Affinity Maturation of a Cyclic Peptide Handle for Therapeutic Antibodies Using Deep Mutational Scanning.

Meditopes are cyclic peptides that bind in a specific pocket in the antigen-binding fragment of a therapeutic antibody such as cetuximab. Provided their moderate affinity can be enhanced, meditope peptides could be used as specific non-covalent and paratope-independent handles in targeted drug delivery, molecular imaging, and therapeutic drug monitoring. Here we show that the affinity of a recently reported meditope for cetuximab can be substantially enhanced using a combination of yeast display and deep mutational scanning. Deep sequencing was used to construct a fitness landscape of this protein-peptide interaction, and four mutations were identified that together improved the affinity for cetuximab 10-fold to 15 nm Importantly, the increased affinity translated into enhanced cetuximab-mediated recruitment to EGF receptor-overexpressing cancer cells. Although in silico Rosetta simulations correctly identified positions that were tolerant to mutation, modeling did not accurately predict the affinity-enhancing mutations. The experimental approach reported here should be generally applicable and could be used to develop meditope peptides with low nanomolar affinity for other therapeutic antibodies.

Kinetic Analysis as a Tool to Distinguish Pathway Complexity in Molecular Assembly: An Unexpected Outcome of Structures in Competition.

While the sensitive dependence of the functional characteristics of self-assembled nanofibers on the molecular structure of their building blocks is well-known, the crucial influence of the dynamics of the assembly process is often overlooked. For natural protein-based fibrils, various aggregation mechanisms have been demonstrated, from simple primary nucleation to secondary nucleation and off-pathway aggregation. Similar pathway complexity has recently been described in synthetic supramolecular polymers and has been shown to be intimately linked to their morphology. We outline a general method to investigate the consequences of the presence of multiple assembly pathways, and show how kinetic analysis can be used to distinguish different assembly mechanisms. We illustrate our combined experimental and theoretical approach by studying the aggregation of chiral bipyridine-extended 1,3,5-benzenetricarboxamides (BiPy-1) in n-butanol as a model system. Our workflow consists of nonlinear least-squares analysis of steady-state spectroscopic measurements, which cannot provide conclusive mechanistic information but yields the equilibrium constants of the self-assembly process as constraints for subsequent kinetic analysis. Furthermore, kinetic nucleation-elongation models based on one and two competing pathways are used to interpret time-dependent spectroscopic measurements acquired using stop-flow and temperature-jump methods. Thus, we reveal that the sharp transition observed in the aggregation process of BiPy-1 cannot be explained by a single cooperative pathway, but can be described by a competitive two-pathway mechanism. This work provides a general tool for analyzing supramolecular polymerizations and establishing energetic landscapes, leading to mechanistic insights that at first sight may seem unexpected and counterintuitive.

DNA Input Classification by a Riboregulator-Based Cell-Free Perceptron.

The ability to recognize molecular patterns is essential for the continued survival of biological organisms, allowing them to sense and respond to their immediate environment. The design of synthetic gene-based classifiers has been explored previously; however, prior strategies have focused primarily on DNA strand-displacement reactions. Here, we present a synthetic in vitro transcription and translation (TXTL)-based perceptron consisting of a weighted sum operation (WSO) coupled to a downstream thresholding function. We demonstrate the application of toehold switch riboregulators to construct a TXTL-based WSO circuit that converts DNA inputs into a GFP output, the concentration of which correlates to the input pattern and the corresponding weights. We exploit the modular nature of the WSO circuit by changing the output protein to the Escherichia coli σ28-factor, facilitating the coupling of the WSO output to a downstream reporter network. The subsequent introduction of a σ28 inhibitor enabled thresholding of the WSO output such that the expression of the downstream reporter protein occurs only when the produced σ28 exceeds this threshold. In this manner, we demonstrate a genetically implemented perceptron capable of binary classification, i.e., the expression of a single output protein only when the desired minimum number of inputs is exceeded.

Cell-Free Characterization of Coherent Feed-Forward Loop-Based Synthetic Genetic Circuits.

Regulatory pathways inside living cells employ feed-forward architectures to fulfill essential signal processing functions that aid in the interpretation of various types of inputs through noise-filtering, fold-change detection and adaptation. Although it has been demonstrated computationally that a coherent feed-forward loop (CFFL) can function as noise filter, a property essential to decoding complex temporal signals, this motif has not been extensively characterized experimentally or integrated into larger networks. Here we use post-transcriptional regulation to implement and characterize a synthetic CFFL in an Escherichia coli cell-free transcription-translation system and build larger composite feed-forward architectures. We employ microfluidic flow reactors to probe the response of the CFFL circuit using both persistent and short, noise-like inputs and analyze the influence of different circuit components on the steady-state and dynamics of the output. We demonstrate that our synthetic CFFL implementation can reliably repress background activity compared to a reference circuit, but displays low potential as a temporal filter, and validate these findings using a computational model. Our results offer practical insight into the putative noise-filtering behavior of CFFLs and show that this motif can be used to mitigate leakage and increase the fold-change of the output of synthetic genetic circuits.

Light-Activated Signaling in DNA-Encoded Sender-Receiver Architectures.

Collective decision making by living cells is facilitated by exchange of diffusible signals where sender cells release a chemical signal that is interpreted by receiver cells. A variety of nonliving artificial cell models have been developed in recent years that mimic various aspects of diffusion-based intercellular communication. However, localized secretion of diffusive signals from individual protocells, which is critical for mimicking biological sender-receiver systems, has remained challenging to control precisely. Here, we engineer light-responsive, DNA-encoded sender-receiver architectures, where protein-polymer microcapsules act as cell mimics and molecular communication occurs through diffusive DNA signals. We prepare spatial distributions of sender and receiver protocells using a microfluidic trapping array and set up a signaling gradient from a single sender cell using light, which activates surrounding receivers through DNA strand displacement. Our systematic analysis reveals how the effective signal range of a single sender is determined by various factors including the density and permeability of receivers, extracellular signal degradation, signal consumption, and catalytic regeneration. In addition, we construct a three-population configuration where two sender cells are embedded in a dense array of receivers that implement Boolean logic and investigate spatial integration of nonidentical input cues. The results offer a means for studying diffusion-based sender-receiver topologies and present a strategy to achieve the congruence of reaction-diffusion and positional information in chemical communication systems that have the potential to reconstitute collective cellular patterns.

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression.

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

Cell-free microcompartmentalised transcription-translation for the prototyping of synthetic communication networks.

Recent efforts in synthetic biology have shown the possibility of engineering distributed functions in populations of living cells, which requires the development of highly orthogonal, genetically encoded communication pathways. Cell-free transcription-translation (TXTL) reactions encapsulated in microcompartments enable prototyping of molecular communication channels and their integration into engineered genetic circuits by mimicking critical cell features, such as gene expression, cell size, and cell individuality within a community. In this review, we discuss the uses of cell-free transcription-translation reactions for the development of synthetic genetic circuits, with a special focus on the use of microcompartments supporting this reaction. We highlight several studies where molecular communication between non-living microcompartments and living cells have been successfully engineered.

Sigma Factor-Mediated Tuning of Bacterial Cell-Free Synthetic Genetic Oscillators.

Cell-free transcription-translation provides a simplified prototyping environment to rapidly design and study synthetic networks. Despite the presence of a well characterized toolbox of genetic elements, examples of genetic networks that exhibit complex temporal behavior are scarce. Here, we present a genetic oscillator implemented in an E. coli-based cell-free system under steady-state conditions using microfluidic flow reactors. The oscillator has an activator-repressor motif that utilizes the native transcriptional machinery of E. coli: the RNAP and its associated sigma factors. We optimized a kinetic model with experimental data using an evolutionary algorithm to quantify the key regulatory model parameters. The functional modulation of the RNAP was investigated by coupling two oscillators driven by competing sigma factors, allowing the modification of network properties by means of passive transcriptional regulation.

Colorful protein-based fluorescent probes for collagen imaging.

Real-time visualization of collagen is important in studies on tissue formation and remodeling in the research fields of developmental biology and tissue engineering. Our group has previously reported on a fluorescent probe for the specific imaging of collagen in live tissue in situ, consisting of the native collagen binding protein CNA35 labeled with fluorescent dye Oregon Green 488 (CNA35-OG488). The CNA35-OG488 probe has become widely used for collagen imaging. To allow for the use of CNA35-based probes in a broader range of applications, we here present a toolbox of six genetically-encoded collagen probes which are fusions of CNA35 to fluorescent proteins that span the visible spectrum: mTurquoise2, EGFP, mAmetrine, LSSmOrange, tdTomato and mCherry. While CNA35-OG488 requires a chemical conjugation step for labeling with the fluorescent dye, these protein-based probes can be easily produced in high yields by expression in E. coli and purified in one step using Ni2+-affinity chromatography. The probes all bind specifically to collagen, both in vitro and in porcine pericardial tissue. Some first applications of the probes are shown in multicolor imaging of engineered tissue and two-photon imaging of collagen in human skin. The fully-genetic encoding of the new probes makes them easily accessible to all scientists interested in collagen formation and remodeling.