A cell-free biosensor for multiplexed and sensitive detection of biological warfare agents.

The rapid and precise detection of pathogenic agents is critical for public health and societal stability. The detection of biological warfare agents (BWAs) is especially vital within military and counter-terrorism contexts, essential in defending against biological threats. Traditional methods, such as polymerase chain reaction (PCR), are limited by their need for specific settings, impacting their adaptability and versatility. This study introduces a cell-free biosensor for BWA detection by converting the 16S rRNA of targeted pathogens into detectable functional protein molecules. The modular nature of this approach allows for the flexible configuration of pathogen detection, enabling the simultaneous identification of multiple pathogenic 16S rRNAs through customized reporter proteins for each targeted sequence. Furthermore, we demonstrate how this method integrates with techniques utilizing retroreflective Janus particles (RJPs) for facile and highly sensitive pathogen detection. The cell-free biosensor, employing RJPs to measure the reflection of non-chromatic white light, can detect 16S rRNA from BWAs at femtomolar levels, corresponding to tens of colony-forming units per milliliter of pathogenic bacteria. These findings represent a significant advancement in pathogen detection, offering a more efficient and accessible alternative to conventional methodologies.

Sensitive Methods to Detect Single-Stranded Nucleic Acids of Food Pathogens Based on Cell-Free Protein Synthesis and Retroreflection Signal Detection.

Cell-free protein synthesis (CFPS) has recently gained considerable attention as a new platform for developing methods to detect various molecules, ranging from small chemicals to biological macromolecules. Retroreflection has been used as an alternative signal to develop analytical methods because it can be detected by using a simple instrument comprising a white light source and a camera. Here, we report a novel reporter protein that couples the capability of CFPS and the simplicity of retroreflection signal detection. The design of the reporter was based on two pairs of protein-peptide interactions, SpyCatcher003-SpyTag003 and MDM2-PMI(N8A). MDM2-MDM2-SpyCatcher003 was decided as the reporter protein, and the two peptides, SpyTag003 and PMI(N8A), were immobilized on the surfaces of retroreflective Janus particles and microfluidic chips, respectively. The developed retroreflection signal detection system was combined with a previously reported CFPS reaction that can transduce the presence of a single-stranded nucleic acid into protein synthesis. The resulting methods were applied to detect 16S rRNAs of several foodborne pathogens. Concentration-dependent relationships were observed over a range of 10° fM to 102 pM, with the limits of detection being single-digit femtomolar concentrations. Considering the designability of the CFPS system for other targets, the retroreflection signal detection method will enable the development of novel methods to detect various molecules.

Assay methods based on proximity-enhanced reactions for detecting non-nucleic acid molecules.

Accurate and reliable detection of biological molecules such as nucleic acids, proteins, and small molecules is essential for the diagnosis and treatment of diseases. While simple homogeneous assays have been developed and are widely used for detecting nucleic acids, non-nucleic acid molecules such as proteins and small molecules are usually analyzed using methods that require time-consuming procedures and highly trained personnel. Recently, methods using proximity-enhanced reactions (PERs) have been developed for detecting non-nucleic acids. These reactions can be conducted in a homogeneous liquid phase via a single-step procedure. Herein, we review three assays based on PERs for the detection of non-nucleic acid molecules: proximity ligation assay, proximity extension assay, and proximity proteolysis assay.

Coupling hCG-based protease sensors with a commercial pregnancy test strip for simple analyses of protease activities.

Proteases play an essential role in many cellular processes, and consequently, abnormalities in their activities are related to various diseases. Methods have been developed to measure the activity of these enzymes, but most involve sophisticated instruments or complicated procedures, which hampers the development of a point-of-care test (POCT). Here, we propose a strategy for developing simple and sensitive methods to analyze protease activity using commercial pregnancy test strips that detect human chorionic gonadotropin (hCG). hCG was engineered to have site-specific conjugated biotin and a peptide sequence, which can be cleaved by a target protease, between hCG and biotin. hCG protein was immobilized on streptavidin-coated beads, resulting in a protease sensor. The hCG-immobilized beads were too large to flow through the membrane of the hCG test strip and yielded only one band in the control line. When the peptide linker was hydrolyzed by the target protease, hCG was released from the beads, and the signal appeared in both the control and test lines. Three protease sensors for matrix metalloproteinase-2, caspase-3, and thrombin were constructed by replacing the protease-cleavable peptide linker. The combination of the protease sensors and a commercial pregnancy strip enabled the specific detection of each protease in the picomolar range, with a 30-min incubation of the hCG-immobilized beads and samples. The modular design of the protease sensor and simple assay procedure will facilitate the development of POCTs for various protease disease markers.

Non-Canonical Amino Acid-Based Engineering of (R)-Amine Transaminase.

Non-canonical amino acids (ncAAs) have been utilized as an invaluable tool for modulating the active site of the enzymes, probing the complex enzyme mechanisms, improving catalytic activity, and designing new to nature enzymes. Here, we report site-specific incorporation of p-benzoyl phenylalanine (pBpA) to engineer (R)-amine transaminase previously created from d-amino acid aminotransferase scaffold. Replacement of the single Phe88 residue at the active site with pBpA exhibits a significant 15-fold and 8-fold enhancement in activity for 1-phenylpropan-1-amine and benzaldehyde, respectively. Reshaping of the enzyme's active site afforded an another variant F86A/F88pBpA, with 30% higher thermostability at 55°C without affecting parent enzyme activity. Moreover, various racemic amines were successfully resolved by transaminase variants into (S)-amines with excellent conversions (∼50%) and enantiomeric excess (>99%) using pyruvate as an amino acceptor. Additionally, kinetic resolution of the 1-phenylpropan-1-amine was performed using benzaldehyde as an amino acceptor, which is cheaper than pyruvate. Our results highlight the utility of ncAAs for designing enzymes with enhanced functionality beyond the limit of 20 canonical amino acids.

Development of one-step isothermal methods to detect RNAs using hairpin-loop signal converters and proximity proteolysis reaction.

Ribonucleic acids (RNAs) provide valuable information for biological systems and act as important indicators of disease states. RNAs are diverse in size and structure, and various strategies have been proposed for the detection of nucleic acids; however, developing them into point-of-care (POC) tests has been challenging as most of them consist of complex time-consuming steps. Here, we propose a strategy to assay RNAs using a hairpin-loop (HP) converter and proximity proteolysis reaction (PPR). Interaction between the loop part of HP and its target exposes a single strand of nucleotides, which acts as the template for PPR. A pair of protease and zymogen-conjugated nucleic acids associates with the adjacent regions of the template, resulting in an enhanced proteolysis reaction between protease and zymogen. The activated zymogen then generates a color signal through the hydrolysis of a chromogenic substrate. The combination of HP converter and PPR allowed the same pair of protease- and zymogen-nucleic acids to be used for different RNAs. Guidelines were provided for designing HP converters based on computational analyses and experimental characterizations. This strategy using an HP converter and PPR has been successfully applied to develop simple isothermal methods for the detection of various RNAs, including several microRNAs and KRAS mRNA, in the picomolar range in 1 h. The simplicity of designing HP converters and the beneficial properties of PPR as POC tests would enable the development of novel methods to detect RNAs under low-resource conditions.

Towards Engineering an Orthogonal Protein Translation Initiation System.

In the last two decades, methods to incorporate non-canonical amino acids (ncAAs) into specific positions of a protein have advanced significantly; these methods have become general tools for engineering proteins. However, almost all these methods depend on the translation elongation process, and strategies leveraging the initiation process have rarely been reported. The incorporation of a ncAA specifically at the translation initiation site enables the installation of reactive groups for modification at the N-termini of proteins, which are attractive positions for introducing abiological groups with minimal structural perturbations. In this study, we attempted to engineer an orthogonal protein translation initiation system. Introduction of the identity elements of Escherichia coli initiator tRNA converted an engineered Methanococcus jannaschii tRNATyr into an initiator tRNA. The engineered tRNA enabled the site-specific incorporation of O-propargyl-l-tyrosine (OpgY) into the amber (TAG) codon at the translation initiation position but was inactive toward the elongational TAG codon. Misincorporation of Gln was detected, and the engineered system was demonstrated only with OpgY. We expect further engineering of the initiator tRNA for improved activity and specificity to generate an orthogonal translation initiation system.

One-pot colorimetric detection of molecules based on proximity proteolysis reaction.

Various types of molecules serve as biomarkers of diseases, and numerous methods have been reported to detect and quantify them. Recently, research efforts have been made to develop point-of-care (POC) tests, which contribute to early diagnoses of diseases, particularly in resource-limited settings. An assay performed in a homogeneous phase is an obvious route to develop these methods. Here, simple homogeneous methods based on proximity proteolysis reactions (PPR) are reported to detect biological molecules. A typical PPR system has been designed such that the proteolysis reaction between protease and zymogen is enhanced in the presence of a target analyte. The activated zymogen generates a color signal by hydrolyzing a chromophore. A protease and zymogen are linked to target binders using specific hybridization between complementary single-stranded DNAs, and several molecules, including proteins, antibodies, aptamers, and small molecules, are used as target binders. The developed assay methods successfully detected several kinds of analytes at subnanomolar concentrations with the one-step procedure and color signal. The modular design of the PPR-based assay will enable the development of simple POC diagnostics for various biomarkers.

Recent Advances in Biocatalysis with Chemical Modification and Expanded Amino Acid Alphabet.

The two main strategies for enzyme engineering, directed evolution and rational design, have found widespread applications in improving the intrinsic activities of proteins. Although numerous advances have been achieved using these ground-breaking methods, the limited chemical diversity of the biopolymers, restricted to the 20 canonical amino acids, hampers creation of novel enzymes that Nature has never made thus far. To address this, much research has been devoted to expanding the protein sequence space via chemical modifications and/or incorporation of noncanonical amino acids (ncAAs). This review provides a balanced discussion and critical evaluation of the applications, recent advances, and technical breakthroughs in biocatalysis for three approaches: (i) chemical modification of cAAs, (ii) incorporation of ncAAs, and (iii) chemical modification of incorporated ncAAs. Furthermore, the applications of these approaches and the result on the functional properties and mechanistic study of the enzymes are extensively reviewed. We also discuss the design of artificial enzymes and directed evolution strategies for enzymes with ncAAs incorporated. Finally, we discuss the current challenges and future perspectives for biocatalysis using the expanded amino acid alphabet.

Viscoelastic particle focusing in human biofluids.

Saliva and blood plasma are non-Newtonian viscoelastic fluids that play essential roles in the transport of particulate matters (e.g., food and blood cells). However, whether the viscoelasticity of such biofluids alters the dynamics of suspended particles is still unknown. In this study, we report that under pressure-driven microflows of both human saliva and blood plasma, spherical particles laterally migrate and form a focused stream along the channel centerline by their viscoelastic properties. We observed that the particle focusing varied among samples on the basis of sampling times/donors, thereby demonstrating that the viscoelasticity of the human biofluids can be affected by their compositions. We showed that the particle focusing, observed in bovine submaxillary mucin solutions, intensified with the increase in mucin concentration. We expect that the findings from this study will contribute to the understanding of the physiological roles of viscoelasticity of human biofluids.

Methods to generate site-specific conjugates of antibody and protein.

Antibody-protein conjugates have been useful tools for studying biological systems and also played important roles in developing therapeutics and diagnostics. In particular, because of the increased interest in therapeutics of complexity higher than monoclonal antibodies, various methods have been reported for generating bispecific antibodies, immunotoxins, and antibody-enzyme conjugates in which antibodies are site-specifically conjugated with other proteins. Compared with conjugating antibodies with synthetic molecules, controlling the modification sites is difficult in the antibodies conjugated with protein cargos due to the presence of several reactive groups in both molecules. Enzymatic reactions are often used to generate antibody-protein conjugates owing to their high specificity for both reactants and products. Chemical modifications involving genetic introduction of natural or unnatural amino acid residues have also been used for site-specific conjugation of antibodies. Recent studies have developed methods to modify native antibodies using peptides having affinity for antibodies, and these methods do not need antibody engineering for conjugation reactions. In this review, we have summarized enzymatic and chemical approaches to generate site-specific antibody-protein conjugates.

Recent Progress in the Understanding and Engineering of Coenzyme B12-Dependent Glycerol Dehydratase.

Coenzyme B12-dependent glycerol dehydratase (GDHt) catalyzes the dehydration reaction of glycerol in the presence of adenosylcobalamin to yield 3-hydroxypropanal (3-HPA), which can be converted biologically to versatile platform chemicals such as 1,3-propanediol and 3-hydroxypropionic acid. Owing to the increased demand for biofuels, developing biological processes based on glycerol, which is a byproduct of biodiesel production, has attracted considerable attention recently. In this review, we will provide updates on the current understanding of the catalytic mechanism and structure of coenzyme B12-dependent GDHt, and then summarize the results of engineering attempts, with perspectives on future directions in its engineering.

Hydrogen-Bond Free Energy of Local Biological Water.

Here, we propose an experimental methodology based on femtosecond-resolved fluorescence spectroscopy to measure the hydrogen (H)-bond free energy of water at protein surfaces under isothermal conditions. A demonstration was conducted by installing a non-canonical isostere of tryptophan (7-azatryptophan) at the surface of a coiled-coil protein to exploit the photoinduced proton transfer of its chromophoric moiety, 7-azaindole. The H-bond free energy of this biological water was evaluated by comparing the rates of proton transfer, sensitive to the hydration environment, at the protein surface and in bulk water, and it was found to be higher than that of bulk water by 0.4 kcal mol-1 . The free-energy difference is dominated by the entropic cost in the H-bond network among water molecules at the hydrophilic and charged protein surface. Our study opens a door to accessing the energetics and dynamics of local biological water to give insight into its roles in protein structure and function.

Construction of an immunotoxin via site-specific conjugation of anti-Her2 IgG and engineered Pseudomonas exotoxin A.

BACKGROUND: Immunotoxins consisting of a toxin from bacteria or plants and a targeting module have been developed as potent anti-cancer therapeutics. The majority of them, especially those in preclinical or clinical testing stages, are fusion proteins of a toxin and antibody fragment. Immunotoxins based on full-length antibodies are less studied, even though the fragment crystallizable (Fc) domain plays an important role in regulating the concentration of immunoglobulin G (IgG) in the serum and in antibody-mediated immune responses against pathogens. RESULTS: We devised a method to site-specifically conjugate IgG and another protein using a cysteine residue introduced into the IgG and a bio-orthogonally reactive unnatural amino acid incorporated into the other protein. The human epidermal growth factor receptor 2 (Her2)-targeting IgG, trastuzumab, was engineered to have an unpaired cysteine in the heavy chain, and an unnatural amino acid with the azido group was incorporated into an engineered Pseudomonas exotoxin A (PE24). The two protein molecules were conjugated site-specifically using a bifunctional linker having dibenzocyclooctyne and maleimide groups. Binding to Her2 and interaction with various Fc receptors of trastuzumab were not affected by the conjugation with PE24. The trastuzumab-PE24 conjugate was cytotoxic to Her2-overexpressing cell lines, which involved the inhibition of cellular protein synthesis due to the modification of elongation factor-2. CONCLUSIONS: We constructed the site-specifically conjugated immunotoxin based on IgG and PE24, which induced target-specific cytotoxicity. To evaluate the molecule as a cancer therapeutic, animal studies are planned to assess tumor regression, half-life in blood, and in vivo immunogenicity. In addition, we expect that the site-specific conjugation method can be used to develop other antibody-protein conjugates for applications in therapeutics and diagnostics.

Linear and Rationally Designed Stapled Peptides Abrogate TLR4 Pathway and Relieve Inflammatory Symptoms in Rheumatoid Arthritis Rat Model.

A mounting evidence exists for the despicable role of the aberrant immune response in the pathogenesis of rheumatoid arthritis (RA), where toll-like receptor 4 (TLR4) can activate synovial fibroblasts that lead to the chronic inflammation and joint destruction, thus making TLR4 a potent drug target in RA. We report that novel TLR4-antagonizing peptide, PIP2, inhibits the induction of inflammatory biomarkers in vitro as well as in vivo. Systemically, PIP2 inhibits the lipopolysaccharide (LPS)-elicited TNF-α, IL-6, and IL-12p40 in a mouse model. The rationally designed cyclic derivative, cPIP2, is capable of inhibiting LPS-induced proinflammatory cytokines at significantly lower concentration as compared to PIP2 (PIP2 IC50 = 20 µM, cPIP2 IC50 = 5 µM). Finally, cPIP2 was able to relieve the inflammatory symptoms and synovial tissue destruction in the RA rat model. Cumulatively, these data suggest that PIP2 and cPIP2 hold strong promise for the development of peptide-based immunotherapeutics that could be of great value in curbing TLR-related immune complications including RA.

Normal stress difference-driven particle focusing in nanoparticle colloidal dispersion.

Colloidal dispersion has elastic properties due to Brownian relaxation process. However, experimental evidence for the elastic properties, characterized with normal stress differences, is elusive in shearing colloidal dispersion, particularly at low Péclet numbers (Pe < 1). Here, we report that single micrometer-sized polystyrene (PS) beads, suspended in silica nanoparticle dispersion (8 nm radius; 22%, v/v), laterally migrate and form a tightly focused stream by the normal stress differences, generated in pressure-driven microtube flow at low Pe. The nanoparticle dispersion was expected to behave as a Newtonian fluid because of its ultrashort relaxation time (2 µs), but large shear strain experienced by the PS beads causes the notable non-Newtonian behavior. We demonstrate that the unique rheological properties of the nanoparticle dispersion generate the secondary flow in perpendicular to mainstream in a noncircular conduit, and the elastic properties of blood plasma-constituting protein solutions are elucidated by the colloidal dynamics of protein molecules.

Identification of peptide inhibitors of matrix metalloproteinase 1 using an in-house assay system for the enzyme.

Matrix metalloproteinases (MMPs) are zinc-dependent proteases involved in the degradation of extracellular matrix proteins. As one of the isoforms, MMP-1 breaks down collagen, and its activity is known to be important in wound healing. Its timely and adequate level of expression is pivotal because MMP-1 is also involved in the damage or aging of skins as well as in certain types of cancers. Thus, both assaying the MMP-1 activity and developing its inhibitors are of great importance. We here developed an in-house assay system that gave us the high degree of freedom in screening peptide inhibitors of MMP-1. The assay system utilized a circularly permutated fusion of ß-lactamase and its inhibitory protein through an MMP-1-sensitive linker so that the activity of MMP-1 could be translated into that of ß-lactamase. As a proof of concept, we applied the developed assay system to initial screens of MMP-1 inhibitors and successfully identified one lead peptide that inhibited the collagenase activity of the enzyme.

Interfacing a Personal Glucose Meter with Cell-Free Protein Synthesis for Rapid Analysis of Amino Acids.

We developed a method to analyze amino acids using a personal glucose meter (PGM). In this method, the principles of protein biosynthesis were interfaced with the sensing mechanism of a PGM to enable simple and ubiquitous measurement of amino acids. A reaction mixture for cell-free protein synthesis was designed to synthesize a bacterial invertase in response to exogenous addition of a specific amino acid. The invertase synthesized upon addition of an assay sample containing the amino acid of interest was used to convert sucrose into glucose, which was detected using a PGM. The titers of the amino acid in assay samples were precisely represented by the readouts of a PGM. In addition to the convenience provided by use of a PGM, the accuracy and reproducibility of this method were comparable to those of standard high-performance liquid chromatography based methods.

Biomimetic Scaffolds for Bone Tissue Engineering.

The use of biomimetic scaffolds for bone tissue engineering has been studied for a long time. Biomimetic scaffolds can assist and accelerate bone regeneration that is similar to that of authentic tissue, which represents the environment of cells in a living organism. Currently, numerous biomaterials have been reported for use as a biomimetic scaffold. This review focuses on the design of biomimetic scaffolds, kinds of biomaterials and methods used to fabricate biomimetic scaffolds, growth factors used with biomimetic scaffold for bone regeneration, mobilization of biological agents into biomimetic scaffolds, and studies on (pre)clinical bone regeneration from biomimetic scaffolds. Then, future prospects for biomimetic scaffolds are discussed.

Nucleic Acid Detection by a Target-Assisted Proximity Proteolysis Reaction.

Nucleic acid analysis plays an important role in diagnosing diseases as well as understanding biology. Despite advances in technology, there is still a need to develop a rapid and simple method to detect specific nucleic acids, especially in remote locations and low-resource cases. Here, we proposed a proximity proteolysis reaction in which the reaction between protease and zymogen is enhanced in the presence of a target molecule. The pair of proteins was site-specifically modified with oligonucleotides, and the conjugates were used to develop a method of detecting nucleic acids. Target DNA and RNA could be detected in less than 1 h at sub-nanomolar concentrations based on an absorbance signal. The assay method was resistant to interference by biological matrixes, and its sensitivity could be improved when combined with an isothermal nucleic acid amplification method. The results demonstrated the feasibility of this proximity proteolysis reaction as a new platform technology for detecting specific nucleic acid sequences.