LuxSit Pro and sPro: Next-Generation Designed Luciferases for Bioluminescent Reporting and Complementation-Based Detection

Vergara, R.; Wagner, J.; Harman, J. L.; Kamalian, P.; Lara, N.; Fischer, E. S.; Bernardes, G. J. L.; Quijano-Rubio, A.; Silva, D.-A.

doi:10.1101/2025.07.21.665742

bioRxiv · 10.1101/2025.07.21.665742

LuxSit Pro and sPro: Next-Generation Designed Luciferases for Bioluminescent Reporting and Complementation-Based Detection

Vergara, R.·Wagner, J.·Harman, J. L.·Kamalian, P.·Lara, N.·Fischer, E. S.·Bernardes, G. J. L.·Quijano-Rubio, A.·Silva, D.-A.

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Abstract

Bioluminescent systems are widely used as molecular reporters in life sciences due to their low background, broad dynamic range, and the key advantage of not requiring external excitation. However, natural luciferases often lack the brightness, stability, and long-lasting light emission needed to support a wide range of high-sensitivity applications. Here, we describe our next-generation luciferase solution: LuxSit Pro and its substrate, Luxterazine (LTZ). LuxSit Pro is a de novo luciferase optimized for steady brightness and structural stability through an iterative combination of computational and experimental methods. Composed of only 117 amino acids, LuxSit Pro is currently the smallest highest-performing known luciferase, exhibiting exceptional brightness and biochemical stability with robust recombinant expression in both mammalian and bacterial systems. Notably, LuxSit Pro was deliberately designed devoid of lysine and cysteine residues which enormously expands its potential applications. This property renders the reporter largely resistant to post-translational modifications such as ubiquitination and also enables site-directed chemical modifications, as lysines or cysteines can be reintroduced at specific sites to serve as chemical handles for augmenting or adapting the enzymes function. The novel synthetic substrate, Luxterazine (LTZ), is a compound that enables LuxSit Pro to deliver bright, steady, blue light emission ([~]490 nm) and offers improved solubility compared to existing Coelenterazine analogs. Finally, we also developed LuxSit sPro, a highly sensitive two-component complementation system for the quantitative detection of molecular interactions. The broad potential applications of LuxSit Pro and sPro, in combination with Luxterazine, are poised to drive the development of next-generation reporters and biosensor systems for the research and in vitro diagnostics fields.

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Vergara, R., Wagner, J., Harman, J. L., Kamalian, P., Lara, N., Fischer, E. S., Bernardes, G. J. L., Quijano-Rubio, A., Silva, D.-A.. 2025-07-24. LuxSit Pro and sPro: Next-Generation Designed Luciferases for Bioluminescent Reporting and Complementation-Based Detection. https://doi.org/10.1101/2025.07.21.665742

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