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.

View original source ↗Export metadata ↓

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.

Explore related subjects

molecular biology Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

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

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Integrated Spatial Metabolomics and Proteomics from the Same Tissue Section Using a Conductive ITO-PET Slide

Integrating spatial metabolomics and spatial proteomics on the same tissue section remains challenging because matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) and laser capture microdissection (LCM)-based proteomics impose different requirements on sample slides. Here, we developed and systematically evaluated a conductive indium tin oxide-coated polyethylene terephthalate (ITO-PET) slide that enables sequential MALDI-MSI and LCM-liquid chromatography-mass spectrometry (LCM-LC-MS) analysis of the same tissue section. Using mouse brain tissue as a model, ITO-PET provided MALDI-MSI performance closely comparable to conventional ITO-glass, including spectral concordance (Pearson correlation, R = 0.90), ion detection coverage, metabolite annotation, signal intensity distribution, and preservation of spatial molecular patterns. Following MALDI-MSI, the ITO-PET slide enabled cutting-mode LCM and yielded proteomic signal intensities and numbers of identified protein groups comparable to those obtained with conventional PEN-glass slides. Across different tissue sampling areas, proteomic signal intensity distributions, precursor ion counts, and protein group identifications remained broadly comparable before and after MALDI-MSI, with substantial overlap in identified protein groups. Similar patterns were observed in mouse kidney, lung, spleen, and liver tissues, further supporting the applicability of the workflow across different tissue types. By combining the electrical conductivity required for MALDI-MSI with the mechanical properties required for LCM cutting, the ITO-PET slide addresses a major material incompatibility between the two analytical modalities and enables sequential spatial metabolomic and proteomic analysis from the same tissue section. This workflow provides a practical analytical platform for obtaining complementary molecular information from spatially limited biological specimens.

molecular biology↗

bioRxiv2026

Aβ42-Driven α-synuclein Fibril Polymorphism and Distinct Intracellular Aggregation

The frequent coexistence of -synuclein (-syn) and amyloid-{beta} (A{beta}) aggregates in neurodegenerative diseases suggests that heterotypic interactions between these amyloidogenic proteins may influence disease progression, yet their molecular consequences remain poorly understood. Here, we investigated how distinct aggregation states of A{beta}42, monomers and preformed fibrils (PFFs), modulate -syn fibril formation, structure, and downstream neuronal pathology. Thioflavin T kinetics showed that A{beta}42 monomers delayed -syn fibril formation, whereas A{beta}42 PFFs exhibited a trend toward accelerated aggregation, indicating aggregation state-dependent effects on -syn aggregation. Negative-stain TEM, proteinase K digestion, and solid-state NMR spectroscopy further demonstrated that both A{beta}42 monomers and PFFs altered -syn fibril structure, generating distinct fibril conformations depending on the A{beta}42 concentration and aggregation state. To determine whether these conformational differences influence pathological activity, -syn PFF variants generated in the presence of different concentrations of A{beta}42 monomers or PFFs were applied to dopaminergic neuronal cells. -Syn fibrils formed in the presence of A{beta}42 PFFs showed greater capacity to induce intraneuronal -syn aggregation than -syn PFFs, whereas fibrils formed in the presence of A{beta}42 monomers exhibited similar or reduced seeding capacity relative to -syn PFFs. Together, our findings demonstrate that heterotypic interactions with A{beta}42 reshape -syn aggregation pathways and fibril conformations, generating structurally distinct -syn fibril populations with different neuronal seeding activities. These results provide a molecular framework for understanding how cross-talk between amyloidogenic proteins may contribute to structural and pathological heterogeneity in mixed neurodegenerative diseases.

molecular biology↗

bioRxiv2026

Insulators As Dynamic, Tunable Regulators of Enhancer-Promoter Coordination in Living Drosophila Embryos

The three-dimensional organization of the genome enables enhancers and promoters to interact across vast distances and direct transcription. Yet whether architectural elements, such as insulators, serve as rigid, passive barriers or as dynamic, active organizers of this communication remains unclear. Here, using single-cell, live imaging of a Drosophila transgene in which a single enhancer regulates two equidistant promoters, we confirm that the enhancer engages both promoters simultaneously and show that coordinated bursting is intrinsically more productive than uncoordinated activity. Flanking this system with insulators increases coordinated bursting frequency and transcriptional output, indicating that insulator-mediated looping promotes multi-way enhancer-promoter interaction. Further, bidirectionally-paired, homotypic insulators produce stronger coordination than unidirectional pairs. Inserting an intermediate insulator to generate competing loop configurations, together with two-state promoter modeling, we show that these chromatin loops are highly dynamic. This work reframes insulators as active, tunable regulators that shape the frequency, coordination, and productivity of enhancer-promoter communication.

molecular biology↗