An expanded substrate scope for cross-chiral ligation enables efficient synthesis of long <scp>l</scp> -RNAs

Despite the growing interest in mirror-image l-oligonucleotides, both as a robust nucleic acid analogue and as an artificial genetic polymer, their broader adoption in biochemical research and medicine remains hindered by challenges associated with the synthesis of long sequences, especially for l-RNA. Herein, we present a novel strategy for assembling long l-RNAs <i>via</i> the joining of two or more shorter fragments using cross-chiral ligase ribozymes together with new substrate activation chemistry. We show that 5'-monophosphorylated l-RNA, which is readily prepared by solid-phase synthesis, can be activated by chemical attachment of a 5'-adenosine monophosphate (AMP) or diphosphate (ADP), yielding 5'-adenosyl(di- or tri-)phosphate l-RNA. The activation reaction is performed in mild aqueous conditions, proceeds efficiently with short or large l-RNA, and, yielding few byproducts, requires little or no further purification after activation. Importantly, both groups, when added to l-RNA, are compatible with ribozyme-mediated ligation, with the 5'-adenosyltriphosphate permitting rapid and efficient joining of two long l-RNA strands. This is exemplified by the assembly of a 129-nt l-RNA molecule <i>via</i> a single cross-chiral ligation event. Overall, by relying on ribozymes that can be readily prepared by <i>in vitro</i> transcription and l-RNA substrates that can be activated through simple chemistry, these methods are expected to make long l-RNAs more accessible to a wider range of researchers and facilitate the expansion of l-ON-based technologies.