mRNA Display Pipeline for Protein Biosensor Construction

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.