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bioRxiv · 10.1101/2025.02.07.637080

Kinetic characterization of three human DExD/H-box RNA helicases

Li, F.·Chan, U. H.·Perez, J. G.·Zeng, H.·Chau, I.·Li, Y.·Seitova, A.·Halabelian, L.
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Abstract

Human DExD/H-box RNA helicases are ubiquitous molecular motors that unwind and rearrange RNA secondary structures in an ATP-dependent manner. These enzymes play essential roles in nearly all aspects of RNA metabolism. While their biological functions are well-characterized, the kinetic mechanisms remain relatively understudied in vitro. In this study, we describe the development and optimization of a bioluminescence-based assay to kinetically characterize three human RNA helicases: MDA5, LGP2, and DDX1. The assays were conducted using annealed 24-mer RNA (blunt-ended double-stranded RNA) or double-stranded RNA (ds-RNA) with a 25- nt 3' overhang. These findings establish a robust and high-throughput in vitro assay suitable for a 384-well format, enabling the discovery and characterization of inhibitors targeting MDA5, LGP2, and DDX1. This work provides a valuable resource for advancing our understanding of these helicases and their therapeutic potential in Alzheimers disease.

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Li, F., Chan, U. H., Perez, J. G., Zeng, H., Chau, I., Li, Y., Seitova, A., Halabelian, L.. 2025-02-07. Kinetic characterization of three human DExD/H-box RNA helicases. https://doi.org/10.1101/2025.02.07.637080

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