SI-MEPSI · When Biotechnology Meets Chemistry: Metabolic and Chemical Stable Isotope Labelling of RNA Building Blocks for the Modular Synthesis of (2H/13C/15N)-N1-Methylpseudouridine (m1Ψ) compounds

When Biotechnology Meets Chemistry: Metabolic and Chemical Stable Isotope Labelling of RNA Building Blocks for the Modular Synthesis of (2H/13C/15N)-N1-Methylpseudouridine (m1Ψ) compounds

The conversion of renewable resources into valuable products is one of the main challenges of our society. In this project, we started from inorganic reagents and utilised CO2 fixation to convert relatively inexpensive materials into fine chemicals by combining the strengths of biotechnology, chemistry, and enzymatic synthesis. In the end, we produced valuable stable-isotope-labelled (SI-labelled) ribonucleic acid (RNA) derivatives as the main products for the applications in nuclear magnetic resonance (NMR) spectroscopy. For example, labelling RNAs with stable isotopes can remove unwanted signals and reduce the complexity of NMR data. As a result, it is much easier to study biological systems and structures relevant for advancing the field of RNA therapeutics. However, the accessibility of SI-labelled RNA biomolecules is limited because the common strategies of obtaining them are often very expensive, especially for RNAs containing modifications. Here, we thus focused on developing an affordable method of isotopically labelling two in-demand RNA modifications: pseudouridine and N1-methylpseudouridine. These two RNA modifications were the key players in developing effective mRNA vaccines during the COVID-19 pandemic. In particular, N1-methylpseudouridine enabled high vaccine efficiency and decreased immunogenicity. To facilitate prospective biological NMR studies of these modified RNAs, the overall target of this project was to profitably label pseudouridine-based derivatives with stable isotopes, produce them on a gram scale, and introduce them to the market. To achieve our objectives, we first investigated H2/O2/CO2-based autotrophic fermentations with the bacterium Cupriavidus necator as a way to profitably label biomolecules with stable isotopes. Since C. necator can grow on a mixture of gases in an aqueous medium containing only inorganic salts, even isotopically labelled analogues of these materials are relatively inexpensive compared to the value of the obtained products. However, such fermentations are traditionally performed under explosive mixtures of H2/O2 gases and require very expensive equipment and extensive safety measures. Hence, only small bioreactors can be used and only multi-gram quantities of SI-labelled biomass can be produced under these conditions. We thus optimised the gas ratios in the fermentation using more sustainable, non-explosive conditions to allow the upscaling of our production of SI-labelled biomolecules. By fulfilling this aim, we produced enough SI-labelled biomass for obtaining multi-gram quantities of SI-labelled RNAs. We then converted these RNA molecules to individual RNA building blocks (nucleotides) and in turn used these as starting materials in a straightforward, versatile, and convergent chemo-enzymatic synthesis of pseudouridine-based derivatives.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз