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The increased availability of user-friendly and accessible computational tools for biomolecular modeling would expand the reach and application of biomolecular engineering and design. For protein modeling, one key challenge is to reduce the complexities of 3D protein folds to sets of parametric equations that nonetheless capture the salient features of these structures accurately. At present, this is possible for a subset of proteins, namely, repeat proteins. The α-helical coiled coil provides one such example, which represents ≈ 3-5% of all known protein-encoding regions of DNA. Coiled coils are bundles of α helices that can be described by a small set of structural parameters. Here we describe how this parametric description can be implemented in an easy-to-use web application, called CCBuilder 2.0, for modeling and optimizing both α-helical coiled coils and polyproline-based collagen triple helices. This has many applications from providing models to aid molecular replacement for X-ray crystallography, in silico model building and engineering of natural and designed protein assemblies, and through to the creation of completely de novo "dark matter" protein structures. CCBuilder 2.0 is available as a web-based application, the code for which is open-source and can be downloaded freely. http://coiledcoils.chm.bris.ac.uk/ccbuilder2. Lay summary We have created CCBuilder 2.0, an easy to use web-based application that can model structures for a whole class of proteins, the α-helical coiled coil, which is estimated to account for 3-5% of all proteins in nature. CCBuilder 2.0 will be of use to a large number of protein scientists engaged in fundamental studies, such as protein structure determination, through to more-applied research including designing and engineering novel proteins that have potential applications in biotechnology.

Molecular hydrogen is a promising currency in the future energy economy due to the uncertain availability of finite fossil fuel resources and environmental effects from their combustion. It also has important uses in the production of fertilizers and platform chemicals as well as in upgrading conventional fuels. Conventional methods for producing molecular hydrogen from natural gas produce carbon dioxide and use a finite resource as feedstock. However, these issues can be overcome by using light energy from the Sun combined with microorganisms and their molecular machinery capable of photosynthesis. In the presence of light, the proteins involved in photosynthesis coupled with appropriate catalysts in higher plants, algae, and cyanobacteria can produce molecular hydrogen, and optimization via genetic modifications and biomolecular engineering further improves production rates. In this review, we will discuss techniques that have been utilized to improve rates of hydrogen production in biological systems based on the protein machinery of photosynthesis coupled with appropriate catalysts. We will also suggest areas for improvement and future directions for work in the field.

Peptide nucleic acids (PNAs) are polyamide analogues of nucleic acids, very effective in terms of affinity and selectivity in DNA/RNA recognition. As other supramolecular entities, the PNA structure has been an interesting scaffold for the development of new molecules, aimed to DNA and RNA recognition, with improved or completely new properties. This review describes recent work, with the aim of describing how the design of these molecules has evolved in recent years, using increasingly effective tools, from simple crystal structure analysis to molecular dynamics and metadynamics. Modified PNA with additional modules appended, either on the backbone or on the nucleobase, are described. Polyfunctional molecules with both backbone and nucleobase modification are then considered. Finally, recent examples of architectures obtained by conjugation of PNAs to inorganic nanostructures as cargo systems for diagnostics and nano-biotechnology are presented.

Immobilized antibody systems are the key to develop efficient diagnostics and separations tools. In the last decade, developments in the field of biomolecular engineering and crosslinker chemistry have greatly influenced the development of this field. With all these new approaches at our disposal, several new immobilization methods have been created to address the main challenges associated with immobilized antibodies. Few of these challenges that we have discussed in this review are mainly associated to the site-specific immobilization, appropriate orientation, and activity retention. We have discussed the effect of antibody immobilization approaches on the parameters on the performance of an immunoassay.

We used the molecular modeling program Rosetta to identify clusters of amino acid substitutions in antibody fragments (scFvs and scAbs) that improve global protein stability and resistance to thermal deactivation. Using this methodology, we increased the melting temperature (T(m)) and resistance to heat treatment of an antibody fragment that binds to the Clostridium botulinum hemagglutinin protein (anti-HA33). Two designed antibody fragment variants with two amino acid replacement clusters, designed to stabilize local regions, were shown to have both higher T(m) compared to the parental scFv and importantly, to retain full antigen binding activity after 2 hours of incubation at 70 °C. The crystal structure of one thermostabilized scFv variants was solved at 1.6 Å and shown to be in close agreement with the RosettaAntibody model prediction.

Materials with novel and enhanced functionalities can be obtained by modifying cellulose with a range of biomolecules. This functionalization can deliver tailored cellulose-based materials with enhanced physical and chemical properties and control of biological interactions that match specific applications. One of the foundations for the success of such biomaterials is to efficiently control the capacity to combine relevant biomolecules into cellulose materials in such a way that the desired functionality is attained. In this context, our main goal was to develop bi-functional biomolecular constructs for the precise modification of cellulose hydrogels with bioactive molecules of interest. The main idea was to use biomolecular engineering techniques to generate and purify different recombinant fusions of carbohydrate binding modules (CBMs) with significant biological entities. Specifically, CBM-based fusions were designed to enable the bridging of proteins or oligonucleotides with cellulose hydrogels. The work focused on constructs that combine a family 3 CBM derived from the cellulosomal-scaffolding protein A from Clostridium thermocellum (CBM3) with the following: (i) an N-terminal green fluorescent protein (GFP) domain (GFP-CBM3); (ii) a double Z domain that recognizes IgG antibodies; and (iii) a C-terminal cysteine (CBM3C). The ability of the CBM fusions to bind and/or anchor their counterparts onto the surface of cellulose hydrogels was evaluated with pull-down assays. Capture of GFP-CBM3 by cellulose was first demonstrated qualitatively by fluorescence microscopy. The binding of the fusion proteins, the capture of antibodies (by ZZ-CBM3), and the grafting of an oligonucleotide (to CBM3C) were successfully demonstrated. The bioactive cellulose platform described here enables the precise anchoring of different biomolecules onto cellulose hydrogels and could contribute significatively to the development of advanced medical diagnostic sensors or specialized biomaterials, among others.

Engineering materials using supramolecular principles enables generalizable and modular platforms that have tunable chemical, mechanical, and biological properties. Applying this bottom‐up, molecular engineering‐based approach to therapeutic design affords unmatched control of emergent properties and functionalities. In preparing responsive materials for biomedical applications, the dynamic character of typical supramolecular interactions facilitates systems that can more rapidly sense and respond to specific stimuli through a fundamental change in material properties or characteristics, as compared to cases where covalent bonds must be overcome. Several supramolecular motifs have been evaluated toward the preparation of “smart” materials capable of sensing and responding to stimuli. Triggers of interest in designing materials for therapeutic use include applied external fields, environmental changes, biological actuators, applied mechanical loading, and modulation of relative binding affinities. In addition, multistimuli‐responsive routes can be realized that capture combinations of triggers for increased functionality. In sum, supramolecular engineering offers a highly functional strategy to prepare responsive materials. Future development and refinement of these approaches will improve precision in material formation and responsiveness, seek dynamic reciprocity in interactions with living biological systems, and improve spatiotemporal sensing of disease for better therapeutic deployment.

Halogenation is widespread in Nature, as witnessed by the several thousands of naturally occurring halogenated compounds. This review seeks to provide an overview on the pathways and multiple roles of halogenation in Nature, as a source of inspiration to chemists. Remarkably, several halogenation pathways were recently found in humans, sparking an increasing interest into halogens as biomolecular recognition sites. A description of the molecular mechanisms underlining the role of halogenation in the biomolecular context is provided, highlighting how the strategic use of halogen-driven interactions can be exploited to tune the properties of both small compounds and macromolecules. The complexity, versatility and bio-orthogonality of halogen-driven interactions are indeed key figures of merit in promoting novel opportunities for halogenation in a range of applications, spanning from drug design to self-assembly and biomolecular engineering. Specifically, the most relevant and impactful recent contributions in the field of peptide and protein science are presented and critically discussed, with a particular emphasis on the existing opportunities to control their conformational and self-assembly properties towards novel therapeutics and functional materials.

Accessible point-of-care technologies that can provide immunoassay and molecular modalities could dramatically enhance diagnostics, particularly for infectious disease control in low-resource settings. Solid-state nanopores are simple and durable sensors with low-energy instrumentation requirements. While nanopore sensors have demonstrated efficacy for nucleic acid targets, selective detection and quantification of target proteins from sample background has not been demonstrated. We present a simple approach for electronic detection and quantification of target proteins that combines novel biomolecular engineering methods, a portable reader device and disposable nanopore test strips. The target of interest can be varied by swapping the binding domain on our engineered detection reagent, which eficiently binds in the bulk-phase to the target and subsequently generates a unique signature when passing through the pore. We show modularity of the detection reagent for two HIV antibodies, TNFα and tetanus toxin as targets. A saliva swab-to-result is demonstrated for clinically relevant HIV antibody levels (0.4–20 mg/liter) in under 60 seconds. While other strip-like assays are qualitative, the presented method is quantitative and sets the stage for simultaneous immunoassay and molecular diagnostic functionality within a single portable platform.

Prelude The early chapters of this book introduce some of the chemicals that are important in human biology. In fact, it is possible to think of the human body as an elaborate bag of chemicals. In the subspecialty called biomolecular engineering (or bio-technology), biomedical engineers examine the changes in chemical components within a biological system and develop methods for modifying these chemicals or their interactions. The concept of introducing chemicals to induce a change in a biological system is familiar; for example, we all have some experience with taking purified chemicals such as acetaminophen or ibuprofen as drugs to relieve pain. But new biological tools now make it possible to consider more complex chemical interventions such as gene therapy (in which a new deoxyribonucleic acid [DNA] sequence is introduced to allow expression of a new genetic activity). The concept of the human body as a bag of chemicals is convenient and comforting. Chemicals can be produced; chemicals can be added or replaced; chemicals are (sometimes) inexpensive. In fact, several decades ago, it was widely reported that the chemicals in the human body were worth about $1. Other investigators estimate the value at closer to $6 million (particularly if the chemicals are purchased from scientific suppliers). Regardless of the dollar value, we often imagine that our bodies can be supplemented, mended, and improved through the addition of chemicals. But the body contains large numbers of many different kinds of chemicals, and as in many complex systems, unexpected patterns can emerge from the sum of the parts. Although the body is composed of chemicals, we don't understand the identity of all of the chemicals in the system, the forces that hold these chemicals together, or the network of interactions that mold the chemicals into an organism. Yet we often attempt to heal with chemicals. This is the challenge of drug delivery (Figure 13.1). Biomolecular engineering includes drug delivery, but also many similar areas of inquiry in which chemical engineering principles are applied to biomedical engineering (BME) problems.

Antibodies are vital for human health because of their ability to function as nature’s drugs by protecting the body from infection. In recent decades, antibodies have been used as pharmaceutics for targeted therapy in patients with cancer, autoimmune diseases, and cardiovascular diseases. Capturing the dynamic structure of antibodies and characterizing antibody fluctuation is critical for gaining a deeper understanding of their structural characteristics and for improving drug development. Current techniques for studying three-dimensional (3D) structural heterogeneity and variability of proteins have limitations in ascertaining the dynamic structural behavior of antibodies and antibody-antigen complexes. Here, we review current techniques used to study antibody structures with a focus on the recently developed individual-particle electron tomography (IPET) technique. IPET, as a particle-by-particle methodology for 3D structural characterization, has shown advantages in studying structural variety and conformational changes of antibodies, providing direct imaging data for biomolecular engineering to improve development and clinical application of synthetic antibodies.

A central question in protein evolution is the extent to which naturally occurring proteins sample the space of folded structures accessible to the polypeptide chain. Repeat proteins composed of multiple tandem copies of a modular structure unit are widespread in nature and have critical roles in molecular recognition, signalling, and other essential biological processes. Naturally occurring repeat proteins have been re-engineered for molecular recognition and modular scaffolding applications. Here we use computational protein design to investigate the space of folded structures that can be generated by tandem repeating a simple helix-loop-helix-loop structural motif. Eighty-three designs with sequences unrelated to known repeat proteins were experimentally characterized. Of these, 53 are monomeric and stable at 95 °C, and 43 have solution X-ray scattering spectra consistent with the design models. Crystal structures of 15 designs spanning a broad range of curvatures are in close agreement with the design models with root mean square deviations ranging from 0.7 to 2.5 Å. Our results show that existing repeat proteins occupy only a small fraction of the possible repeat protein sequence and structure space and that it is possible to design novel repeat proteins with precisely specified geometries, opening up a wide array of new possibilities for biomolecular engineering.

Collagen-based biomedical materials have developed into important, clinically effective materials used in a range of devices that have gained wide acceptance. These devices come with collagen in various formats, including those based on stabilized natural tissues, those that are based on extracted and purified collagens, and designed composite, biosynthetic materials. Further knowledge on the structure and function of collagens has led to on-going developments and improvements. Among these developments has been the production of recombinant collagen materials that are well defined and are disease free. Most recently, a group of bacterial, non-animal collagens has emerged that may provide an excellent, novel source of collagen for use in biomaterials and other applications. These newer collagens are discussed in detail. They can be modified to direct their function, and they can be fabricated into various formats, including films and sponges, while solutions can also be adapted for use in surface coating technologies.

Cellular reprogramming using cocktails of transcription factors (TFs) affirms the epigenetic and developmental plasticity of mammalian cells. It demonstrates the ability of TFs to 'read' genetic information and to rewire regulatory networks in different cellular contexts. Silenced chromatin is not an impediment to the genome engagement by ectopically expressed TFs. Reprogramming TFs have been identified in diverse structural families that lack shared domains or sequence motifs. Interestingly, the reprogramming activity of non-redundant paralogous TFs can be switched with a few point mutations. These findings revealed that the sequence-function relationships influencing reprogramming are tied to subtle features directing genome wide binding. Therefore, endogenous reprogramming TFs are amenable to directed biomolecular engineering that opens up new avenues to optimize cell fate conversions.

In nature, assembled protein structures offer the most complex functional structures. The understanding of the mechanisms ruling protein-protein interactions opens the door to manipulate protein assemblies in a rational way. Proteins are versatile scaffolds with great potential as tools in nanotechnology and biomedicine because of their chemical, structural, and functional versatility. Currently, bottom-up self-assembly based on biomolecular interactions of small and well-defined components, is an attractive approach to biomolecular engineering and biomaterial design. Specifically, repeat proteins are simplified systems for this purpose. In this work, we provide an overview of fundamental concepts of the design of new protein interfaces. We describe an experimental approach to form higher order architectures by a bottom-up assembly of repeated building blocks. For this purpose, we use designed consensus tetratricopeptide repeat proteins (CTPRs). CTPR arrays contain multiple identical repeats that interact through a single inter-repeat interface to form elongated superhelices. Introducing a novel interface along the CTPR superhelix allows two CTPR molecules to assemble into protein nanotubes. We apply three approaches to form protein nanotubes: electrostatic interactions, hydrophobic interactions, and π-π interactions. We isolate and characterize the stability and shape of the formed dimers and analyze the nanotube formation considering the energy of the interaction and the structure in the three different models. These studies provide insights into the design of novel protein interfaces for the control of the assembly into more complex structures, which will open the door to the rational design of nanostructures and ordered materials for many potential applications in nanotechnology.

Virus-like particles (VLPs) are repetitive organizations of viral proteins assembled in an appropriate physicochemical environment. VLPs can stimulate both innate and adaptive immune responses, due to their particulate structure enabling uptake by antigen presenting cells. These characteristics have led to successful development of VLP-vaccine products, and will ensure their vast potential in years to come. Future success of VLP therapeutic products will be determined by advances in their bioengineering, and also by the development of tools to design for their stability, function and application. This review focuses on approaches for VLP assembly in controlled chemical environments in vivo and in vitro, and the application of computational tools for improved chemical sequence design, and fundamental understanding of assembly.

This paper outlines the re-design of learning of Water Quality Analysis using problem-based learning (PBL) in a Year 2 module Chemical Engineering Laboratory (CEL) in Chemical and Biomolecular Engineering (CBE) diploma. By constructing an authentic problem in partnership with National Parks Board (NParks), the PBL approach has been shown to be constructive in multiple ways, ranging from creating integrative learning in a real-world context to shaping students to be self-directed and confident problem solvers. This integrative learning design avoids the compartmentalization of teaching of knowledge and concepts, hands-on technical skills and non-engineering “softskills” in separate lecture, practical and general education sessions, respectively. At the same time, it would be critically useful in preparing graduates to handle complex, industry-real issues and situations.

Novel methods of molecular modeling help solving urgent problems in drug design, directed evolution of biocatalysts and biosensors, and a lot of other research fields. Implementation of such methods to organophosphorus hydrolase being perfect research object that hydrolyzes dangerous neurotoxic organophosphates could intensify development of antidote and protective preparations to treat poisoning. Structures of enzyme-polyelectrolyte complexes (EPCs) based on hexahistidine-tagged organophosphorus hydrolase (His6-OPH) with different biopolymers (various modifications of polyglutamic and polyaspartic acid, as well as hydroxyethyl starch and succinylated gelatin) were simulated at different pH using molecular docking. A number of EPCs with expected “positive” effect on maintaining the maximum level of His6-OPH activity, and some “negative” options were produced, and their catalytic performance was studied. The theoretical results were experimentally confirmed for four of the six “positive” options. EPCs obtained possessed up to 20–40% higher catalytic efficiency in hydrolysis reactions of Paraoxon and Parathion-methyl as compared with that of the native His6-OPH. The results obtained may be a good proof of concept for implementation of molecular docking to calculate model complexes of proteins with (bio)polymers of 6.4–105.5 kg/mol. Also, the approach used here could be interesting as alternative or addition to the directed modifications of enzymes to alter their catalytic characteristics.

Biomolecular engineering can be used to purposefully manipulate biomolecules, such as peptides, proteins, nucleic acids and lipids, within the framework of the relations among their structures, functions and properties, as well as their applicability to such areas as developing novel biomaterials, biosensing, bioimaging, and clinical diagnostics and therapeutics. Nanotechnology can also be used to design and tune the sizes, shapes, properties and functionality of nanomaterials. As such, there are considerable overlaps between nanotechnology and biomolecular engineering, in that both are concerned with the structure and behavior of materials on the nanometer scale or smaller. Therefore, in combination with nanotechnology, biomolecular engineering is expected to open up new fields of nanobio/bionanotechnology and to contribute to the development of novel nanobiomaterials, nanobiodevices and nanobiosystems. This review highlights recent studies using engineered biological molecules (e.g., oligonucleotides, peptides, proteins, enzymes, polysaccharides, lipids, biological cofactors and ligands) combined with functional nanomaterials in nanobio/bionanotechnology applications, including therapeutics, diagnostics, biosensing, bioanalysis and biocatalysts. Furthermore, this review focuses on five areas of recent advances in biomolecular engineering: (a) nucleic acid engineering, (b) gene engineering, (c) protein engineering, (d) chemical and enzymatic conjugation technologies, and (e) linker engineering. Precisely engineered nanobiomaterials, nanobiodevices and nanobiosystems are anticipated to emerge as next-generation platforms for bioelectronics, biosensors, biocatalysts, molecular imaging modalities, biological actuators, and biomedical applications.

Azobenzene has been widely used as a photoregulator due to its reversible photoisomerization, large structural change between E and Z isomers, high photoisomerization yield, and high chemical stability. On the other hand, some azobenzene derivatives can be used as universal quenchers for many fluorophores. Nucleic acid is a good candidate to be modified because it is not only the template of gene expression but also widely used for building well-organized nanostructures and nanodevices. Because the size and polarity distribution of the azobenzene molecule is similar to a nucleobase pair, the introduction of azobenzene into nucleic acids has been shown to be an ingenious molecular design for constructing light-switching biosystems or light-driven nanomachines. Here we review recent advances in azobenzene-modified nucleic acids and their applications for artificial regulation of gene expression and enzymatic reactions, construction of photoresponsive nanostructures and nanodevices, molecular beacons, as well as obtaining structural information using the introduced azobenzene as an internal probe. In particular, nucleic acids bearing multiple azobenzenes can be used as a novel artificial nanomaterial with merits of high sequence specificity, regular duplex structure, and high photoregulation efficiency. The combination of functional groups with biomolecules may further advance the development of chemical biotechnology and biomolecular engineering.

Control over the functionality of interfaces through biomolecular engineering is a central tool for nanoscale technology as well as many current applications of biology. In this work we designed fusion proteins that combined the surface adhesion and interfacial activity of a hydrophobin-protein together with the high affinity biotin-binding capability of an avidin-protein. We found that an overall architecture that was based on a circularly permuted version of avidin, dual-chain avidin, and hydrophobin gave a highly functional combination. The protein was produced in the filamentous fungus Trichoderma reesei and was efficiently purified using an aqueous two-phase partitioning procedure. The surface adhesive properties were widely different compared to wild-type avidin. Functional characterization showed that the protein assembled on hydrophobic surfaces as a thin layer even at very low concentrations and efficiently bound a biotinylated compound. The work shows how the challenge of creating a fusion protein with proteins that form multimers can be solved by structural design and how protein self-assembly can be used to efficiently functionalize interfaces.

Fungi are a prolific source of bioactive compounds, some of which have been developed as essential medicines and life-enhancing drugs. Genome sequencing has revealed that fungi have the potential to produce considerably more natural products (NPs) than are typically observed in the laboratory. Recently, there have been significant advances in the identification, understanding, and engineering of fungal biosynthetic gene clusters (BGCs). This review briefly describes examples of the engineering of fungal NP biosynthesis at the global, pathway, and enzyme level using in vivo and in vitro approaches and refers to the range and scale of heterologous expression systems available, developments in combinatorial biosynthesis, progress in understanding how fungal BGCs are regulated, and the applications of these novel biosynthetic enzymes as biocatalysts.

Folding-based electrochemical sensors utilizing structure-switching aptamers are specific, selective, sensitive, and widely applicable to the detection of a variety of target analytes. The specificity is achieved by the binding properties of an electrode-bound RNA or DNA aptamer biorecognition element. Signaling in this class of sensors arises from changes in electron transfer efficiency upon target-induced changes in the conformation/flexibility of the aptamer probe. These changes can be readily monitored electrochemically. Because of this signaling mechanism, there are several approaches to maximizing the analytical attributes (i.e., sensitivity, limit of detection, and observed binding affinity) of the aptamer sensor. Here, we present a systematic study of several approaches, including electrochemical interrogation parameters and biomolecular engineering of the aptamer sequence, to develop a sensor for the detection of aminoglycoside antibiotics. Specifically, through a combination of optimizing the electrochemical signal and engineering the parent 26-nucleotide RNA aptamer sequence to undergo larger conformation changes, we develop several improved sensors. These sensors exhibit binding affinities ranging from 220 nM to 42 μM, as much as a 100-fold improved limit of detection in comparison to previously reported sensors, and a variety of linear ranges including the therapeutic window for tobramycin. These data demonstrate that rational engineering of the aptamer structure to create large conformation changes upon target binding leads to improved sensor performance. We believe that the sensor design guidelines outlined here represent a general strategy for developing new aptamer folding-based electrochemical sensors.

Si-Shen Feng Author for correspondence: Department of Chemical & Biomolecular Engineering, Department of Bioengineering, & Nanoscience & Nanotechnology Initiative (NUSNNI/NanoCore), National University of Singapore, Block E3, 05–29, 2 Engineering Drive 3, 117576, Singapore Tel.: +65 6576 3835 Fax: +65 6779 1936 chefess@nus.edu.sg Star-shaped block polymers as a molecular biomaterial for nanomedicine development

Nanomedicine is an emerging field that integrates nanotechnology, biomolecular engineering, life sciences and medicine; it is expected to produce major breakthroughs in medical diagnostics and therapeutics. Nano-scale structures and devices are compatible in size with proteins and nucleic acids in living cells. Therefore, the design, characterization and application of nano-scale probes, carriers and machines may provide unprecedented opportunities for achieving a better control of biological processes, and drastic improvements in disease detection, therapy, and prevention. Recent advances in nanomedicine include the development of nanoparticle-based probes for molecular imaging, nano-carriers for drug/gene delivery, multi-functional nanoparticles for theranostics, and molecular machines for biological and medical studies. This article provides an overview of the nanomedicine field, with an emphasis on nanoparticles for imaging and therapy, as well as engineered nucleases for genome editing. The challenges in translating nanomedicine approaches to clinical applications are discussed.

Biomolecular engineering enables synthesis of improved proteins through synergistic fusion of modules from unrelated biomolecules. Modularization of peptide antigen from an unrelated pathogen for presentation on a modular virus-like particle (VLP) represents a new and promising approach to synthesize safe and efficacious vaccines. Addressing a key knowledge gap in modular VLP engineering, this study investigates the underlying fundamentals affecting the ability of induced antibodies to recognize the native pathogen. Specifically, this quality of immune response is correlated to the peptide antigen module structure. We modularized a helical peptide antigen element, helix 190 (H190) from the influenza hemagglutinin (HA) receptor binding region, for presentation on murine polyomavirus VLP, using two strategies aimed to promote H190 helicity on the VLP. In the first strategy, H190 was flanked by GCN4 structure-promoting elements within the antigen module; in the second, dual H190 copies were arrayed as tandem repeats in the module. Molecular dynamics simulation predicted that tandem repeat arraying would minimize secondary structural deviation of modularized H190 from its native conformation. In vivo testing supported this finding, showing that although both modularization strategies conferred high H190-specific immunogenicity, tandem repeat arraying of H190 led to a strikingly higher immune response quality, as measured by ability to generate antibodies recognizing a recombinant HA domain and split influenza virion. These findings provide new insights into the rational engineering of VLP vaccines, and could ultimately enable safe and efficacious vaccine design as an alternative to conventional approaches necessitating pathogen cultivation.

The ability to synthesize, characterize, and manipulate DNA forms the foundation of a range of advanced disciplines including genomics, molecular biology, and biomolecular engineering. In particular for the latter field, DNA has proven useful as a structural or functional component in nanoscale self-assembled structures, antisense therapeutics, microarray diagnostics, and biosensors. Such applications frequently require DNA to be modified and conjugated to other macromolecules, including proteins, polymers, or fatty acids, in order to equip the system with properties required for a particular application. However, conjugation of DNA to large molecular components using classical chemistries often suffers from suboptimal yields. Here, we report the use of terminal deoxynucleotidyl transferase (TdT) for direct enzymatic ligation of native DNA to nucleotide triphosphates coupled to proteins and other large macromolecules. We demonstrate facile synthesis routes for a range of NTP-activated macromolecules and subsequent ligation to the 3' hydroxyl group of oligodeoxynucleotides using TdT. The reaction is highly specific and proceeds rapidly and essentially to completion at micromolar concentrations. As a proof of principle, parallelly labeled oligonucleotides were used to produce nanopatterned DNA origami structures, demonstrating rapid and versatile incorporation of non-DNA components into DNA nanoarchitectures.

The recent recognition that halogen bonding (XB) plays important roles in the recognition and assembly of biological molecules has led to new approaches in medicinal chemistry and biomolecular engineering. When designing XBs into strategies for rational drug design or into a biomolecule to affect its structure and function, we must consider the relationship between this interaction and the more ubiquitous hydrogen bond (HB). In this review, we explore these relationships by asking whether and how XBs can replace, compete against or behave independently of HBs in various biological systems. The complex relationships between the two interactions inform us of the challenges we face in fully utilizing XBs to control the affinity and recognition of inhibitors against their therapeutic targets, and to control the structure and function of proteins, nucleic acids and other biomolecular scaffolds.

International audience; Mycotoxin contamination of food and feed is now ubiquitous. Exposures to mycotoxin via contact or ingestion can potentially induce adverse health outcomes. Affordable mycotoxin-monitoring systems are highly desired but are limited by (a) the reliance on technically challenging and costly molecular recognition by immuno-capture technologies; and (b) the lack of predictive tools for directing the optimization of alternative molecular recognition modalities. Our group has been exploring the development of ochratoxin detection and monitoring systems using the peptide NFO4 as the molecular recognition receptor in fluorescence, electrochemical and multimodal biosensors. Using ochratoxin as the model mycotoxin, we share our perspective on addressing the technical challenges involved in biosensor fabrication, namely: (a) peptide receptor design; and (b) performance evaluation. Subsequently, the scope and utility of molecular modeling and simulation (MMS) approaches to address the above challenges are described. Informed and enabled by phage display, the subsequent application of MMS approaches can rationally guide subsequent biomolecular engineering of peptide receptors, including bioconjugation and bioimmobilization approaches to be used in the fabrication of peptide biosensors. MMS approaches thus have the potential to reduce biosensor development cost, extend product life cycle, and facilitate multi-analyte detection of mycotoxins, each of which positively contributes to the overall affordability of mycotoxin biosensor monitoring systems.

Natural products are complex small molecules produced by an array of organisms and which humans have put to diverse uses, including as pesticides, antibiotics, and anticancer agents. While a wide range has already been uncovered it is clear that there are many more potential biosynthetic pathways for which the products have never been studied. Using the tools of synthetic biology allows us to manipulate these pathways and produce the desired compounds. The genes involved can also be transferred into other organisms which can be more easily manipulated. Recent advances in understanding these complex systems have also informed the targeted manipulation of biosynthetic pathways to produce specific modifications of the final natural product. In the future it may be possible, rather than relying on chemical synthesis, to design and build artificial pathways to synthesize completely new compounds as desired.

Many important biological processes – ranging from simple metabolism to complex cognition – take place deep inside living organisms, yet our ability to study them in this context is very limited. Technologies such as fluorescent proteins and optogenetics enable exquisitely precise imaging and control of cellular function in small, translucent specimens using visible light, but are limited by the poor penetration of such light into larger tissues. In contrast, most non-invasive technologies such as magnetic resonance imaging (MRI) and ultrasound – while based on energy forms that penetrate tissue effectively – lack the needed molecular precision. Our work attempts to bridge this gap by engineering new molecular technologies that connect penetrant energy to specific aspects of cellular function in vivo. In this talk, I will describe molecular reporters for non-invasive imaging using MRI and ultrasound developed by adapting and engineering naturally occurring proteins. These proteins have physical properties, such as paramagnetism or self-assembly into hollow nanostructures, that allow them to be sensitively detected with MRI and ultrasound. By engineering them at the genetic level, we have adapted these natural constructs into noninvasive molecular reporters of biological processes ranging from gene expression to chemical neurotransmission and metabolism. In addition, I will describe recent work on the use of penetrant forms of energy to control cellular function within the body.

Proteins are the key biomolecules in almost all the physiological and pathological processes that are occurring in the cell. Functionality of proteins is related to its conformation, which is ultimately dictated by their unique amino acid sequence. In the current chapter, a brief overview will be provided on the nature of amino acids, structural characteristics and functional versatility of proteins. A glimpse on various interactions present in stabilizing the protein fold and the methods for unraveling their atomic level structures are also discussed. Finally, we present the role of engineered synthetic proteins for the welfare of humanity with huge potential in research and industrial sectors including biotechnological and biomedical fields under the emerging concepts of biomolecular/protein engineering.

Synthetic Biology offers qualitatively new perspectives on the benefits of industrially harnessed biological processes. The ability to modify and reprogramme natural biology increases the scope of tailored bioprocesses and yields attractive prospects beyond conventional Biotechnology. The present review summarises the major achievements and categorises them according to a hierarchy of system levels. Similar structures are known in the engineering sciences and might prove useful for the future development of Synthetic Biology. The hierarchy encompasses several levels of detail. Biological (macro-)molecules present the most detailed level (parts), followed by compartmentalised or non-compartmentalised modules (devices). In the next level, parts and devices are combined into functional cells and further into cellular communities. The manifold interactions between biological entities of the same hierarchical level or between different levels are accounted for by networks, primarily metabolic pathways and regulatory circuits. Networks of different types are represented as a superordinate hierarchical level that achieves full system integration. On all these levels, extensive and sound scientific foundations exist regarding experimental but also theoretical methods. These have led to diverse manifestations of Synthetic Biology on the parts and devices levels. Investigations involving synthetic components on the systems scale represent the most difficult and remain limited in number. A main challenge lies with the quantitative prediction of interactions between different entities across different scales. Systems-theoretical approaches provide important tools to analyse complex biological behaviour and can support the design of artificial biological systems. A promising strategy is seen in an efficient modularisation that reduces biological systems to a limited set of functional modules with well-characterised interfaces. For the design of synthetic biological systems the interactions across these interfaces should be standardised to reduce complexity. Yet, the identification of modules and standardised interaction routes remains a non-trivial problem. Furthermore, an appropriate platform that efficiently describes replication and evolutionary processes has to be developed in order to extend the achievements of Synthetic Biology into designed biological processes.