Aurora 2.0: A fluorogenic dye library for expanding the capability of protein-adaptive differential scanning fluorimetry (paDSF)
Differential Scanning Fluorimetry (DSF) is a biophysical assay that is used to estimate protein stability in vitro. In a DSF experiment, the increased fluorescence of a solvatochromatic dye, such as Sypro Orange, is used to detect the unfolding of a protein during heating. However, Sypro Orange is o...
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Elsevier
2025-09-01
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| Series: | SLAS Discovery |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2472555225000528 |
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| author | Annemarie F. Charvat Kayleigh Mason-Chalmers Aneta Grabinska-Rogala Shloka Shivakumar Zachary Gale-Day Taiasean Wu Zoe Millbern Jonathan B. Grimm Emma C. Carroll K․ Peter R․ Nilsson Luke D. Lavis Nelson R. Vinueza Jason E. Gestwicki |
| author_facet | Annemarie F. Charvat Kayleigh Mason-Chalmers Aneta Grabinska-Rogala Shloka Shivakumar Zachary Gale-Day Taiasean Wu Zoe Millbern Jonathan B. Grimm Emma C. Carroll K․ Peter R․ Nilsson Luke D. Lavis Nelson R. Vinueza Jason E. Gestwicki |
| author_sort | Annemarie F. Charvat |
| collection | DOAJ |
| description | Differential Scanning Fluorimetry (DSF) is a biophysical assay that is used to estimate protein stability in vitro. In a DSF experiment, the increased fluorescence of a solvatochromatic dye, such as Sypro Orange, is used to detect the unfolding of a protein during heating. However, Sypro Orange is only compatible with a minority of proteins (< 30 %), limiting the scope of this method. We recently reported that protein-adaptive DSF (paDSF) can partially solve this problem, wherein the protein is initially pre-screened against ∼300 chemically diverse dyes, termed the Aurora collection. While this approach significantly improves the number of targets amenable to DSF, it still fails to produce protein-dye pairs for some proteins. Here, we report the expansion of the dye collection to Aurora 2.0, which includes a total of 517 structurally diverse molecules and multiple new chemotypes. To assess performance, these dyes were screened against a panel of ∼100 proteins, which were selected, in part, to represent the most challenging targets (e.g. small size). From this effort, Aurora 2.0 achieved an impressive success rate of 94 %, including producing dyes for some targets that were not matched in the original collection. These findings support the idea that larger, more chemically diverse libraries improve the likelihood of detecting melting transitions across a wider range of proteins. We propose that Aurora 2.0 makes paDSF an increasingly powerful method for studying protein stability, ligand binding and other biophysical properties in high throughput. |
| format | Article |
| id | doaj-art-51d922a421ae450fa2d587d9ba35e4b4 |
| institution | Kabale University |
| issn | 2472-5552 |
| language | English |
| publishDate | 2025-09-01 |
| publisher | Elsevier |
| record_format | Article |
| series | SLAS Discovery |
| spelling | doaj-art-51d922a421ae450fa2d587d9ba35e4b42025-08-20T03:43:44ZengElsevierSLAS Discovery2472-55522025-09-013510025910.1016/j.slasd.2025.100259Aurora 2.0: A fluorogenic dye library for expanding the capability of protein-adaptive differential scanning fluorimetry (paDSF)Annemarie F. Charvat0Kayleigh Mason-Chalmers1Aneta Grabinska-Rogala2Shloka Shivakumar3Zachary Gale-Day4Taiasean Wu5Zoe Millbern6Jonathan B. Grimm7Emma C. Carroll8K․ Peter R․ Nilsson9Luke D. Lavis10Nelson R. Vinueza11Jason E. Gestwicki12Institute for Neurodegenerative Diseases, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA 94158, USAInstitute for Neurodegenerative Diseases, University of California, San Francisco, San Francisco, CA 94158, USAInstitute for Neurodegenerative Diseases, University of California, San Francisco, San Francisco, CA 94158, USAInstitute for Neurodegenerative Diseases, University of California, San Francisco, San Francisco, CA 94158, USAInstitute for Neurodegenerative Diseases, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA 94158, USAInstitute for Neurodegenerative Diseases, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA 94158, USADepartment of Textile Engineering, Chemistry and Science, North Carolina State University, Raleigh, NC 27695, USAJanelia Research Campus, Howard Hughes Medical InstituteInstitute for Neurodegenerative Diseases, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA 94158, USA; Department of Chemistry, San José State University, San Jose, CA 95112, USAScience for Life Laboratory, KTH—Royal Institute of Technology SE-171 21 Stockholm, SwedenJanelia Research Campus, Howard Hughes Medical InstituteDepartment of Textile Engineering, Chemistry and Science, North Carolina State University, Raleigh, NC 27695, USAInstitute for Neurodegenerative Diseases, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA 94158, USA; Corresponding author at: University of California San Francisco, Sandler Center, 675 Nelson Rising Lane, Room 311, San Francisco CA 94158, USA.Differential Scanning Fluorimetry (DSF) is a biophysical assay that is used to estimate protein stability in vitro. In a DSF experiment, the increased fluorescence of a solvatochromatic dye, such as Sypro Orange, is used to detect the unfolding of a protein during heating. However, Sypro Orange is only compatible with a minority of proteins (< 30 %), limiting the scope of this method. We recently reported that protein-adaptive DSF (paDSF) can partially solve this problem, wherein the protein is initially pre-screened against ∼300 chemically diverse dyes, termed the Aurora collection. While this approach significantly improves the number of targets amenable to DSF, it still fails to produce protein-dye pairs for some proteins. Here, we report the expansion of the dye collection to Aurora 2.0, which includes a total of 517 structurally diverse molecules and multiple new chemotypes. To assess performance, these dyes were screened against a panel of ∼100 proteins, which were selected, in part, to represent the most challenging targets (e.g. small size). From this effort, Aurora 2.0 achieved an impressive success rate of 94 %, including producing dyes for some targets that were not matched in the original collection. These findings support the idea that larger, more chemically diverse libraries improve the likelihood of detecting melting transitions across a wider range of proteins. We propose that Aurora 2.0 makes paDSF an increasingly powerful method for studying protein stability, ligand binding and other biophysical properties in high throughput.http://www.sciencedirect.com/science/article/pii/S2472555225000528Thermal shift assayThermofluorProtein stabilityBinding assayHigh throughput screeningFluorescent probes |
| spellingShingle | Annemarie F. Charvat Kayleigh Mason-Chalmers Aneta Grabinska-Rogala Shloka Shivakumar Zachary Gale-Day Taiasean Wu Zoe Millbern Jonathan B. Grimm Emma C. Carroll K․ Peter R․ Nilsson Luke D. Lavis Nelson R. Vinueza Jason E. Gestwicki Aurora 2.0: A fluorogenic dye library for expanding the capability of protein-adaptive differential scanning fluorimetry (paDSF) SLAS Discovery Thermal shift assay Thermofluor Protein stability Binding assay High throughput screening Fluorescent probes |
| title | Aurora 2.0: A fluorogenic dye library for expanding the capability of protein-adaptive differential scanning fluorimetry (paDSF) |
| title_full | Aurora 2.0: A fluorogenic dye library for expanding the capability of protein-adaptive differential scanning fluorimetry (paDSF) |
| title_fullStr | Aurora 2.0: A fluorogenic dye library for expanding the capability of protein-adaptive differential scanning fluorimetry (paDSF) |
| title_full_unstemmed | Aurora 2.0: A fluorogenic dye library for expanding the capability of protein-adaptive differential scanning fluorimetry (paDSF) |
| title_short | Aurora 2.0: A fluorogenic dye library for expanding the capability of protein-adaptive differential scanning fluorimetry (paDSF) |
| title_sort | aurora 2 0 a fluorogenic dye library for expanding the capability of protein adaptive differential scanning fluorimetry padsf |
| topic | Thermal shift assay Thermofluor Protein stability Binding assay High throughput screening Fluorescent probes |
| url | http://www.sciencedirect.com/science/article/pii/S2472555225000528 |
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