Methods:

• Utilized mutagenesis-based evolution on a parental l-aptamer targeting the HIV-1 TAR RNA hairpin.
• Employed deep sequencing to analyze sequence changes and aptamer folding.
• Performed RNA pulldown experiments to assess target selectivity in complex contexts.

Main Results:

• Generated two evolved l-aptamers that, with the parent, form an orthogonal set distinguishing single-nucleotide RNA hairpin differences.
• Deep sequencing revealed extensive aptamer fold remodeling, not incremental changes, is crucial for retargeting.
• Orthogonal l-aptamers demonstrated strict selectivity in competitive environments with complex RNA structures.

Conclusions:

• Mutagenesis-based evolution is an effective strategy for rapidly reprogramming cross-chiral l-aptamers.
• Cross-chiral aptamer recognition relies on global RNA architecture rather than local sequence complementarity.
• This work provides a framework for developing adaptable and multiplexable RNA-targeting strategies.