DNA基因探针由于其可编程性和生物适应性而受到了广泛关注，成为生化分析的多功能工具。然而，大多数现有的DNA基因探针依赖荧光作为信号输出，当应用于复杂的生物材料（如活体细胞或组织）时，可能会出现自发荧光和散射等问题。在本文中，我们报告了生物发光核酸（bioLUNA）传感器的开发，该传感器提供了独立于激光激发的比色成像功能，可应用于体内目标成像。该系统基于计算建模和突变研究，使用环状重排Nano-luciferase（NLuc）和HaloTag之间的基因融合，使蛋白质能够与DNA酶结合。在存在Zn2+时，DNA酶传感器释放带有荧光标记的链，导致荧光素与发光素之间的生物发光共振能量转移（BRET）减少。因此，这个过程引起生物发光信号的比色变化。我们证明了这种bioLUNA传感器能够在体内成像外源性Zn2+和正常上皮前列腺及前列腺肿瘤中内源性Zn2+的流出。这项工作将DNA酶传感器扩展到了使用生物发光，从而丰富了广泛的生物医学应用的核酸传感器工具箱。 ©2023 Wiley-VCH Verlag GmbH.

DNA-based probes have gained significant attention as versatile tools for biochemical analysis, benefiting from their programmability and biocompatibility. However, most existing DNA-based probes rely on fluorescence as the signal output, which can be problematic due to issues like autofluorescence and scattering when applied in complex biological materials such as living cells or tissues. Herein, we report the development of bioluminescent nucleic acid (bioLUNA) sensors that offer laser excitation-independent and ratiometric imaging of the target in vivo. The system is based on computational modelling and mutagenesis investigations of a genetic fusion between circular permutated Nano-luciferase (NLuc) and HaloTag, enabling the conjugation of the protein with a DNAzyme. In the presence of Zn2+ , the DNAzyme sensor releases the fluorophore-labelled strand, leading to a reduction in bioluminescent resonance energy transfer (BRET) between the luciferase and fluorophore. Consequently, this process induces ratiometric changes in the bioluminescent signal. We demonstrated that this bioLUNA sensor enabled imaging of both exogenous Zn2+ in vivo and endogenous Zn2+ efflux in normal epithelial prostate and prostate tumors. This work expands the DNAzyme sensors to using bioluminescence and thus has enriched the toolbox of nucleic acid sensors for a broad range of biomedical applications.© 2023 Wiley-VCH Verlag GmbH.