During the reporting period, we established a ModB‑based enzymatic platform for nucleic acid–protein conjugation and completed Objective 1 “Design of RNAylated proteins in vitro.” We defined design principles for ModB‑mediated RNAylation/DNAylation and showed that ModB covalently attaches NAD‑derived nucleotides to proteins via a natural N‑glycosidic bond at defined arginine residues.
Using an imidazolide chemistry, we generated NGD‑, NCD‑ and NUD‑capped RNAs and demonstrated efficient, one‑step transfer of all NXD‑RNAs to proteins, with NCD‑RNA yielding up to fourfold higher RNAylation than NAD‑RNA. We further showed that NXD‑capped DNAs are accepted as substrates, extending the platform to DNAylation as a new modality for site‑specific DNA–protein conjugates.
To generalise the method, we engineered an RNAylation tag based on rS1 domain II and a modular SpyTag R3K–rS1 DII / SpyCatcher system that enforces a single, defined modification site on virtually any protein of interest. We characterised stability and reversibility, demonstrating cap‑dependent ARH1 cleavage and strong protection of RNA against XRN1 and decay in human cell lysates, while identifying limited plasma stability as a target for future optimisation.
Technically, we established a two‑step denaturing IMAC/DEAE purification pipeline yielding highly pure conjugates and used it to produce DNAylated GFP‑rL2 that can be delivered into HEK293T cells with preserved co‑localisation of protein and DNA. These advances are documented in a peer‑reviewed research article, a comprehensive preprint and supported by an international patent application on RNAylation‑based conjugates.
End‑of‑project outcomes will comprise: a validated ModB‑based RNAylation/DNAylation platform (including NXD‑caps, tags and protocols), scalable purification and delivery workflows, and experimentally grounded design rules enabling broad use of site‑specific nucleic acid–protein conjugates in synthetic biology, diagnostics and nucleic‑acid‑based therapeutics.
