Ligase‐Catalyzed Transcription and Reverse Transcription of XNA by T3 DNA Ligase

The growing utility of xeno-nucleic acids (XNAs) lies in their ability to extend the reach of genetic chemistry beyond the limits imposed by natural polymers. XNAs, with their diverse chemical backbones, resist enzymatic degradation and yet retain the capacity for sequence-defined information, and have found broad applications in biotechnology. The approach described herein provides a systematic method for the transliteration between XNAs and DNAs. This article delineates the ligase-catalyzed oligonucleotide polymerization (LOOPER) process as applied to the transcription and reverse transcription of XNA libraries using T3 DNA ligase. Two complementary procedures are presented. Basic Protocol 1 details the assembly of XNA polymers through the ligase-mediated templated ligation of 5'-phosphorylated trinucleotide anticodons bearing XNA modifications, exemplified here by locked nucleic acids (LNAs). Basic Protocol 2 describes the reverse transcription of XNA sequences into cDNA using unmodified DNA 5'-phosphorylated trinucleotide anticodons. Together, these protocols enable a bidirectional exchange between DNA and chemically diverse XNA species, a prerequisite for the application of SELEX and other evolutionary methodologies to noncanonical backbones. This ligase-based framework dispenses with substrate biases that can often be present with polymerases, allowing high-fidelity transliteration (>95%) across a variety of modified nucleotides. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Ligase-catalyzed oligonucleotide polymerization (LOOPER) for XNA synthesis Basic Protocol 2: Ligase-catalyzed oligonucleotide polymerization (LOOPER) for cDNA synthesis from XNA templates.