Picomolar-Level Sensing of Cannabidiol by Metal Nanoparticles Functionalized with Chemically Induced Dimerization Binders.

Simple and fast detection of small molecules is critical for health and environmental monitoring. Methods for chemical detection often use mass spectrometers or enzymes; the former relies on expensive equipment, and the latter is limited to those that can act as enzyme substrates. Affinity reagents like antibodies can target a variety of small-molecule analytes, but the detection requires the successful design of chemically conjugated targets or analogs for competitive binding assays. Here, we developed a generalizable method for the highly sensitive and specific in-solution detection of small molecules, using cannabidiol (CBD) as an example. Our sensing platform uses gold nanoparticles (AuNPs) functionalized with a pair of chemically induced dimerization (CID) nanobody binders (nanobinders), where CID triggers AuNP aggregation and sedimentation in the presence of CBD. Despite moderate binding affinities of the two nanobinders to CBD (equilibrium dissociation constants KD of ∼6 and ∼56 µM), a scheme consisting of CBD-AuNP preanalytical incubation, centrifugation, and electronic detection (ICED) was devised to demonstrate a high sensitivity (limit of detection of ∼100 picomolar) in urine and saliva, a relatively short sensing time (∼2 h), a large dynamic range (5 logs), and a sufficiently high specificity to differentiate CBD from its analog, tetrahydrocannabinol. The high sensing performance was achieved with the multivalency of AuNP sensing, the ICED scheme that increases analyte concentrations in a small assay volume, and a portable electronic detector. This sensing system is readily applicable for wide molecular diagnostic applications.

Picomolar-Level Sensing of Cannabidiol by Metal Nanoparticles Functionalized with Chemically Induced Dimerization Binders.

Simple and fast detection of small molecules is critical to health and environmental monitoring. Methods for chemical detection often use mass spectrometers or enzymes; the former relies on expensive equipment and the latter is limited to those that can act as enzyme substrates. Affinity reagents like antibodies can target a variety of small-molecule analytes, but the detection requires successful design of chemically conjugated targets or analogs for competitive binding assays. Here, we developed a generalizable method for highly sensitive and specific in-solution detection of small molecules, using cannabidiol (CBD) as an example. Our sensing platform uses gold nanoparticles (AuNPs) functionalized with a pair of chemically induced dimerization (CID) nanobody binders (nano-binders), where CID triggers AuNPs aggregation and sedimentation in the presence of CBD. Despite moderate binding affinities of the two nano-binders to CBD (KDs of ~6 and ~56 µM), a scheme consisting of CBD-AuNP pre-analytical incubation, centrifugation, and electronic detection (ICED) was devised to demonstrate a high sensitivity (limit of detection of ~100 picomolar) in urine and saliva, a relatively short assay time (~2 hours), a large dynamic range (5 logs), and a sufficiently high specificity to differentiate CBD from its analog, tetrahydrocannabinol. The high sensing performance was achieved with the multivalency of AuNP sensing, the ICED scheme that increases analyte concentrations in a small assay volume, and a portable electronic detector. This sensing system is readily coupled to other binders for wide molecular diagnostic applications.

Identification of Anti-TNFα VNAR Single Domain Antibodies from Whitespotted Bambooshark (Chiloscyllium plagiosum).

Tumor necrosis factor α (TNFα), an important clinical testing factor and drug target, can trigger serious autoimmune diseases and inflammation. Thus, the TNFα antibodies have great potential application in diagnostics and therapy fields. The variable binding domain of IgNAR (VNAR), the shark single domain antibody, has some excellent advantages in terms of size, solubility, and thermal and chemical stability, making them an ideal alternative to conventional antibodies. This study aims to obtain VNARs that are specific for mouse TNF (mTNF) from whitespotted bamboosharks. After immunization of whitespotted bamboosharks, the peripheral blood leukocytes (PBLs) were isolated from the sharks, then the VNAR phage display library was constructed. Through phage display panning against mTNFα, positive clones were validated through ELISA assay. The affinity of the VNAR and mTNFα was measured using ELISA and Bio-Layer Interferometry. The binding affinity of 3B11 VNAR reached 16.7 nM. Interestingly, one new type of VNAR targeting mTNF was identified that does not belong to any known VNAR type. To understand the binding mechanism of VNARs to mTNFα, the models of VNARs-mTNFα complexes were predicted by computational modeling combining HawkDock and RosettaDock. Our results showed that four VNARs' epitopes overlapped in part with that of mTNFR. Furthermore, the ELISA assay shows that the 3B11 potently inhibited mTNFα binding to mTNFR. This study may provide the basis for the TNFα blockers and diagnostics applications.

Combinatorial Approaches for Efficient Design of Photoswitchable Protein-Protein Interactions as In Vivo Actuators.

Light switchable two-component protein dimerization systems offer versatile manipulation and dissection of cellular events in living systems. Over the past 20 years, the field has been driven by the discovery of photoreceptor-based interaction systems, the engineering of light-actuatable binder proteins, and the development of photoactivatable compounds as dimerization inducers. This perspective is to categorize mechanisms and design approaches of these dimerization systems, compare their advantages and limitations, and bridge them to emerging applications. Our goal is to identify new opportunities in combinatorial protein design that can address current engineering challenges and expand in vivo applications.

Defining molecular glues with a dual-nanobody cannabidiol sensor.

"Molecular glue" (MG) is a term coined to describe the mechanism of action of the plant hormone auxin and subsequently used to characterize synthetic small molecule protein degraders exemplified by immune-modulatory imide drugs (IMiDs). Prospective development of MGs, however, has been hampered by its elusive definition and thermodynamic characteristics. Here, we report the crystal structure of a dual-nanobody cannabidiol-sensing system, in which the ligand promotes protein-protein interaction in a manner analogous to auxin. Through quantitative analyses, we draw close parallels among the dual-nanobody cannabidiol sensor, the auxin perception complex, and the IMiDs-bound CRL4CRBN E3, which can bind and ubiquitinate "neo-substrates". All three systems, including the recruitment of IKZF1 and CK1α to CRBN, are characterized by the lack of ligand binding activity in at least one protein partner and an under-appreciated preexisting low micromolar affinity between the two proteinaceous subunits that is enhanced by the ligand to reach the nanomolar range. These two unifying features define MGs as a special class of proximity inducers distinct from bifunctional compounds and can be used as criteria to guide target selection for future rational discovery of MGs.

Synthetic nanobody-functionalized nanoparticles for accelerated development of rapid, accessible detection of viral antigens.

Successful control of emerging infectious diseases requires accelerated development of fast, affordable, and accessible assays for wide implementation at a high frequency. This paper presents a design for an in-solution assay pipeline, featuring nanobody-functionalized nanoparticles for rapid, electronic detection (Nano2RED) of Ebola and COVID-19 antigens. Synthetic nanobody binders with high affinity, specificity, and stability are selected from a combinatorial library and site-specifically conjugated to gold nanoparticles (AuNPs). Without requiring any fluorescent labelling, washing, or enzymatic amplification, these multivalent AuNP sensors reliably transduce antigen binding signals upon mixing into physical AuNP aggregation and sedimentation processes, displaying antigen-dependent optical extinction readily detectable by spectrometry or portable electronic circuitry. With Ebola virus secreted glycoprotein (sGP) and a SARS-CoV-2 spike protein receptor binding domain (RBD) as targets, Nano2RED showed a high sensitivity (the limit of detection of ∼10 pg /mL, or 0.13 pM for sGP and ∼40 pg/mL, or ∼1.3 pM for RBD in diluted human serum), a high specificity, a large dynamic range (∼7 logs),and fast readout within minutes. The rapid detection, low material cost (estimated <$0.01 per test), inexpensive and portable readout system (estimated <$5), and digital data output, make Nano2RED a particularly accessible assay in screening of patient samples towards successful control of infectious diseases.

Creating Red Light-Switchable Protein Dimerization Systems as Genetically Encoded Actuators with High Specificity.

Protein dimerization systems controlled by red light with increased tissue penetration depth are a highly needed tool for clinical applications such as cell and gene therapies. However, mammalian applications of existing red light-induced dimerization systems are hampered by limitations of their two components: a photosensory protein (or photoreceptor) which often requires a mammalian exogenous chromophore and a naturally occurring photoreceptor binding protein typically having a complex structure and nonideal binding properties. Here, we introduce an efficient, generalizable method (COMBINES-LID) for creating highly specific, reversible light-induced heterodimerization systems independent of any existing binders to a photoreceptor. It involves a two-step binder screen (phage display and yeast two-hybrid) of a combinatorial nanobody library to obtain binders that selectively engage a light-activated form of a photoswitchable protein or domain not the dark form. Proof-of-principle was provided by engineering nanobody-based, red light-induced dimerization (nanoReD) systems comprising a truncated bacterial phytochrome sensory module using a mammalian endogenous chromophore, biliverdin, and light-form specific nanobodies. Selected nanoReD systems were biochemically characterized, exhibiting low dark activity and high induction specificity, and further demonstrated for the reversible control of protein translocation and activation of gene expression in mice. Overall, COMBINES-LID opens new opportunities for creating genetically encoded actuators for the optical manipulation of biological processes.

Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library.

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the dissection and manipulation of biological pathways. Currently, only a limited number of chemically induced dimerization (CID) systems exist and engineering new ones with desired sensitivity and selectivity for specific small-molecule ligands remains a challenge in the field of protein engineering. We here describe a high throughput screening method, combinatorial binders-enabled selection of CID (COMBINES-CID), for the de novo engineering of CID systems applicable to a large variety of ligands. This method uses the two-step selection of a phage-displayed combinatorial nanobody library to obtain 1) "anchor binders" that first bind to a ligand of interest and then 2) "dimerization binders" that only bind to anchor binder-ligand complexes. To select anchor binders, a combinatorial library of over 109 complementarity-determining region (CDR)-randomized nanobodies is screened with a biotinylated ligand and hits are validated with the unlabeled ligand by bio-layer interferometry (BLI). To obtain dimerization binders, the nanobody library is screened with anchor binder-ligand complexes as targets for positive screening and the unbound anchor binders for negative screening. COMBINES-CID is broadly applicable to select CID binders with other immunoglobulin, non-immunoglobulin, or computationally designed scaffolds to create biosensors for in vitro and in vivo detection of drugs, metabolites, signaling molecules, etc.

COMBINES-CID: An Efficient Method for De Novo Engineering of Highly Specific Chemically Induced Protein Dimerization Systems.

Chemically induced dimerization (CID) systems, in which two proteins dimerize only in the presence of a small molecule ligand, offer versatile tools for small molecule sensing and actuation. However, only a handful of CID systems exist and creating one with the desired sensitivity and specificity for any given ligand is an unsolved problem. Here, we developed a combinatorial binders-enabled selection of CID (COMBINES-CID) method broadly applicable to different ligands. We demonstrated a proof-of-principle by generating nanobody-based heterodimerization systems induced by cannabidiol with high ligand selectivity. We applied the CID system to a sensitive sandwich enzyme-linked immunosorbent assay-like assay of cannabidiol in body fluids with a detection limit of ∼0.25 ng/mL. COMBINES-CID provides an efficient, cost-effective solution for expanding the biosensor toolkit for small molecule detection.

Why the c-Fos/c-Jun complex is extremely conserved: An in vitro evolution exploration by combining cDNA display and proximity ligation.

Transcriptional regulation involves a series of sophisticated protein-protein and protein-DNA interactions (PPI and PDI). Some transcriptional complexes, such as c-Fos/c-Jun and their binding DNA fragments, have been conserved over the past one billion years. Considering the thermodynamic principle for transcriptional complex formation, we hypothesized that the c-Fos/c-Jun complex may represent a thermodynamic summit in the evolutionary space. To test this, we invented a new method, termed One-Pot-seq, which combines cDNA display and proximity ligation to analyse PPI/PDI complexes simultaneously. We found that the wild-type c-Fos/c-Jun complex is indeed the most thermodynamically stable relative to various mutants of c-Fos/c-Jun and binding DNA fragments. Our method also provides a universal approach to detect transcriptional complexes and explore transcriptional regulation mechanisms.

ATP selection in a random peptide library consisting of prebiotic amino acids.

Based upon many theoretical findings on protein evolution, we proposed a ligand-selection model for the origin of proteins, in which the most ancient proteins originated from ATP selection in a pool of random peptides. To test this ligand-selection model, we constructed a random peptide library consisting of 15 types of prebiotic amino acids and then used cDNA display to perform six rounds of in vitro selection with ATP. By means of next-generation sequencing, the most prevalent sequence was defined. Biochemical and biophysical characterization of the selected peptide showed that it was stable and foldable and had ATP-hydrolysis activity as well.

Reconstructing a flavodoxin oxidoreductase with early amino acids.

Primitive proteins are proposed to have utilized organic cofactors more frequently than transition metals in redox reactions. Thus, an experimental validation on whether a protein constituted solely by early amino acids and an organic cofactor can perform electron transfer activity is an urgent challenge. In this paper, by substituting "late amino acids (C, F, M, T, W, and Y)" with "early amino acids (A, L, and V)" in a flavodoxin, we constructed a flavodoxin mutant and evaluated its characteristic properties. The major results showed that: (1) The flavodoxin mutant has structural characteristics similar to wild-type protein; (2) Although the semiquinone and hydroquinone flavodoxin mutants possess lower stability than the corresponding form of wild-type flavodoxin, the redox potential of double electron reduction Em,7 (fld) reached -360 mV, indicating that the flavodoxin mutant constituted solely by early amino acids can exert effective electron transfer activity.

Improved annotation of a plant pathogen genome Xanthomonas oryzae pv. oryzae PXO99A.

Many bacterial genomes have been sequenced and stored in public databases now, of which Reference Sequence (RefSeq) is the most widely used one. However, the annotation in RefSeq is still unsatisfactory. The present analysis is focused on the re-annotation of an important plant pathogen genome Xanthomonas oryzae pv. oryzae PXO99A (Xoo PXO99A), which is the causal agent of bacterial blight on rice. Based on the parameters of 28 nucleotide frequencies and support vector machine algorithm, 41 originally annotated hypothetical genes were recognized as noncoding sequences, which were further supported by principal component analysis and other evidence. Ten of them were tested with reverse transcription-polymerase chain reaction experiments (RT-PCR), and all of them were confirmed to be noncoding sequences. Furthermore, 197 potential new genes not annotated in RefSeq were both recognized by two ab initio gene finding programs. Most of them only have sequence similarities with part of the known genes in other species, so they are unlikely to be protein-coding genes. Twelve potential new genes have high full-length sequence similarities with function-known genes, which are very likely to be true protein-coding genes. All the 12 potential genes were tested with RT-PCR, and 11 of them (92%) were successfully amplified in cDNA template. The RT-PCR experiments confirm that our theoretical prediction has high accuracy. The improvement of Xoo PXO99A annotation is helpful for the research of lifestyle, metabolism, and pathogenicity of this important plant pathogen. The improved annotation can be obtained from http://211.69.128.148/Xoo .

rac-Ethyl 2-amino-3-hy-droxy-3-[4-(methyl-sulfon-yl)phen-yl]propano-ate.

In the title compound, C(12)H(17)NO(5)S, the orientations of the 2-ethyl-2-amino-3-hy-droxy-propano-ate group and the 4-methyl-sulfonyl moiety towards the aromatic ring are periplanar and (-)-anti-clinal, respectively. In the crystal packing, the dominant inter-action is O-H⋯N hydrogen bonding, which generates a chain running along [100]. N-H⋯O and C-H⋯O interactions are also observed.

2-Aza-niumyl-4-(ethyl-carbamo-yl)butano-ate: the zwitterionic form of the amino acid theanine.

In the title zwitterion, C(7)H(14)N(2)O(3), the ethyl-amino and the 5-oxo groups are positionally disordered with occupancy ratios of 0.50:0.50 and 0.70:0.30, respectively. The terminal ethyl -CH(3) group undergoes considerable thermal motion. In the crystal, mol-ecules are linked via N-H⋯O hydrogen bonds, forming a two-dimensional arrangement propagating in the bc plane.