A BRET-based Mpro biosensor containing a nanobody and tandem cleavage sites shows an increased cleavage rate
Here, we report the engineering of a Bioluminescence Resonance Energy Transfer (BRET)-based SARS-CoV-2 main protease (Mpro) biosensor containing the Mpro N-terminal autocleavage sequence in tandem and a nanobody that shows an enhanced rate of Mpro-mediated proteolytic cleavage. Specifically, we desi...
محفوظ في:
| المؤلف الرئيسي: | |
|---|---|
| مؤلفون آخرون: | , , , , , , , , , |
| التنسيق: | article |
| منشور في: |
2025
|
| الموضوعات: | |
| الوصول للمادة أونلاين: | http://dx.doi.org/10.1016/j.snr.2025.100315 https://www.sciencedirect.com/science/article/pii/S2666053925000335 http://hdl.handle.net/10576/65127 |
| الوسوم: |
إضافة وسم
لا توجد وسوم, كن أول من يضع وسما على هذه التسجيلة!
|
| الملخص: | Here, we report the engineering of a Bioluminescence Resonance Energy Transfer (BRET)-based SARS-CoV-2 main protease (Mpro) biosensor containing the Mpro N-terminal autocleavage sequence in tandem and a nanobody that shows an enhanced rate of Mpro-mediated proteolytic cleavage. Specifically, we designed Mpro biosensors containing 2×, 4× and 8× repeats of Mpro N-terminal autocleavage sequences and a combination of Mpro cleavage sequences containing a total of 12 cleavage sites sandwiched between mNeonGreen (mNG) and NanoLuc (NLuc). Gaussian accelerated molecular dynamics (GaMD) simulations of the predicted alpha-helical synthetic Mpro cleavage sequences revealed a dynamic nature of the cleavage sequences, which is critical for their efficient cleavage, and a relatively short end-to-end distances, which is required for high BRET. Live cell assays revealed a cleavage sequence length-dependent resonance energy transfer, except for the 12× -syn cleavage site, and an increased rate of cleavage and a decreased pharmacological inhibitor efficacy for the Mpro biosensor containing 2× cleavage sequences. Further, mutational analysis revealed a requirement for both cleavage sites to be intact for increased cleavage rate. Importantly, the inclusion of an Mpro-binding, but non-inhibiting, NB2E3 nanobody at the N-terminal further increased the cleavage rate of the 2× cleavage sequence-containing Mpro biosensor. We envisage that the NB2E3 nanobody-2× Mpro biosensor engineered here will be useful in drug discovery and functional characterization of Mpro mutants in newly emerging SARS-CoV-2 variants as well as in detecting SARS-CoV-2 infection in a point-of-care testing (POCT) format. |
|---|