Owing to their bioinert property, L-oligonucleotides have been used widely for biotechnology applications including molecular tools and biosensors. However, L-oligonucleotides can only be prepared by solid-phase synthesis, this limits its application to short sequences. Here we reported a straightforward native enzymatic method to prepare long and functional L-oligonucleotide via the ligation of two shorter fragments. Two D-nucleotides were introduced at the 3’ and 5’ end of the acceptor fragment and donor fragment, respectively. The acceptor and 5’ phosphorylated donor fragments were then ligated by T4RNA ligase 1. As a proof-of-principle, we assembled L-RNA sequences including ribozymes and aptamers, which all exhibited similar characteristics to their original design. Moreover, these chimeric ligated L-RNAs remained functional in the presence of serum, showing that introducing four D-nucleotides did not decrease their stability. Finally, we assembled an L-RNA version of a 124-nucleotide fluorescent biosensor, which is one of the longest L-RNA molecules ever made synthetically. Overall, we demonstrate a straightforward approach to prepare long L-oligonucleotides, hoping that this approach will make the L-oligonucleotides application more accessible and versatile.

Assembly of long L-RNA by native RNA ligation

ACS Spring 2022

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