Expanding the Functional Space of Non-Natural Nucleic Acids through Strategic Molecular Evolution

Functional nucleic acids (FNAs) are single-stranded nucleic acid molecules that acquire specific activities by folding into complex three-dimensional structures. Represented by nucleic acid enzymes, aptamers, and riboswitches, FNAs provide powerful tools for probing the chemical and structural potential of nucleic acids, and hold broad promise for applications in biotechnology and medicine. However, their limited structural diversity, paucity of protein-like functional groups, and poor biostability restrict their catalytic and binding capabilities in biologically relevant contexts. Overcoming these intrinsic constraints requires strategies that enhance both functional efficiency and biological robustness. Leveraging recent advances in synthetic genetics, structural biology, and AI-enabled structure prediction, we have established integrated approaches to expand the functional space of FNAs. First, guided by crystallographic analysis, we introduced 2’-fluoroarabinose nucleic acid (FANA) substitutions into the fluorescent DNA aptamer lettuce, yielding FANA-DNA chimeras with ~1.5-2.5-fold enhanced fluorescence intensity. Second, guided by AlphaFold 3-predicted complex structures, we installed dual covalent warheads into the PTK7-binding DNA aptamer sgc8c, achieving stable covalent target engagement that induced sustained PTK7 degradation and potentiated NK cell–mediated tumor killing. Third, we selected and characterized a chimeric nucleic acid enzyme composed of FANA and base-functionalized DNA that catalyzed RNA substrate cleavage through a previously unobserved, non–Watson-Crick mode of enzyme-substrate recognition.Together, these studies improve the functional performance and biological applicability of FNAs while revealing new catalytic and recognition principles, thereby expanding the chemical biology and therapeutic potential of functional nucleic acid-based molecules.