An Expanded Substrate Scope for Cross-Chiral Ligation Enables Efficient Synthesis of Long L-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 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 via 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 biproducts, 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′-adenosyl triphosphate permitting rapid and efficient joining of multiple, long L-RNA strands. This is exemplified by the assembly of a 129-nt L-RNA molecule via a single cross-chiral ligation event. Overall, by relying on ribozymes that can be readily prepared by in vitro 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.