(A-30) Genetically Encoded Boolean Responsiveness for Logically Controlled Protein Release from Hydrogel Biomaterials

Introduction:: Advances towards the faithful recapitulation of biological processes ex vivo are largely predicated on the creation of next-generation constructs that are both dynamic and stimulus-responsive. Previous work from our lab has successfully engineered Boolean YES/OR/AND-based logical circuits into hydrogel biomaterial scaffolds through precise engineering of molecular crosslinks and tethers for controlled degradation ¹ and/or therapeutic delivery ^2,3. Though these methods were uniquely enabling in terms of biomaterial programmability, this initial approach suffered from its requirement on multi-step synthetic schemes that dramatically limited material yields and the method’s overall translatability. In this current work, we report methods to afford such Boolean biomaterials using intrinsically scalable recombinant protein-based methods with no required post-synthetic modification. Inspired by recent advances in protein semisynthesis, ligation chemistries, and biocomputation, we demonstrate the engineering of sequence-defined Boolean responsiveness – compatible with virtually any protease system – in a fully genetically encoded manner.


Materials and Methods:: Highlighting the ease and off-the-shelf applicability of our proposed method, all constructs used in this study were generated through bacterial fermentation processes and purified on an automated AKTA Pure FPLC unit through Immobilized Metal Affinity Chromatography (IMAC) and Size-Exclusion Chromatography (SEC). The purity of all engineered proteins was verified through SDS-PAGE and further validated by mass spectrometry. To demonstrate bio-responsiveness, Boolean YES/OR/AND-type constructs were expressed, purified, and treated with the requisite protease enzyme(s), in tandem or in series; cleavage products were in turn analyzed in the same manner.


Results, Conclusions, and Discussions:: Tethers representing all possible Boolean YES/OR/AND logic geometries from three signal sequences were designed, generated, and validated successfully. This work successfully harnesses advances made across a suite of chemical biology tools and applies them as (re)configurable elements tailored to the creation of logically responsive protein release systems. We successfully recapture all 17 distinct logical Boolean operations from the same starting set of protein assembly chemistries and 3 model proteolytic sequences that are largely absent from the mammalian proteome.

We successfully apply a suite of design and engineering principles derived from chemical biology towards the engineering of user-tunable and stimulus-responsive protein tethers. Our approach is entirely genetically encoded, rendering it particularly amenable for adoption by a variety of labs designing biomaterial scaffolds for disease modeling and treatment. Using our Boolean logic system, we can accommodate the modeling of environments that present any subset of possible signal combinations at target sites, all while keeping an eye towards translatability and ease-of-access. Promisingly, this platform can accommodate any number of potential proteolytic signals by (re)engineering of its initial constituent parts. Beyond its direct applicability and implication in biomaterials, this work represents the first example of a generalizable biocomputation formally engineered into protein chimeras through the genetic encoding and tailoring of resultant polypeptide geometries.


Acknowledgements (Optional): : RTG would like to acknowledge funding support from the John C. Berg Endowed Fellowship. Additional funding for this work was provided by the NIH (R35GM138036) and NSF (DMR 1652141).


References (Optional): : 1. Badeau, B. A., Comerford, M. P., Arakawa, C. K., Shadish, J. A. & Deforest, C. A. Engineered modular biomaterial logic gates for environmentally triggered therapeutic delivery. Nat. Chem. 10, 251–258 (2018).

2. Gawade, P. M., Shadish, J. A., Badeau, B. A. & DeForest, C. A. Logic-Based Delivery of Site-Specifically Modified Proteins from Environmentally Responsive Hydrogel Biomaterials. Adv. Mater. 31, 1–6 (2019).

3. Ruskowitz, E. R., Comerford, M. P., Badeau, B. A. & Deforest, C. A. Logical stimuli-triggered delivery of small molecules from hydrogel biomaterials. Biomater. Sci. 7, 542–546 (2019).