Real-time monitoring of ADAT activity and inhibition using newly developed emissive nucleotides

• Applicant: Dr. Julia Dietzsch
• Subject Area: Biological and Biomimetic Chemistry
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 535949581

tRNA is known to contain a significant amount of modified nucleosides, with inosine being the most abundant one. Here, inosine is introduced through enzymatic deamination by adenosine deaminases acting on tRNA (ADATs). Although it is known that A-to-I editing plays a role in tRNA maturation and strongly impacts human pathologies, many open questions remain including the exact biological function of inosine in the tRNA anticodon loop and the influence of ADAT mutations on the emergence of neurodevelopmental disorders. However, most reported biochemical and molecular methods to monitor adenosine deamination rely on radioisotope labeling or cDNA sequencing, which both suffer from drawbacks such as time-consuming sample preparation and low sensitivity. Therefore, the aim of the proposed project is the development of fluorescence-based methods for real-time monitoring of ADAT activity and inhibition to facilitate the establishment of high throughput screening assays for the discovery of ADAT enhancers and inhibitors. To achieve efficient fluorescence readout, emissive isomorphic nucleoside analogs will directly be incorporated into different positions of the anticodon loop of naturally occurring tRNA constructs. For this specific application, thieno and isothiazolo pyrimidines (thN and tzN) belonging to the emissive nucleobase alphabet developed in the lab of Yitzhak Tor are promising candidates as these nucleobase analogs have been reported to exhibit spectroscopically distinct features upon deamination. The respective nucleosides will be synthesized as phosphoramidite building blocks for incorporation into different positions of the required tRNA constructs by solid phase synthesis or as triphosphates for enzymatic incorporation. Spectroscopic characterization of these modified systems will provide important photophysical parameters and subsequent in vitro deamination experiments are expected to reveal the most suitable position to be modified to efficiently monitor A-to-I editing. Furthermore, subsequent experiments aim to the elucidation of the biochemical role of inosine methylation after deamination as well as to shed light on the biological significance of tRNA fragments arising from anticodon cleavage. In a more advanced stage of the project, the emissive tRNAs will be used for biochemical assays to identify potential ADAT enhancers and inhibitors in vivo with the long-term goal to track tRNA and tRNA fragments spectroscopically. The second part of the proposed projects, which will be carried out in parallel, includes the design, synthesis and characterization of new emissive nucleoside analogs with improved spectroscopic properties. Based on the structure of the antiviral nucleoside Remdesivir, these new compounds are expected to exhibit a deamination-sensitive spectroscopic performance and might therefore be promising candidates to replace thN and tzN nucleotides in in vitro and later in vivo deamination assays.

• DFG Programme: WBP Position