mRNA Display Pipeline for Protein Biosensor Construction
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| Title: | mRNA Display Pipeline for Protein Biosensor Construction |
|---|---|
| Authors: | Cui, Zhenling, Ayva, Cagla Ergun, Liew, Yi Jin, Guo, Zhong, Mutschler, Roxane, Dreier, Birgit, Fiorito, Maria M, Walden, Patricia, Howard, Christopher B, Ely, Fernanda, Plückthun, Andreas, Pretorius, Carel, Ungerer, Jacobus Pj, Buckle, Ashley M, Alexandrov, Kirill |
| Contributors: | University of Zurich, Alexandrov, Kirill |
| Source: | ACS Sens |
| Publisher Information: | American Chemical Society (ACS), 2024. |
| Publication Year: | 2024 |
| Subject Terms: | 1502 Bioengineering, Calmodulin, 3105 Instrumentation, 1507 Fluid Flow and Transfer Processes, 10019 Department of Biochemistry, 570 Life sciences, biology, Humans, 610 Medicine & health, Biosensing Techniques, RNA, Messenger, 1508 Process Chemistry and Technology |
| Description: | Despite the significant potential of protein biosensors, their construction remains a trial-and-error process. The most obvious approach for addressing this is to utilize modular biosensor architectures where specificity-conferring modalities can be readily generated to recognize new targets. Toward this goal, we established a workflow that uses mRNA display-based selection of hyper-stable monobody domains for the target of choice or ribosome display to select equally stable DARPins. These binders were integrated into a two-component allosteric biosensor architecture based on a calmodulin-reporter chimera. This workflow was tested by developing biosensors for liver toxicity markers such as cytosolic aspartate aminotransferase, mitochondrial aspartate aminotransferase, and alanine aminotransferase 1. We demonstrate that our pipeline consistently produced >103 unique binders for each target within a week. Our analysis revealed that the affinity of the binders for their targets was not a direct predictor of the binder's performance in a biosensor context. The interactions between the binding domains and the reporter module affect the biosensor activity and the dynamic range. We conclude that following binding domain selection, the multiplexed biosensor assembly and prototyping appear to be the most promising approach for identifying biosensors with the desired properties. |
| Document Type: | Article Other literature type |
| File Description: | ZORA261909.pdf - application/pdf |
| Language: | English |
| ISSN: | 2379-3694 |
| DOI: | 10.1021/acssensors.3c02471 |
| DOI: | 10.5167/uzh-261909 |
| Access URL: | https://pubmed.ncbi.nlm.nih.gov/38807313 |
| Rights: | CC BY NC ND |
| Accession Number: | edsair.doi.dedup.....2b067d8145890c3d2d3f5ebfcfccb7b5 |
| Database: | OpenAIRE |
Nájsť tento článok vo Web of Science | Abstract: | Despite the significant potential of protein biosensors, their construction remains a trial-and-error process. The most obvious approach for addressing this is to utilize modular biosensor architectures where specificity-conferring modalities can be readily generated to recognize new targets. Toward this goal, we established a workflow that uses mRNA display-based selection of hyper-stable monobody domains for the target of choice or ribosome display to select equally stable DARPins. These binders were integrated into a two-component allosteric biosensor architecture based on a calmodulin-reporter chimera. This workflow was tested by developing biosensors for liver toxicity markers such as cytosolic aspartate aminotransferase, mitochondrial aspartate aminotransferase, and alanine aminotransferase 1. We demonstrate that our pipeline consistently produced >103 unique binders for each target within a week. Our analysis revealed that the affinity of the binders for their targets was not a direct predictor of the binder's performance in a biosensor context. The interactions between the binding domains and the reporter module affect the biosensor activity and the dynamic range. We conclude that following binding domain selection, the multiplexed biosensor assembly and prototyping appear to be the most promising approach for identifying biosensors with the desired properties. |
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| ISSN: | 23793694 |
| DOI: | 10.1021/acssensors.3c02471 |