Caspases are a family of evolutionary conserved cysteine proteases that play key roles in programmed cell death and inflammation. Among the methods for the detection of caspase activity, biosensors based on luciferases have advantages in genetical encoding and convenience in assay. In this study, we constructed a new set of caspase biosensors based on NanoLuc luciferase. This kind of sensors, named NanoLock, work in dark-to-bright model, with the help of a NanoLuc quencher peptide (HiBiT-R/D) mutated from HiBiT. Optimized NanoLock responded to proteases with high signal to noise ratio (S/N), 1233-fold activation by tobacco etch virus protease in HEK293 cells and > 500-fold induction to caspase 3 in vitro. We constructed NanoLocks for the detection of caspase 1, 3, 6, 7, 8, 9, and 10, and assays in HEK293 cells demonstrated that these sensors performed better than commercial kits in the aspect of S/N and convenience. We further established a cell line stably expressing NanoLock-casp 6 and provided a proof-of-concept for the usage of this cell line in the high throughput screening of caspase 6 modulator.

半胱天冬酶是进化保守的半胱氨酸蛋白酶家族，在程序性细胞死亡和炎症中起关键作用。在检测半胱天冬酶活性的方法中，基于荧光素酶的生物传感器在基因编码和测定方便等方面具有优势。在这项研究中，我们构建了一套新的基于 NanoLuc 荧光素酶的半胱天冬酶生物传感器。这种名为 NanoLock 的传感器在从 HiBiT 突变的 NanoLuc 猝灭肽 (HiBiT-R/D) 的帮助下工作在从暗到亮的模型中。优化的 NanoLock 以高信噪比 (S/N) 响应蛋白酶，在 HEK293 细胞中被烟草蚀刻病毒蛋白酶激活 1233 倍，体外对半胱天冬酶 3 的诱导超过 500 倍。我们构建了用于检测 caspase 1、3、6、7、8、9 和 10 的 NanoLock，和 HEK293 细胞中的测定表明，这些传感器在 S/N 和便利性方面优于商业试剂盒。我们进一步建立了稳定表达 NanoLock-casp 6 的细胞系，并为该细胞系在 caspase 6 调节剂的高通量筛选中的应用提供了概念验证。