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...
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2025
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| Online Access: | http://dx.doi.org/10.1016/j.snr.2025.100315 https://www.sciencedirect.com/science/article/pii/S2666053925000335 http://hdl.handle.net/10576/65127 |
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| _version_ | 1857415086794080256 |
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| author | Geethakumari, Anupriya M |
| author2 | Sultana, Asfia Fatima, Asma Uddin, S M Nasir Abdulhakim, Somaiya Mohamed, Amera Rahman, Samiha Al-Buainain, Khaloud Yassine, Hadi M Khatib, Hebah A Al Biswas, Kabir H |
| author2_role | author author author author author author author author author author |
| author_facet | Geethakumari, Anupriya M Sultana, Asfia Fatima, Asma Uddin, S M Nasir Abdulhakim, Somaiya Mohamed, Amera Rahman, Samiha Al-Buainain, Khaloud Yassine, Hadi M Khatib, Hebah A Al Biswas, Kabir H |
| author_role | author |
| dc.creator.none.fl_str_mv | Geethakumari, Anupriya M Sultana, Asfia Fatima, Asma Uddin, S M Nasir Abdulhakim, Somaiya Mohamed, Amera Rahman, Samiha Al-Buainain, Khaloud Yassine, Hadi M Khatib, Hebah A Al Biswas, Kabir H |
| dc.date.none.fl_str_mv | 2025-05-22T09:57:08Z 2025-06-30 |
| dc.format.none.fl_str_mv | application/pdf |
| dc.identifier.none.fl_str_mv | http://dx.doi.org/10.1016/j.snr.2025.100315 26660539 https://www.sciencedirect.com/science/article/pii/S2666053925000335 http://hdl.handle.net/10576/65127 9 |
| dc.language.none.fl_str_mv | en |
| dc.publisher.none.fl_str_mv | Elsevier |
| dc.rights.none.fl_str_mv | http://creativecommons.org/licenses/by/4.0/ info:eu-repo/semantics/openAccess |
| dc.subject.none.fl_str_mv | Biosensor BRET Molecular dynamics simulation Mpro Nanobody SARS-CoV-2 |
| dc.title.none.fl_str_mv | A BRET-based Mpro biosensor containing a nanobody and tandem cleavage sites shows an increased cleavage rate |
| dc.type.none.fl_str_mv | Article info:eu-repo/semantics/publishedVersion info:eu-repo/semantics/article |
| description | 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. |
| eu_rights_str_mv | openAccess |
| format | article |
| id | qu_c2dd9b8c257271f00b12535a64d84693 |
| identifier_str_mv | 26660539 9 |
| language_invalid_str_mv | en |
| network_acronym_str | qu |
| network_name_str | Qatar University repository |
| oai_identifier_str | oai:qspace.qu.edu.qa:10576/65127 |
| publishDate | 2025 |
| publisher.none.fl_str_mv | Elsevier |
| repository.mail.fl_str_mv | |
| repository.name.fl_str_mv | |
| repository_id_str | |
| rights_invalid_str_mv | http://creativecommons.org/licenses/by/4.0/ |
| spelling | A BRET-based Mpro biosensor containing a nanobody and tandem cleavage sites shows an increased cleavage rateGeethakumari, Anupriya MSultana, AsfiaFatima, AsmaUddin, S M NasirAbdulhakim, SomaiyaMohamed, AmeraRahman, SamihaAl-Buainain, KhaloudYassine, Hadi MKhatib, Hebah A AlBiswas, Kabir HBiosensorBRETMolecular dynamics simulationMproNanobodySARS-CoV-2Here, 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.This work is supported by the Undergraduate Research Experience Program (UREP) grant (# 28–264–3–092) awarded by the Qatar National Research Foundation (QNRF), Qatar Foundation and an internal funding from the College of Health & Life Sciences (CHLS), Hamad Bin Khalifa University (HBKU), a member of the Qatar Foundation. Some of the computational research work reported in the manuscript were performed using high-performance computer resources and services provided by the Research Computing group in Texas A&M University at Qatar. Research Computing is funded by the Qatar Foundation for Education, Science and Community Development (http://www.qf.org.qa). A.M.G. was supported by a postdoctoral fellowship from CHLS/HBKU. A.S., A.F., and S.M.N.U. were supported by scholarships from CHLS/HBKU.Elsevier2025-05-22T09:57:08Z2025-06-30Articleinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://dx.doi.org/10.1016/j.snr.2025.10031526660539https://www.sciencedirect.com/science/article/pii/S2666053925000335http://hdl.handle.net/10576/651279enhttp://creativecommons.org/licenses/by/4.0/info:eu-repo/semantics/openAccessoai:qspace.qu.edu.qa:10576/651272025-05-22T19:06:58Z |
| spellingShingle | A BRET-based Mpro biosensor containing a nanobody and tandem cleavage sites shows an increased cleavage rate Geethakumari, Anupriya M Biosensor BRET Molecular dynamics simulation Mpro Nanobody SARS-CoV-2 |
| status_str | publishedVersion |
| title | A BRET-based Mpro biosensor containing a nanobody and tandem cleavage sites shows an increased cleavage rate |
| title_full | A BRET-based Mpro biosensor containing a nanobody and tandem cleavage sites shows an increased cleavage rate |
| title_fullStr | A BRET-based Mpro biosensor containing a nanobody and tandem cleavage sites shows an increased cleavage rate |
| title_full_unstemmed | A BRET-based Mpro biosensor containing a nanobody and tandem cleavage sites shows an increased cleavage rate |
| title_short | A BRET-based Mpro biosensor containing a nanobody and tandem cleavage sites shows an increased cleavage rate |
| title_sort | A BRET-based Mpro biosensor containing a nanobody and tandem cleavage sites shows an increased cleavage rate |
| topic | Biosensor BRET Molecular dynamics simulation Mpro Nanobody SARS-CoV-2 |
| url | http://dx.doi.org/10.1016/j.snr.2025.100315 https://www.sciencedirect.com/science/article/pii/S2666053925000335 http://hdl.handle.net/10576/65127 |