Conjugation of NAD-capped RNAs to proteins by ADP-ribosyltransferases to generate RNA therapeutics

Project Information

NAD-ART

Grant agreement ID: 101114948

DOI

10.3030/101114948

EC signature date 26 September 2023

Start date 1 January 2024

End date 31 December 2028

Funded under

• European Research Council (ERC)

Total cost

€ 1 499 162,50

EU contribution

€ 1 499 162,50

Coordinated by

PHILIPPS UNIVERSITAET MARBURG
Germany

Periodic Reporting for period 1 - NAD-ART (Conjugation of NAD-capped RNAs to proteins by ADP-ribosyltransferases to generate RNA therapeutics)

Reporting period: 2024-01-01 to 2026-06-30

Summary of the context and overall objectives of the project

The ERC project "NAD-ART" investigates a new way for cells to “glue” RNA molecules onto proteins and asks how we can use this natural process to improve future medicines. RNA is often called a genetic messenger, but on its own it is fragile, hard to deliver into cells, and can trigger unwanted immune reactions. By attaching RNA to proteins in a controlled, natural way, we hope to make RNA messages more stable, easier to deliver, and more precisely targeted inside the body.
In the NAD-ART project, we first want to understand in detail how this RNA–protein “glue” works and which enzymes carry it out. Then we will build modular RNA–protein building blocks in the lab and test how well they enter human cells, how long they last, and how safely they act there. In the long term, this knowledge could help develop a new class of RNA-based drugs and vaccines that are more effective and have fewer side effects.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

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.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The project delivers a novel, fully biochemical conjugation strategy that uses a single natural enzyme to generate biologically recognised N‑glycosidic bonds between nucleic acids and proteins at a defined site, overcoming major limitations of chemical and tag‑based methods. Core results include broad ModB substrate promiscuity towards synthetic NXD‑capped RNAs/DNAs, a modular SpyTag R3K–rS1 DII / SpyCatcher system for precise single‑site modification, and cap‑dependent tuning of conjugate stability and enzymatic reversibility.
These findings create a versatile toolbox for interrogating and controlling translation, transcription and genome editing via covalent nucleic‑acid attachment, and they open avenues for next‑generation mRNA/DNA therapeutics, targeted delivery systems and diagnostics. Patent protection and initial industrial interactions indicate clear translational potential and interest in deploying the NAD‑ART platform beyond academia.
For further uptake, key needs include: systematic translation of in vitro conjugate designs into defined cellular functions, optimisation of intracellular trafficking and subcellular targeting, comprehensive profiling of innate immune responses, and scale‑up of conjugate production towards GMP‑compatible workflows. Additional support for process development, regulatory and standardisation aspects, IPR management and early‑stage commercialisation will be important to realise the full impact of RNAylation/DNAylation technology.