Lock, Protect, and Bind: In Vitro Selection of Locked Nucleic Acid‐Modified Aptamers Using a Mutant T7 RNA Polymerase

RNA therapeutics are powerful tools for gene modulation and targeted therapies, but their clinical application is hindered by nuclease degradation and immunogenicity. Incorporating chemical modifications, like locked nucleic acids (LNAs), can enhance nuclease resistance, targeting properties, and thermal stability. Traditionally, LNA incorporation has relied on solid‐phase synthesis of short RNAs. Engineered polymerases capable of incorporating xenonucleic acids (XNAs), including LNA, into longer RNAs have been described. However, their XNA yield is limited by primer and template copy numbers, and the generated DNA‐XNA duplexes can be difficult to purify. We present a novel approach for incorporating LNA‐ATP and LNA‐TTP alongside 2′fluoro (2′F)‐modified pyrimidines via in vitro transcription using a mutant T7 RNA polymerase. This method enables efficient, primer‐independent synthesis and amplification of LNA‐modified RNA with low error rates. To demonstrate its utility, we performed systematic evolution of ligands by exponential enrichment (SELEX) to select LNA‐ and 2′F‐modified aptamers targeting influenza hemagglutinin and human CD40 ligand, two therapeutically relevant proteins. Iterative SELEX cycles yielded aptamers with low‐nanomolar affinities, high specificity, increased thermal stability, and high nuclease resistance. Overall, this approach provides a scalable and versatile platform for generating chemically stabilized RNAs, fully compatible with SELEX, and holds potential for developing next‐generation RNA‐based therapeutics with improved pharmacokinetics.