In Vitro Selection of Threose Nucleic Acid (TNA) Enzymes That Catalyze RNA Transformations

This article provides a comprehensive set of protocols for the in vitro selection of threose nucleic acid (TNA) enzymes capable of catalyzing RNA cleavage or ligation reactions. The article first outlines the generation of random-sequence TNA libraries (typically comprising ∼10¹⁴ unique molecules) covalently attached to their RNA substrates via engineered DNA polymerases. It then describes the procedure for isolating catalytically active TNA sequences through affinity chromatography or gel electrophoresis. These sequences are subsequently reverse-transcribed to cDNA for amplification or sequencing. This set of protocols is primarily designed for selecting RNA-cleaving and RNA ligase TNA enzymes but can be adapted for the discovery of TNA enzymes that act on DNA substrates or enzymes derived from other xeno-nucleic acids, provided that requisite nucleoside triphosphates and compatible tool enzymes are available.© 2025 Wiley Periodicals LLC. Basic Protocol 1: TNA library synthesis Basic Protocol 2: Selection of RNA-cleaving TNA enzyme Alternate Protocol: Selection of RNA ligase TNA enzyme Basic Protocol 3: Reverse transcription of enriched TNA library Basic Protocol 4: Preparation of template DNA for the next round.