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Summary Bispecific antibodies (Bispecifics) demonstrate exceptional clinical potential to address some of the most complex diseases. However, Bispecific production in a single cell often requires the correct pairing of multiple polypeptide chains for desired assembly. This is a considerable hurdle that hinders the development of many immunoglobulin G (IgG)-like bispecific formats. Our approach focuses on the rational engineering of charged residues to facilitate the chain pairing of distinct heavy chains (HC). Here, we deploy structure-guided protein design to engineer charge pair mutations (CPMs) placed in the CH3-CH3′ interface of the fragment crystallizable (Fc) region of an antibody (Ab) to correctly steer heavy chain pairing. When used in combination with our stable effector functionless 2 (SEFL2.2) technology, we observed high pairing efficiency without significant losses in expression yields. Furthermore, we investigate the relationship between CPMs and the sequence diversity in the parental antibodies, proposing a rational strategy to deploy these engineering technologies., Graphical abstract, Highlights • Crystal structures unveil molecular basis of SEFL2.2 and CPM technologies • Next gen structure-guided design of CPMs to steer HC-HC pairing • Top CPMs show high pairing efficiency and optimal expression and stability • Balancing CPM charge distribution minimizes impact of sequence diversity, Biochemistry; Bioengineering; Biomolecular engineering; Structural biology

Aromatic compounds have broad applications and have been the target of biosynthetic processes for several decades. New biomolecular engineering strategies have been applied to improve production of aromatic compounds in recent years, some of which are expected to set the stage for the next wave of innovations. Here, we will briefly complement existing reviews on microbial production of aromatic compounds by focusing on a few recent trends where considerable work has been performed in the last 5 years. The trends we highlight are pathway modularization and compartmentalization, microbial co-culturing, non-traditional host engineering, aromatic polymer feedstock utilization, engineered ring cleavage, aldehyde stabilization, and biosynthesis of non-standard amino acids. Throughout this review article, we will also touch on unmet opportunities that future research could address.

Summary Mitochondrial biogenesis is a cell response to external stimuli which is generally believed to suppress apoptosis. However, during the process of apoptosis, whether mitochondrial biogenesis occurs in the early stage of the apoptotic cells remains unclear. To address this question, we constructed the COX8-EGFP-ACTIN-mCherry HeLa cells with recombinant fluorescent proteins respectively tagged on the nucleus and mitochondria and monitored the mitochondrial changes in the living cells exposed to gamma-ray radiation. Besides in situ detection of mitochondrial fluorescence changes, we also examined the cell viability, nuclear DNA damage, reactive oxygen species (ROS), mitochondrial superoxide, citrate synthase activity, ATP, cytoplasmic and mitochondrial calcium, mitochondrial mass, mitochondrial morphology, and protein expression related to mitochondrial biogenesis, as well as the apoptosis biomarkers. As a result, we confirmed that significant mitochondrial biogenesis took place preceding the radiation-induced apoptosis, and it was closely correlated with the apoptotic cells at late stage. The involved mechanism was also discussed., Graphical abstract, Highlights • Dual fluorescence approach was used for in situ observation of living cell processes • Radiation-induced effects of mitochondrial biogenesis and apoptosis were observed • Relationship between mitochondrial biogenesis and apoptosis was revisited • Assessing early mitochondrial biogenesis is critical for predicting later fate of cells, Biochemistry methods; Biomolecular engineering; Cell biology

Diseases are caused by mutations in DNA sequences. DNA tends to form double-helix structures but in more recent times, it has been found that it can also form non-canonical structures in response to changes in the environment around it. Japanese researchers have now unearthed some exciting findings about the chain reaction that results, potentially leading to diseases such as cancer. Associate Professor Hisae Tateishi-Karimata and Professor Naoki Sugimoto are based at the Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University in Japan, and are collaborating on important work to better understand the complex inner workings of cells in disease. An area of focus for this research is the elucidation of mechanisms of diseases caused by non-canonical structures of DNA and regulation for DNA function.

Protein interactions communicate critical information from the environment into cells to orchestrate functional responses relevant to health and disease. Whereas the natural repertoire of protein interfaces is finite, biomolecular engineering tools provide access to an unlimited scope of potential interactions that can be custom-designed for affinity, specificity, mechanism, or other properties of interest. This review highlights recent developments in protein interface engineering that offer insight into human physiology to inform the design of new pharmaceuticals, with a particular focus on immunotherapeutics. We cover three innovative and translationally promising approaches: (1) reprogramming receptor oligomerization to manipulate signaling pathways; (2) computational protein interface design strategies; and (3) engineering bioorthogonal protein interaction networks.

Background:Drug design and development is a vast field that requires huge investment along with a long duration for providing approval to suitable drug candidates. With the advancement in the field of genomics, the information about druggable targets is being updated at a fast rate which is helpful in finding a cure for various diseases.Methods:There are certain biochemicals as well as physiological advantages of using peptide-based therapeutics. Additionally, the limitations of peptide-based drugs can be overcome by modulating the properties of peptide molecules through various biomolecular engineering techniques. Recent advances in computational approaches have been helpful in studying the effect of peptide drugs on the biomolecular targets. Receptor – ligand-based molecular docking studies have made it easy to screen compatible inhibitors against a target.Furthermore, there are simulation tools available to evaluate stability of complexes at the molecular level. Machine learning methods have added a new edge by enabling accurate prediction of therapeutic peptides.Results:Peptide-based drugs are expected to take over many popular drugs in the near future due to their biosafety, lower off-target binding chances and multifunctional properties.Conclusion:This article summarises the latest developments in the field of peptide-based therapeutics related to their usage, tools, and databases.

Joonbaek Jang received his B.S. from Korea University in 2016. He then moved to the University of Pennsylvania where he obtained his M.S. working with Prof. Raymond J. Gorte. During his master’s degree, Joonbaek used atomic layer deposition to modify metal oxide catalysts for the improvement of industrial catalytic processes. In 2018, he joined the Morales-Guio Lab in the Department of Chemical and Biomolecular Engineering at UCLA. Joonbaek is currently working toward the understanding of electrode/electrolyte interfaces and mass transport effects in electrocatalytic processes for sustainable energy applications. Kangze Shen received his B.S. in Materials Chemistry from University of Science and Technology of China in 2018. His undergraduate research with Prof. Yitai Qian focused on the development of high-performance electrode materials for rechargeable Li-ion and Na-ion batteries. He then joined the Morales-Guio Lab in the Department of Chemical and Biomolecular Engineering at UCLA where he is working toward the understanding of electrocatalytic processes and electrochemical systems engineering in sustainable energy applications. Carlos G. Morales-Guio is an Assistant Professor of Chemical and Biomolecular Engineering at UCLA. The Morales-Guio Lab is interested in electrochemical catalysis, particularly with respect to energy and chemical transformations for sustainable energy applications. The lab is currently focused on understanding fundamentals of mass and charge transport coupled to electrochemical transformations that will guide the scale up of electrocatalytic reactors. Carlos received his B. Eng. degree from Osaka University in 2011 and his M.S. and Ph.D. in Chemistry and Chemical Engineering from EPFL in 2013 and 2016, respectively. Most recently, he was a postdoctoral research fellow at Stanford University before joining UCLA in 2018.

Regulatory mechanisms in biological systems are analogous to process control exerted in many chemical and biomolecular engineering processes. Ribonucleic acid (RNA) has been identified as a ubiquit...

The halogen bond (or X-bond) is a noncovalent interaction that is increasingly recognized as an important design tool for engineering protein-ligand interactions and controlling the structures of proteins and nucleic acids. In the past decade, there have been significant efforts to characterize the structure-energy relationships of this interaction in macromolecules. Progress in the computational modeling of X-bonds in biological molecules, however, has lagged behind these experimental studies, with most molecular mechanics/dynamics-based simulation methods not properly treating the properties of the X-bond. We had previously derived a force field for biological X-bonds (ffBXB) based on a set of potential energy functions that describe the anisotropic electrostatic and shape properties of halogens participating in X-bonds. Although fairly accurate for reproducing the energies within biomolecular systems, including X-bonds engineered into a DNA junction, the ffBXB with its seven variable parameters was considered to be too unwieldy for general applications. In the current study, we have generalized the ffBXB by reducing the number of variables to just one for each halogen type and show that this remaining electrostatic variable can be estimated for any new halogenated molecule through a standard restricted electrostatic potential calculation of atomic charges. In addition, we have generalized the ffBXB for both nucleic acids and proteins. As a proof of principle, we have parameterized this reduced and more general ffBXB against the AMBER force field. The resulting parameter set was shown to accurately recapitulate the quantum mechanical landscape and experimental interaction energies of X-bonds incorporated into DNA junction and T4 lysozyme model systems. Thus, this reduced and generalized ffBXB is more readily adaptable for incorporation into classical molecular mechanics/dynamics algorithms, including those commonly used to design inhibitors against therapeutic targets in medicinal chemistry and materials in biomolecular engineering.

Although natural or artificial modified pyrimidine nucleobases represent important molecules with valuable properties as constituents of DNA and RNA, no systematic analyses of the structural aspects of bromo derivatives of cytosine have appeared so far in the literature. In view of the biochemical and pharmaceutical relevance of these compounds, six different crystals containing proton-transfer derivatives of 5-bromocytosine are prepared and analyzed in the solid-state by single crystal X-ray diffraction. All six compounds are organic salts, with proton transfer occurring to the Nimino atom of the pyridine ring. Experimental results are then complemented with Hirshfeld surface analysis to quantitively evaluate the contribution of different intermolecular interactions in the crystal packing. Furthermore, theoretical calculations, based on different arrangements of molecules extracted from the crystal structure determinations, are carried out to analyze the formation mechanism of halogen bonds (XBs) in these compounds and provide insights into the nature and strength of the observed interactions. The results show that the supramolecular architectures of the six molecular salts involve extensive classical intermolecular hydrogen bonds. However, in all but one proton-transfer adducts, weak to moderate XBs are revealed by C–Br…O short contacts between the bromine atom in the fifth position, which acts as XB donor (electron acceptor). Moreover, the lone pair electrons of the oxygen atom of adjacent pyrimidine nucleobases and/or counterions or water molecules, which acts as XB acceptor (electron donor).

Classical hydrogen bonds have, for many decades, been the dominant non-covalent interaction in the toolbox that chemists and chemical engineers have used to design and control the structures of compounds and molecular assemblies as novel materials. Recently, a set of non-classical non-covalent (NC-NC) interactions have emerged that exploit the properties of the Group IV, V, VI, and VII elements of the periodic table (the tetrel, pnictogen, chalcogen, and halogen bonds, respectively). Our research group has been characterizing the prevalence, geometric constraints, and structure-function relationship specifically of the halogen bond in biological systems. We have been particularly interested in exploiting the biological halogen bonds (or BXBs) to control the structures, stabilities, and activities of biomolecules, including the DNA Holliday junction and enzymes. In this review, we first provide a set of criteria for how to determine whether BXBs or any other NC-NC interactions would have biological relevance. We then navigate the trail of studies that had led us from an initial, very biological question to our current point in the journey to establish BXBs as a tool for biomolecular engineering. Finally, we close with a perspective on future directions for this line of research.

Proteins are one of the most fundamental biomolecules present in living organisms. They are indispensable to the sustenance of these organisms owing to the multitude of functions that they perform. They serve as structural constituents of cells and are involved in cell signaling, transport of biological components, cell adhesion, eliciting immune responses, catalyzing chemical reactions, etc. In order to get better insights into these functional aspects, it is quintessential to understand not only the physicochemical features of proteins, but also their structural organization (starting from primary sequences and ending in quaternary structures), and the various interactions that stabilize the higher-order structures. Many biological functions in the cell are mediated when a protein interacts with one or two other proteins in a phenomenon known as protein–protein interaction (PPI). The chapter concludes by providing an overview of PPIs including their functional importance.

Advanced diagnostic techniques, such as PCR and ELISA, are widely used in well-equipped laboratories. However, they are expensive and are not accessible in resource-limited settings. With growing evidence that point-of-care testing (POCT) improves clinical outcomes, there exists a need for simple, fast, low cost, yet sensitive POCT technique. We present a single pot “molecular adaptor”, which we name DNA split proximity circuit (SPC), that can be used as an isothermal, enzyme-free and wash-free diagnostics platform. SPC consists of a pair of “plug-and-play” DNA initiators readily adaptable to various biomarkers ranging from nucleic acids to protein complexes, which then triggers a signal amplification readout based on Hybridization Chain Reaction (HCR). We first improved the kinetics and the sensitivity of HCR by refining HCR hairpin designs. Using biomarkers related to breast cancer as model systems, we applied the optimized HCR to our SPC system and demonstrated its capability to detect microRNA (miRNA) and cell surface receptors. SPC demonstrated good limit of detection in the femtomolar range within 30 min for all miRNA targets tested. It also showed high specificity and an ability to differentiate between different cell surface protein markers. Being isothermal and enzyme-free, our system is less sensitive to temperature and buffer conditions, making it highly robust, thus showing great potential for bedside use. With applications in biomarker analysis, cell visualization and disease diagnostics, herein we improved and established the potential of SPC as a powerful next-generation POCT assay.

Despite the vital role of vaccines in fighting viral pathogens, effective vaccines are still unavailable for many infectious diseases. The importance of vaccines cannot be overstated during the outbreak of a pandemic, such as the coronavirus disease 2019 (COVID-19) pandemic. The understanding of genomics, structural biology, and innate/adaptive immunity have expanded the toolkits available for current vaccine development. However, sudden outbreaks and the requirement of population-level immunization still pose great challenges in today's vaccine designs. Well-established vaccine development protocols from previous experiences are in place to guide the pipelines of vaccine development for emerging viral diseases. Nevertheless, vaccine development may follow different paradigms during a pandemic. For example, multiple vaccine candidates must be pushed into clinical trials simultaneously, and manufacturing capability must be scaled up in early stages. Factors from essential features of safety, efficacy, manufacturing, and distributions to administration approaches are taken into consideration based on advances in materials science and engineering technologies. In this review, we present recent advances in vaccine development by focusing on vaccine discovery, formulation, and delivery devices enabled by alternative administration approaches. We hope to shed light on developing better solutions for faster and better vaccine development strategies through the use of biomaterials, biomolecular engineering, nanotechnology, and microfabrication techniques.

Biosurfactants are surface-active molecules originated from renewable resources, which are produced by microbial fermentation or chemical/enzymatic catalysis. These molecules present important advantages as compared to petrochemical surfactants, given their resistance to extreme conditions, biodegradability, specificity, and environmental compatibility. Besides that, the high production costs hinder its commercialization. In this way, this article aimed to analyze microbial biosurfactants production, focusing on the optimization of metabolic pathways and production processes, to identify key aspects and provide alternatives to allow a cost-effective production at industrial scale. This was achieved by a broad analysis of biosurfactants properties, applications, and biosynthetic pathways (in terms of yield, cofactors, and energy), in addition to an assessment of production-associated costs. As a result of the present extensive data survey and analysis, key production aspects are disclosed. The metabolic pathway yield analysis demonstrated that production of biosurfactants can be significantly improved (highest theoretical yield was 0.47 gbiosurfactant /gsubstrate ) by the use of biomolecular engineering techniques to generate optimized synthetic pathways. With an alternative proposed pathway for surfactin, yield was improved and imbalance in cofactors and ATP was reduced. Analysis of productive costs indicated that to make rhamnolipids commercial production feasible, the main efforts should focus on lowering substrate costs as well as the identification of energy-efficient unit operations to lower electricity cost, since these parameters accounted for 19.36 and 78.22%, respectively, of the production costs. The data generated by this analysis highlight the need for multidisciplinary collaboration to make rhamnolipids economically feasible, including biomolecular engineering and process intensification.

Many cell behaviors are significantly affected by cell culture geometry, though it remains unclear which geometry from two- to three-dimensional (2D to 3D) culture is appropriate for probing a specific cell function and mimicking native microenvironments. Toward addressing this, we established a 2.5D culture geometry, enabling initial cell spreading while reducing polarization to bridge between 2D and 3D geometries, and examined the responses of wound healing cells, human pulmonary fibroblasts, within it. To achieve this, we used engineered biomimetic hydrogels formed by photopolymerization, creating robust layered hydrogels with spread fibroblasts at the interface. We found that fibroblast responses were similar between 2D and 2.5D culture and different from 3D culture, with some underlying differences in mechanotransduction. These studies established the 2.5D cell culture geometry in conjunction with biomimetic synthetic matrices as a useful tool for investigations of fibroblast activation with relevance to the study of other cell functions and types.

Summary Lanthanide-based, Förster resonance energy transfer (LRET) biosensors enabled sensitive, time-gated luminescence (TGL) imaging or multiwell plate analysis of protein-protein interactions (PPIs) in living cells. We prepared stable cell lines that expressed polypeptides composed of an alpha helical linker flanked by a Tb(III) complex-binding domain, GFP, and two interacting domains at each terminus. The PPIs examined included those between FKBP12 and the rapamycin-binding domain of m-Tor (FRB) and between p53 (1–92) and HDM2 (1–128). TGL microscopy revealed dramatic differences (>500%) in donor- or acceptor-denominated, Tb(III)-to-GFP LRET ratios between open (unbound) and closed (bound) states of the biosensors. We observed much larger signal changes (>2,500%) and Z′-factors of 0.5 or more when we grew cells in 96- or 384-well plates and analyzed PPI changes using a TGL plate reader. The modular design and exceptional dynamic range of lanthanide-based LRET biosensors will facilitate versatile imaging and cell-based screening of PPIs., Graphical Abstract, Highlights • Non-invasive, microscopic imaging or screening of protein-protein interactions • Intracellular assembly of sensor polypeptides with luminescent Tb(III) complexes • High dynamic range with time-gated detection of Tb(III)-to-GFP sensitized emission, Sensor; Molecular Spectroscopy Techniques; Molecular Interaction; Biomolecular Engineering

The rational design of a multi-responsive protein-based supramolecular system that can predictably respond to more than one stimulus remains an essential but highly challenging goal in biomolecular engineering. Herein, we report a novel chemical method for the construction of multi-responsive supramolecular nanoassemblies using custom-designed facially amphiphilic monodisperse protein-dendron bioconjugates. The macromolecular synthons contain a globular hydrophilic protein domain site-specifically conjugated to photo-responsive hydrophobic benzyl-ether dendrons of different generations through oligo(ethylene glycol) linkers of defined length. The size of the protein nanoassemblies can be systematically tuned by choosing an appropriate dendron or linker of defined length. Exposure of protein nanoassemblies to light results in partial rather than complete disassembly of the complex. The newly formed protein nanoparticle no longer responds to light but could be disassembled into constitutive monomers under acidic conditions or by further treatment with a small molecule. More interestingly, the distribution ratio of the assembled versus disassembled states of protein nanoassemblies after photochemical reaction does not depend on dendron generation, the nature of the linker functionality or the identity of the protein, but is heavily influenced by the linker length. In sum, this work discloses a new chemical method for the rational design of a monodisperse multi-responsive protein-based supramolecular system with exquisite control over the disassembly process.

The majority of clinically approved therapeutics target membrane proteins (MPs), highlighting the need for tools to study this important category of proteins. To overcome limitations with recombinant MP expression, whole cell screening techniques have been developed that present MPs in their native conformations. Whereas many such platforms utilize adherent cells, here we introduce a novel suspension cell-based platform termed “biofloating” that enables quantitative analysis of interactions between proteins displayed on yeast and MPs expressed on mammalian cells, without need for genetic fusions. We characterize and optimize biofloating and illustrate its sensitivity advantage compared to an adherent cell-based platform (biopanning). We further demonstrate the utility of suspension cell-based approaches by iterating rounds of magnetic-activated cell sorting (MACS) selections against MP-expressing mammalian cells to enrich for a specific binder within a yeast-displayed antibody library. Overall, biofloating represents a promising new technology that can be readily integrated into protein discovery and development workflows.

The combination of cell culture models with microscale technology has fostered emergence of in vitro cell-based microphysiological models, also known as organ-on-a-chip systems. Body-on-a-chip systems, which are multi-organ systems on a chip to mimic physiological relations, enable recapitulation of organ-organ interactions and potentially whole-body response to drugs, as well as serve as models of diseases. Chemical reaction engineering principles can be applied to understanding complex reactions inside the cell or human body, which can be treated as a multi-reactor system. These systems use physiologically-based pharmacokinetic (PBPK) models to guide the development of microscale systems of the body where organs or tissues are represented by living cells or tissues, and integrated into body-on-a-chip systems. Here, we provide a brief overview on the concept of chemical reaction engineering and how its principles can be applied to understanding and predicting the behavior of body-on-a-chip systems.

One of the central themes of biomolecular engineering is the challenge of exploiting the properties of biological materials. Part of this challenge has been uncovering and harnessing properties of biological components that only emerge following their ordered self-assembly. One biomolecular building block that has received significant interest in the past decade is the M13 bacteriophage. There have been a number of recent attempts to trigger the ordered assembly of M13 bacteriophage into multivirion structures, relying on the innate tendency of M13 to form liquid crystals at high concentrations. These, in general, yield planar two-dimensional materials. Presented here is the production of multivirion assemblies of M13 bacteriophage via the chemical modification of its surface by the covalent attachment of the xanthene-based dye tetramethylrhodamine (TMR) isothiocyanate (TRITC). We show that TMR induces the formation of three-dimensional aster-like assemblies of M13 by providing "adhesive" action between bacteriophage particles through the formation of H-aggregates (face-to-face stacking of dye molecules). We also show that the H-aggregation of TMR is greatly enhanced by covalent attachment to M13 and is enhanced further still upon the ordered self-assembly of M13, leading to the suggestion that M13 could be used to promote the self-assembly of dyes that form J-aggregates, a desirable arrangement of fluorescent dye, which has interesting optical properties and potential applications in the fields of medicine and light harvesting technology.

The alkyne functionality has attracted much interest due to its diverse chemical and biological applications. We recently elucidated an acyl carrier protein (ACP)-dependent alkyne biosynthetic pathway, however, little is known about ACP interactions with the alkyne biosynthetic enzymes, an acyl-ACP ligase (JamA) and a membrane-bound bi-functional desaturase/acetylenase (JamB). Here, we showed that JamB has a more stringent interaction with ACP than JamA. In addition, site directed mutagenesis of a non-cognate ACP significantly improved its compatibility with JamB, suggesting a possible electrostatic interaction at the ACP-JamB interface. Finally, error-prone PCR and screening of a second non-cognate ACP identified hot spots on the ACP that are important for interacting with JamB and yielded mutants which were better recognized by JamB. Our data thus not only provide insights into the ACP interactions in alkyne biosynthesis, but it also potentially aids in future combinatorial biosynthesis of alkyne-tagged metabolites for chemical and biological applications.Topical HeadingBiomolecular Engineering, Bioengineering, Biochemicals, Biofuels, and Food

Artificial multiprotein complexes are sought after reagents for biomolecular engineering. A current limiting factor is the paucity of molecular scaffolds which allow for site-specific multicomponent assembly. Here, we address this limitation by synthesizing bioorthogonal elastin-like polypeptide (ELP) scaffolds containing periodic noncanonical l-azidohomoalanine amino acids in the guest residue position. The nine azide ELP guest residues served as conjugation sites for site-specific modification with dibenzocyclooctyne (DBCO)-functionalized single-domain antibodies (SdAbs) through strain-promoted alkyne-azide cycloaddition (SPAAC). Sortase A and ybbR tags at the C- and N-termini of the ELP scaffold provided two additional sites for derivatization with small molecules and peptides by Sortase A and 4`-phosphopantetheinyl transferase (Sfp), respectively. These functional groups are chemically bioorthogonal, mutually compatible, and highly efficient, thereby enabling synthesis of multi-antibody ELP complexes in a one-pot reaction. We demonstrate application of this material for enhancing the performance of sandwich immunoassays of the recombinant protein mCherry. In undiluted human plasma, surfaces modified with multi-antibody ELP complexes showed between 2.3- and 14.3-fold improvement in sensitivity and ∼30-40% lower limits of detection as compared with nonspecifically adsorbed antibodies. Dual-labeled multi-antibody ELP complexes were further used for cytometric labeling and analysis of live eukaryotic cells. These results demonstrate how multiple antibodies complexed onto bioorthogonal protein-based polymers can be used to enhance immunospecific binding interactions through multivalency effects.

Nanostructures built from biomolecules such as proteins, DNA and RNA are attracting attention in many areas of biological and materials sciences. Such nanoscale engineering was pioneered with proteins, yet the use of DNA is rapidly gaining traction. What are the advantages of the different biopolymers and which is best suited for a given molecular structure, function or application? In this Review, we evaluate the different structural properties of proteins and nucleic acids, as well as possible designs and synthetic routes for functional nanostructures. By comparing protein engineering and DNA nanotechnology, we highlight molecular architectures that are relevant in biotechnology, biomedicine and synthetic biology research, and identify emerging areas for research such as hybrid materials composed of protein and DNA/RNA. Proteins, DNA and RNA can be used to build functional nanostructures. This Review compares protein and DNA/RNA in terms of biochemical properties and ease of engineering in three major areas of application: biomolecular recognition, biocatalysis and structural support.

Fundamental advances in the field of biomolecular engineering present researchers with the unprecedented opportunity to apply molecular design strategies to modify naturally occurring proteins and generate new molecules for targeted disease therapy. In particular, the development of immunotherapeutics such as cytokine-based and antibody-based drugs has been empowered by evolving technologies and insights from protein engineering. In this review, we highlight specific examples that demonstrate the growing influence of biomolecular engineering on the discovery and design of immunotherapeutics, with particular emphasis on three strategies in biologics development: (1) Cytokine engineering, (2) Cytokine-targeted antibody design, and (3) Fine-tuning the disease-targeting mechanisms of antibody drugs. These strategies have already yielded significant success in the clinic and promise to continue informing and improving the drug development landscape.

Synthetic biologists have applied biomolecular engineering approaches toward the goal of novel biological devices and have shown progress in diverse areas of medicine and biotechnology. Especially promising is the application of synthetic biological devices towards a novel class of molecular diagnostics. As an example, a de-novo-designed riboregulator called toehold switch, with its programmability and compatibility with field-deployable devices showed promising in vitro applications for viral RNA detection such as Zika and Corona viruses. However, the in vivo application of high-performance RNA sensors remains challenging due to the secondary structure of long mRNA species. Here, we introduced ‘Helper RNAs’ that can enhance the functionality of toehold switch sensors by mitigating the effect of secondary structures around a target site. By employing the helper RNAs, previously reported mCherry mRNA sensor showed improved fold-changes in vivo. To further generalize the Helper RNA approaches, we employed automatic design pipeline for toehold sensors that target the essential genes within the pks island, an important target of biomedical research in connection with colorectal cancer. The toehold switch sensors showed fold-changes upon the expression of full-length mRNAs that apparently depended sensitively on the identity of the gene as well as the predicted local structure within the target region of the mRNA. Still, the helper RNAs could improve the performance of toehold switch sensors in many instances, with up to 10-fold improvement over no helper cases. These results suggest that the helper RNA approaches can further assist the design of functional RNA devices in vivo with the aid of the streamlined automatic design software developed here. Further, our solutions for screening and stabilizing single-stranded region of mRNA may find use in other in vivo mRNA-sensing applications such as cas13 crRNA design, transcriptome engineering, and trans-cleaving ribozymes.

International audience; ERK1/2 involvement in cell death remains unclear, although many studies have demonstrated the importance of ERK1/2 dynamics in determining cellular re- sponses. To untangle how ERK1/2 contributes to two cell death programs, we investigated ERK1/2 signaling dynamics during hFasL-induced apoptosis and TNF-induced necroptosis in L929 cells. We observed that ERK1/2 inhibition sensi- tizes cells to apoptosis while delaying necroptosis. By monitoring ERK1/2 activity by live-cell imaging using an improved ERK1/2 biosensor (EKAR4.0), we reported differential ERK1/2 signaling dynamics between cell survival, apoptosis, and nec- roptosis. We also decrypted a temporally shifted amplitude- and frequency-modu- lated (AM/FM) ERK1/2 activity profile in necroptosis versus apoptosis. ERK1/2 inhibition, which disrupted ERK1/2 signaling dynamics, prevented TNF and IL-6 gene expression increase during TNF-induced necroptosis. Using an inducible cell line for activated MLKL, the final executioner of necroptosis, we showed ERK1/2 and its distinctive necroptotic ERK1/2 activity dynamics to be positioned downstream of MLKL.