Materials Synthetic Biology

The pH dependence of the absorption and (time-resolved) fluorescence of two red-shifted fluorescent proteins, mCardinal and mNeptune, was investigated. Decay-associated spectra were measured following fluorescence excitation at 470 nm in PBS buffer with a pH that ranged from 5.5 to 8.0. The fluorescence of both proteins shows two different decay components. mCardinal exhibits an increase in the long-lived fluorescence component with acidification from 1.34 ns at pH 8.0 to 1.62 ns at pH 5.5. An additional fast decay component with 0.64 ns at pH 8.0 up to 1.1 ns at pH 5.5 was found to be blue-shifted compared to the long-lived component. The fluorescence lifetime of mNeptune is insensitive to pH. DAS of mCardinal were simulated assuming a coupled two-level system to describe the 1S state of the chromophore within two different conformations of the protein. MD simulations were conducted to correlate the experimentally observed pH-induced change in the lifetime in mCardinal with its molecular properties. While the chromophores of both protein variants are stabilized by the same number of hydrogen bonds, it was found that the chromophore in mCardinal exhibits more water contacts compared to mNeptune. In mCardinal, interaction between the chromophore and Glu-145 is reduced as compared to mNeptune, but interaction with Thr-147 which is Ser-147 in mNeptune is stronger in mCardinal. Therefore, the dynamics of the excited-state proton transfer (ESPT) might be different in mCardinal and mNeptune. The pH dependency of ESPT is suggested as a key mechanism for pH sensitivity.

Optogenetics is a versatile and powerful tool for the control and analysis of cellular signaling processes. The activation of cellular receptors by light using optogenetic switches usually requires genetic manipulation of cells. However, this considerably limits the application in primary, nonengineered cells, which is crucial for the study of physiological signaling processes and for controlling cell fate and function for therapeutic purposes. To overcome this limitation, we developed a system for the light-dependent extracellular activation of cell surface receptors of nonengineered cells termed OptoREACT (Optogenetic Receptor Activation) based on the light-dependent protein interaction of A. thaliana phytochrome B (PhyB) with PIF6. In the OptoREACT system, a PIF6-coupled antibody fragment binds the T cell receptor (TCR) of Jurkat or primary human T cells, which upon illumination is bound by clustered phytochrome B to induce receptor oligomerization and activation. For clustering of PhyB, we either used tetramerization by streptavidin or immobilized PhyB on the surface of cells to emulate the interaction of a T cell with an antigen-presenting cell. We anticipate that this extracellular optogenetic approach will be applicable for the light-controlled activation of further cell surface receptors in primary, nonengineered cells for versatile applications in fundamental and applied research.

Phase separation of biomolecules into condensates is a key mechanism in the spatiotemporal organization of biochemical processes in cells. However, the impact of the material properties of biomolecular condensates on important processes, such as the control of gene expression, remains largely elusive. Here, the material properties of optogenetically induced transcription factor condensates are systematically tuned, and probed for their impact on the activation of target promoters. It is demonstrated that transcription factors in rather liquid condensates correlate with increased gene expression levels, whereas stiffer transcription factor condensates correlate with the opposite effect, reduced activation of gene expression. The broad nature of these findings is demonstrated in mammalian cells and mice, as well as by using different synthetic and natural transcription factors. These effects are observed for both transgenic and cell-endogenous promoters. The findings provide a novel materials-based layer in the control of gene expression, which opens novel opportunities in optogenetic engineering and synthetic biology.

A novel approach for controlling translation initiation in mammalian cells is demonstrated based on the conditional attachment of eukaryotic translation initiation factor-binding proteins to the 3′ UTR of mRNAs via small molecule-, light-, or protein-responsive interactions. The technology overcomes limitations of previously used transcription-based switches and was shown to be functional in managing diabetes or tumor growth in preclinical animal models.

The site-specific and covalent conjugation of proteins on solid supports and in hydrogels is the basis for the synthesis of biohybrid materials offering broad applications. Current methods for conjugating proteins to desired targets are often challenging due to unspecific binding, unstable (noncovalent) coupling, or expensive and difficult-to-synthesize ligand molecules. Here, is presented PenTag, an approach for the bioorthogonal, highly specific, and covalent conjugation of a protein to its ligand for various applications in materials sciences. Penicillin-binding protein 3 (PBP3) is engineered and shows that this protein can be used for the stable and spontaneous conjugation of proteins to dyes, polymers, or solid supports. PenTag as a crosslinking tool is applied for synthesizing stimuli-responsive hydrogels or for the development of a biohybrid material system performing computational operations emulating a 4:2 encoder. Based on this broad applicability and the use of a small, cheap, and easy-to-functionalize ligand and a stable, soluble recombinant protein, is seen PenTag as a versatile approach toward biohybrid material synthesis.

Synthetic biology applies concepts from electrical engineering and information processing to endow cells with computational functionality. Transferring the underlying molecular components into materials and wiring them according to topologies inspired by electronic circuit boards has yielded materials systems that perform selected computational operations. However, the limited functionality of available building blocks is restricting the implementation of advanced information-processing circuits into materials. Here, a set of protease-based biohybrid modules the bioactivity of which can either be induced or inhibited is engineered. Guided by a quantitative mathematical model and following a design-build-test-learn (DBTL) cycle, the modules are wired according to circuit topologies inspired by electronic signal decoders, a fundamental motif in information processing. A 2-input/4-output binary decoder for the detection of two small molecules in a material framework that can perform regulated outputs in form of distinct protease activities is designed. The here demonstrated smart material system is strongly modular and can be used for biomolecular information processing for example in advanced biosensing or drug delivery applications.

Molecular optogenetics utilizes genetically encoded, light-responsive protein switches to control the function of molecular processes. Over the last two years, there have been notable advances in the development of novel optogenetic switches, their utilization in elucidating intricate signaling pathways, and their progress toward practical applications in biotechnological processes, material sciences, and therapeutic applications. In this review, we discuss these areas, offer insights into recent developments, and contemplate future directions.

Circumventing the limitations of current bioassays, we introduce a light-controlled assay, OptoAssay, toward wash- and pump-free point-of-care diagnostics. Extending the capabilities of standard bioassays with light-dependent and reversible interaction of optogenetic switches, OptoAssays enable a bidirectional movement of assay components, only by changing the wavelength of light. Demonstrating exceptional versatility, the OptoAssay showcases its efficacy on various substrates, delivering a dynamic bioassay format. The applicability of the OptoAssay is successfully demonstrated by the calibration of a competitive model assay, resulting in a superior limit of detection of 8 pg ml−1, which is beyond those of conventional ELISA tests. In the future, combined with smartphones, OptoAssays could obviate the need for external flow control systems such as pumps or valves and signal readout devices, enabling on-site analysis in resource-limited settings.

PeCa is a rare entity with rising incidence rates due to increased infections with human papillomaviruses (HPV). The distinct subtypes of PeCa with an individual pathogenesis demand biomarkers for a precise patient risk assessment regarding disease progression and therapeutic susceptibility. We recently identified promising candidates associated with an HPV-instructed tumor microenvironment (TME) using HPV-positive PeCa cell lines and tissue microarrays (TMA). The capacity of HPV + p63 + PeCa cells to release neutrophil-attracting CXCL-8 provided a molecular link explaining the infiltration of CD15 + myeloid cells in PeCa specimens. The candidate biomarkers HPV, p63, CD15, DKK1, and CD147 linked a tumor-promoting TME with a higher TNM classification reflecting more aggressive and metastasizing cancers. Based on immune-reactive scores (IRS) from TMA staining for these biomarkers, we calculated correlations and conducted association analyses to assess the degree of relationship between all biomarkers. We then conducted Kaplan–Meier survival estimates and Cox regression analyses to delineate the impact on PeCa patient survival. There is a notable predictive potential regarding the survival of patients with biomarker profiles beyond the potency of the individual biomarker. From all candidate biomarkers and biomarker profiles, the combination of CD147 and infiltrating CD15 + cells linked to an active HPV-driven transformation displayed cancer-immune dynamics with dismal prognosis for patients. After deciphering relevant interdependencies, the HPV + CD147 + CD15 + status was the most potent profile predicting metastasis-free survival of PeCa patients. The results of this report underscore the need for analysis of the TME and the development of multi-parameter composite scores that reflect fundamental cancer-immune relationships to tailor therapeutic interventions based on actual cancer immune dynamics.

Neutrophils play a crucial role in the tumor microenvironment (TME) of head and neck squamous cell carcinomas (HNSCC) and significantly influence treatment outcomes. Phenotypic and functional properties of neutrophils adapt to the TME with distinct subsets modulating disease progression and therapeutic interventions. Here, we evaluated phenotypic and functional differences of neutrophils derived from HNSCC patients and healthy donors. We observed significant phenotypic differences between neutrophils from healthy donors and HNSCC patient-derived neutrophils. Gender and tumor stage influenced neutrophil phenotypes and their ability to lyse tumor cells through antibody-dependent cell-mediated cytotoxicity (ADCC). Patients with advanced HNSCC and males may benefit less from neutrophil-centered immunotherapy. An engineered IgA2 antibody specific for the epidermal growth factor receptor (EGFR) demonstrated superior efficacy in activating neutrophils for ADCC compared to Panitumumab using healthy and patient-derived neutrophils, underscoring the potential of the IgA isotype as a therapeutic alternative. The distinct behavior and antibody-isotype dependent ADCC competence of CD177+/- neutrophils of healthy but not HNSCC donors warrants further exploration. Our study emphasizes the importance of personalized immunotherapy treatments that consider the characteristics of neutrophils, patient demographics, and the type of antibody to improve ADCC and ultimately enhance treatment outcomes for HNSCC.