The generation and validation of a de novo-designed titin binder for use in fluorescence microscopy

Abstract Advances in the generation of proteins in silico has enabled the efficient design of target-specific binders. Here, we demonstrate the use of a de novo-designed protein targeting the C-terminal domain, M10, of the giant muscle protein titin in situ and image its localisation using fluorescence microscopy, a widely used technique that has relied on antibodies or similar binders to identify the presence and location of targets in their native environment. Confocal microscopy of muscle sections shows the binder localizes to the M-band of the sarcomere – where M10 is found – and fails to label muscle in competition experiments and in mutant muscle where M10 is absent. This new approach to identifying the presence and location of targets in their native environment avoids the need to generate binders in vivo or in vitro which can have technical, financial and ethical challenges.