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There are numerous evaluations of natural products, of which majority are food bioactives, performed up to date for their various health beneficial activities via targeting specific proteins. However, the direct identification of a targeted protein remains unexplored for natural occurring compounds. Proteolysis targeting chimera (PROTAC) is a type of bifunctional chimeric molecules that can directly degrade the binding proteins targeted by bioactive molecules in an ubiquitin-proteasome pathway. As the agents in protein degradation dependent on ubiquitin ligase, the bifunctional molecule connects the target protein ligand and E3 ligase ligand together via an appropriate linker. It is highly selective and efficient to induce the ubiquitin-mediated degradation of targeted binding proteins. Therefore, it has been demonstrated that the PROTAC technology has broad application in the modulation of the target protein level. In this review, we outlined the advances in PROTAC combined molecule compounds, summarized its quantitative structure-activity relationship, and finally reviewed the methods applied in identifying the target proteins of natural products. We hope it will provide an insightful application of PROTAC techniques in the target protein identification of natural products including food bioactive molecules.

There is an urgent need for reliable biosensors to detect nucleic acid of interest in clinical samples. We propose that the accuracy of the present nucleic acid-sensing method can be advanced by avoiding false-positive identifications derived from nonspecific interactions (e.g., nonspecific binding, probe degradation). The challenge is to exploit biosensors that can distinguish false-positive from true-positive samples in nucleic acid screening. In the present study, by learning from the enzymatic cycle in nature, we raise an allostery tool displaying invertible positive/negative cooperativity for reversible or cyclic activity control of the biosensing probe. We demonstrate that the silencing and regeneration of a positive (or negative) allosteric effector can be carried out through toehold displacement or an enzymatic reaction. We, thus, have developed several dynamic biosensors that can repeatedly measure a single nucleic acid sample. The ability to distinguish a false-positive from a true-positive signal is ascribed to the nonspecific interaction presenting equivalent signal variations, while the specific target binding exhibits diverse signal variations according to repeated measurements. Given its precise identification, such consequent dynamic biosensors offer exciting opportunities in physiological and pathological diagnosis.

This two-part preclinical study aims to evaluate prostate specific membrane antigen (PSMA) as a valuable target for expression-based imaging applications and to determine changes in target binding in function of varying apparent molar activities (MAapp) of [18F]AlF-PSMA-11. For the evaluation of PSMA expression levels, male NOD/SCID mice bearing prostate cancer (PCa) xenografts of C4-2 (PSMA+++), 22Rv1 (PSMA+) and PC-3 (PSMA−) were administered [18F]AlF-PSMA-11 with a medium MAapp (20.24 ± 3.22 MBq/nmol). SUVmean and SUVmax values were respectively 3.22 and 3.17 times higher for the high versus low PSMA expressing tumors (p app, C4-2 and 22Rv1 xenograft bearing mice underwent additional [18F]AlF-PSMA-11 imaging with a high (211.2 ± 38.9 MBq/nmol) and/or low MAapp (1.92 ± 0.27 MBq/nmol). SUV values showed a significantly increasing trend with higher MAapp. Significant changes were found for SUVmean and SUVmax between the high versus low MAapp and medium versus low MAapp (both p app (p = 0.055 and 0.25, respectively). The effect of varying MAapp was more pronounced in low expressing tumors and PSMA expressing tissues (e.g. salivary glands and kidneys). Overall, administration of a high MAapp increases the detection of low expression tumors while also increasing uptake in PSMA expressing tissues, possibly leading to false positive findings. In radioligand therapy, a medium MAapp could reduce radiation exposure to dose-limiting organs with only limited effect on radionuclide accumulation in the tumor.

Anti-CRISPR (Acr) proteins are phage-borne inhibitors of the CRISPR-Cas immune system in archaea and bacteria. AcrIIC2 from prophages of Neisseria meningitidis disables the nuclease activity of typ...

Introduction The 2019-novel coronavirus disease (COVID-19) is an intractable global health challenge resulting in an aberrant rate of morbidity and mortality worldwide. The mode of entry for SARS-CoV-2 into host cells occurs through clathrin-mediated endocytosis. As part of the efforts to mitigate COVID-19 infections, rapid and accurate detection methods, as well as smart vaccine and drug designs with SARS-CoV-2 targeting capabilities are critically needed. This systematic review aimed to present a good mapping between the structural and functional characteristics of aptamers and their potential applications in COVID-19 theranostics. Methods In this study, extensive discussions into the potential development of aptameric systems as robust theranostics for rapid mitigation of the virulent SARS-CoV-2 was made. Information required for this study were extracted from a systematic review of literature in PubMed, SCOPUS, Web of Science (WOS), and other official related reports from reputable organisations. Results The global burden of COVID-19 pandemic was discussed including the progress in rapid detection, repurposing of existing antiviral drugs, and development of prophylactic vaccines. Aptamers have highly specific and stable target binding characteristics which can be generated and engineered with less complexity for COVID-19 targeted theranostic applications. Conclusions There is an urgent need to develop safe innovative biomedical technologies to mitigate the dire impact of COVID-19 on public health worldwide. Research advances into aptameric systems bode well with the fact that they can be engineered for the development of effective and affordable diagnostics, therapeutics and prophylactic vaccines for SARS-CoV-2 and other infectious pathogens.

Mitochondria are subcellular organelles that perform a variety of critical biological functions, including ATP production and acting as hubs of immune and apoptotic signalling. Mitochondrial dysfunction has been extensively linked to the pathology of multiple neurodegenerative disorders, resulting in significant investment from the drug discovery community. Despite extensive efforts, there remains no disease modifying therapies for neurodegenerative disorders. This manuscript aims to review the compounds historically used to modulate the mitochondrial network through the lens of modern medicinal chemistry, and to offer a perspective on the evidence that relevant exposure was achieved in a representative model and that exposure was likely to result in target binding and engagement of pharmacology. We hope this manuscript will aid the community in identifying those targets and mechanisms which have been convincingly (in)validated with high quality chemical matter, and those for which an opportunity exists to explore in greater depth.

A localized positive charge on IgG (referred to as a "charge patch") shows an adverse effect on pharmacokinetics (PK), so it would seem to be best practice to avoid charge patches during the discovery stage and closely monitor charge interactions during the development process. In certain circumstances, however, charge patches are required for target binding, in which case completely removing charge patches is not feasible. Therefore, quantitative measurement of a charge patch and its impact on PK is critical to the success of therapeutic antibody development. In this article, we generated mutations of a recombinant human antibody (referred to as mAb1) with disrupted charge patches to investigate how charge patches on IgG antibodies impact both target-binding affinity and PK-related factors. We conclude that it is important to modulate the size of the charge patch in order to balance target-binding affinity and PK.

DEL selections are binding assays conducted with mixtures of chemically diverse DNA-tagged ligands and a screening target. DEL selections use DNA sequence counts to measure target binding, where ideally higher affinity ligands will have higher counts than weaker affinity ligands. However, there is not always a clear relationship between DNA sequence count (assay signal) and binding affinity. This disconnect may be due to the fidelity of library chemistry, where reactions often do not go to completion, and also to repetitive rounds of binding and elution that are standard practice in most DEL selection experiments. We describe here a strategy that addresses both of these issues and provides a means to calculate ligand affinity from primary selection data. The reaction yields of selected compounds during DEL library synthesis can also be predicted with this method.

Two major moieties in genome editing are required for precise genetic changes: the locator moiety for target binding and the effector moiety for genetic engineering. By taking advantage of CRISPR/Cas, which consists of different modules for independent target binding and cleavage, a spectrum of precise and versatile genome editing technologies have been developed for broad applications in biomedical research, biotechnology, and therapeutics. Here, we briefly summarize the progress of genome editing systems from a view of both locator and effector moieties and highlight the advance of newly reported CRISPR-conjugated base editing and prime editing systems. We also underscore distinct mechanisms of off-target effects in CRISPR-conjugated systems and further discuss possible strategies to reduce off-target mutations in the future.

Proximity-dependent hybridization chain reaction (HCR) has shown great potential in sensing biomolecules on the cell surface. However, the requirement of two adjacent bioevents occurring simultaneously limits its application. To solve the problem, split aptamers with target binding ability were introduced to combine with split triggers for initiating HCR, thus producing a novel dual-split aptamer probe (DSAP). By employing cancer-related receptors as models, in situ HCR on a cancer cell surface induced by recognition-driven remodeling of the DSAP was demonstrated. The DSAP consisted of two sequences. Each contained two segments; one derived from split aptamers and the other originated in split triggers. In the presence of target cells, split aptamers reassembled on the cell surface under the "induced-fit effect", thus forcing two split triggers close to each other. The remodeled DSAP worked as an intact trigger, which opened the H1 hairpin probe and then hybridized with the H2 hairpin probe, thus initiating HCR to produce an activated fluorescence signal. As a proof of concept, human liver cancer SMMC-7721 cells and their split ZY11 aptamer were used to construct the DSAP. Results indicated that the DSAP realized sensitive analysis of target cells, permitting the actual detection of 20 cells in the buffer. Moreover, the specific identification of target cells in mixed cell samples and the quantitative analysis of target cells in serum were also achieved. The DSAP strategy is facile and universal, which not only would expand the application range of HCR but also might be developed as a multitarget detection technique for bioanalysis.

Target-mediated drug disposition (TMDD) is often observed for targeted therapeutics, and manifests as decreases in clearance and volume of distribution with increasing dose as a result of saturable, high affinity target binding. In the present work, we demonstrate that classically defined TMDD is just one of the characteristic features of the system. In fact, for molecules with rapid non-specific elimination relative to target-mediated elimination, binding to target may actually lead to improved exposure at sub-saturating doses. This feature, which we refer to as target-mediated exposure enhancement (TMEE), produces the opposite trend to classical TMDD, i.e., with increasing dose levels, clearance and volume of distribution will also increase. The general model of TMDD was able to well-characterize the pharmacokinetics of two molecules that display TMEE, ALX-0081 and linagliptin. Additional fittings using the commonly reported TMDD model approximations revealed that both the quasi-equilibrium and quasi-steady-state approximations were able to well-describe TMEE; however, the Michaelis-Menten approximation was unable to describe this behavior. With the development of next-generation therapeutics with high affinity for target and rapid non-specific elimination, such as antibody fragments and peptides, this previously unexplored limit of TMDD is anticipated to become increasingly relevant for describing pharmacokinetics of investigational therapeutics.

Aptamer switches that respond sensitively to pH could enhance control over molecular devices, improving their diagnostic and therapeutic efficacy. Previous designs have inserted pH-sensitive DNA motifs into aptamer sequences. Unfortunately, their performance was limited by the motifs’ intrinsic pH-responses and could not be tuned to operate across arbitrary pH ranges. Here, we present a methodology for converting virtually any aptamer into a molecular switch with pH-selective binding properties — in acidic, neutral, or alkaline conditions. Our design inserts two orthogonal motifs that can be manipulated in parallel to tune pH-sensitivity without altering the aptamer sequence itself. From a single ATP aptamer, we engineer pH-controlled target binding under diverse conditions, achieving pH-induced selectivity in affinity of up to 1,000-fold. Importantly, we demonstrate the design of tightly regulated aptamers with strong target affinity over only a narrow pH range. Our approach offers a highly generalizable strategy for integrating pH-responsiveness into molecular devices., Previous design strategies for pH sensitive aptamers were not readily tunable across pH ranges. Here the authors present a general method to convert aptamers into pH-responsive switches using two orthogonal motifs.

The development of Lateral Flow Immunochromatography Assay can be divided into two levels; standardizing membrane characteristics and optimizing molecular level immunoassay reaction between analyte and detector molecules. In the preliminary phase the reaction specificity of capture and detector antibodies with the analyte has to be checked with other techniques like ELISA. Molarity and pH of conjugation buffer have prime importance in the immunoreaction among analyte and antibodies. Epitope mapping of the capture and detector antibodies is also recommended to confirm the specificity of the assay. Standardization of membrane characteristics directly relates to the sensitivity of the assay through its porosity, hydrophobicity, protein holding/releasing capacity and wicking rate. Under optimised condition a perfect Lateral Flow Immunochromatography Assay should have high on-rate (target binding efficiency), low off-rate (target releasing efficiency) and low Cross-reactivity. In this manuscript, we share our experience, especially on developmental strategies and troubleshooting, that we have experienced during Lateral Flow Immunochromatography Assay kit development.

Super-resolution imaging allows for visualization of cellular structures on a nanoscale level. DNA-PAINT (DNA Point Accumulation In Nanoscale Topology) is a super-resolution method that depends on the binding and unbinding of DNA imager strands. The current DNA-PAINT technique suffers from slow acquisition due to the low binding rate of the imager strands. Here we report on a method where imager strands are loaded into a protein, Argonaute (Ago), that allows for faster binding. Ago pre-orders the DNA imager strand into a helical conformation, allowing for 10 times faster target binding. Using a 2D DNA origami structure, we demonstrate that Ago-assisted DNA-PAINT (Ago-PAINT) can speed up the current DNA-PAINT technique by an order of magnitude while maintaining the high spatial resolution. We envision this tool to be useful not only for super-resolution imaging, but also for other techniques that rely on nucleic-acid interactions.

Aldehyde is a versatile chemical handle for protein modification. Although many methods have been developed to label proteins with aldehyde, target-specific methods amenable to endogenous proteins are limited. Here, we report a simple affinity probe strategy to introduce aldehydes to native proteins. Notably, the probe contains a latent aldehyde functionality that is only exposed upon target binding, thereby enabling a one-pot labeling procedure.

In drug discovery, it is essential to accurately measure drug-target binding affinity. Here, we revisit the fact that target binding kinetics impact the measurement of affinity, using a case study: development of corticotropin-releasing factor antagonists. Slow dissociation of the drug-target complex results in affinity assays being far from equilibrium, which results in erroneous estimates of affinity. This scenario can impair prediction of human dosing, assessment of target selectivity, identification of high-affinity ligands and determination of SAR. We describe strategies to detect lack of equilibration in affinity assays and methods to correctly measure affinity of slowly dissociating compounds. These considerations will facilitate drug discovery by ensuring reliable measurement of drug-target binding affinity.

A rapid and simple methodology for the biomolecular analysis of single cells and microenvironments via a stick-and-peel plasmonic sensing platform is reported. Substrate-bound assemblies of plasmonic gold nanoparticles linked by reconfigurable oligonucleotides undergo disassembly upon target binding. Changes in the light scattering intensity of thousands of discrete nanoparticle assemblies are extrapolated concomitantly to yield the mapping of local target concentrations. The methodology is completely free of labelling, purification and separation steps. We quantified the intracellular ATP levels for two ovarian cancer cell lines to elucidate the differences and cellular distribution, and demonstrated the potential of the stick-and-peel platform for mapping the microenvironment of a 2D heterogeneous surface. The portable and economical analytical platform may broaden the affordability and applicability of single-cell based analyses and enable new opportunities in clinical care such as on-site molecular pathology.

We report a platform utilizing a reporter enzyme, which produces a chromogenic indigo precipitate that preferentially localizes within a hydrogel microparticle. The 3D network of the hydrogel maintains the rapid target binding kinetics found in solution, while multiplexed target detection is achieved through shape-encoding of the particles. Moreover, the precipitate-laden hydrogels can be imaged with a simple phone camera setup. We used this system to detect microRNA (miRNA) down to 0.22 fmol. We then showed the compatibility of this system with real samples by performing multiplexed miRNA measurements from total RNA from matched colon cancer and normal adjacent tissue.

Background Aptamers, single-stranded DNAs or RNAs, can be selected from a library containing random sequences using a method called Systematic Evolution of Ligands by EXponential Enrichment (SELEX). In SELEX, monitoring the enriching statuses of aptamer candidates during the process is a key step until today. Conformational change of an aptamer caused by target-binding in gel can be used to indicate its statuses of binding. Results In this study, an easy-to-implement gel-based diffusion method (GBDM) was developed to monitor the interaction between enriched aptamer candidates and their targets. In order to prove the concept, characterization of aptamers targeting their targets including protein (thrombin) and non-protein molecules (acetamiprid, ATP, atrazine, profenofos and roxithromycin), respectively, were performed using mini gels. Our method has advantages over the common methods including easy performed with labor- and time- saving in experimental operation. The concept has been proven by monitoring enrichment of dynamic aptamer candidate libraries targeting a small molecule 2,2-bis(4-chlorophenyl) acetic acid (DDA) during SELEX process. A mini gel cassette was designed and fabricated by our laboratory to make mini agarose gels for diffusion with different directions. Conclusions These results indicate that GBDM, in particular, chasing diffusion is suitable for monitoring the interaction between enriched aptamer candidates and their targets. These pioneering efforts are helpful for novel aptamer selection by breaking through the technical bottleneck of aptamer development and helpful for development of novel aptasensors.

ConspectusAlthough antibodies are a powerful tool for molecular biology and clinical diagnostics, there are many emerging applications for which nucleic acid-based aptamers can be advantageous. However, generating high-quality aptamers with sufficient affinity and specificity for biomedical applications is a challenging feat for most research laboratories. In this Account, we describe four techniques developed in our lab to accelerate the discovery of high quality aptamer reagents that can achieve robust binding even for challenging molecular targets. The first method is particle display, in which we convert solution-phase aptamers into aptamer particles that can be screened via fluorescence-activated cell sorting (FACS) to quantitatively isolate individual aptamer particles based on their affinity. This enables the efficient isolation of high-affinity aptamers in fewer selection rounds than conventional methods, thereby minimizing selection biases and reducing the emergence of artifacts in the final aptamer pool. We subsequently developed the multi-parametric particle display (MPPD) method, which employs two-color FACS to isolate aptamer particles based on both affinity and specificity, yielding aptamers that exhibit excellent target binding even in complex matrices like serum. The third method is a click chemistry-based particle display (click-PD) that enables the generation and high-throughput screening of “non-nattural” aptamers with a wide range of base modifications. We have shown that these base-modified aptamers can achieve robust affinity and specificity for targets that have proven challenging or inaccessible with natural nucleotide-based aptamer libraries. Lastly, we describe the non-natural aptamer array (N2A2) platform, in which a modified benchtop sequencing instrument is used to characterize base-modified aptamers in a massively parallel fashion, enabling the efficient identification of molecules with excellent affinity and specificity for their targets. This system first generates aptamer clusters on the flow-cell surface that incorporate alkyne-modified nucleobases, and then performs a click reaction to couple those nucleobases to an azide-modified chemical moiety. This yields a sequence-defined array of tens of millions of base-modified sequences, which can then be characterized in a high-throughput fashion. Collectively, we believe that these advancements are helping to make aptamer technology more accessible, efficient, and robust, thereby enabling the use of these affinity reagents for a wider range of molecular recognition and detection-based applications.

Background As antibiotic resistance creates a significant global health threat, we need not only to accelerate the development of novel antibiotics but also to develop better treatment strategies using existing drugs to improve their efficacy and prevent the selection of further resistance. We require new tools to rationally design dosing regimens from data collected in early phases of antibiotic and dosing development. Mathematical models such as mechanistic pharmacodynamic drug-target binding explain mechanistic details of how the given drug concentration affects its targeted bacteria. However, there are no available tools in the literature that allow non-quantitative scientists to develop computational models to simulate antibiotic-target binding and its effects on bacteria. Results In this work, we have devised an extension of a mechanistic binding-kinetic model to incorporate clinical drug concentration data. Based on the extended model, we develop a novel and interactive web-based tool that allows non-quantitative scientists to create and visualize their own computational models of bacterial antibiotic target-binding based on their considered drugs and bacteria. We also demonstrate how Rifampicin affects bacterial populations of Tuberculosis bacteria using our vCOMBAT tool. Conclusions The vCOMBAT online tool is publicly available at https://combat-bacteria.org/.

Although many quinolones have shown promise as potent antimalarials, their clinical development has been slow due to poor performance in vivo. Insights into structural modifications that can improve their therapeutic potential will be very valuable in this vibrant area of research. Our studies involving a library of quinolones which vary in substitution pattern at N1, C3, C6 and C7 positions have shown that the presence of adenine moiety at C7 can bring a noticeable improvement in activity compared to other heterocyclic groups at this location. The most potent compound emerged from this study showed IC50 values of 0.38 μM and 0.75 μM against chloroquine-sensitive and -resistant (W2) strains, respectively. Docking analysis in the Qo site of cytochrome bc1 complex revealed the contribution of a key H-bonding interaction from the adenine unit in target binding. This corroborates with compound-induced loss of mitochondrial functions. These findings not only open avenues for further exploration of antimalarial potential of adenine-modified quinolones, but also suggests broader opportunities during lead-optimization against other antimalarial targets.

Aptamers are promising biorecognition elements for sensors. However, aptamer-based assays often lack the requisite levels of sensitivity and/or selectivity because they typically employ structure-switching aptamers with attenuated affinity and/or utilize reporters that require aptamer labeling or which are susceptible to false positives. Dye-displacement assays offer a label-free, sensitive means for overcoming these issues, wherein target binding liberates a dye that is complexed with the aptamer, producing an optical readout. However, broad utilization of these assays has been limited. Here, we demonstrate a rational approach to develop colorimetric cyanine dye-displacement assays that can be broadly applied to DNA aptamers regardless of their structure, sequence, affinity, or the physicochemical properties of their targets. Our approach should accelerate the development of mix-and-measure assays that could be applied for diverse analytical applications.

Micro-cantilever sensors are a known reliable tool for gas sensing in industrial applications. We have demonstrated the application of cantilever sensors on the detection of a meat freshness volatile biomarker (cadaverine), for determination of meat and fish precise expiration dates. For achieving correct target selectivity, the cantilevers need to be functionalized with a cadaverine-selective binder, based on a cyclam-derivative. Cantilever surface properties such as surface energy strongly influence the binder morphology and material clustering and, therefore, target binding. In this paper, we explore how chemical and physical surface treatments influence cantilever surface, binder morphology/clustering and binding capabilities. Sensor measurements with non-controlled surface properties are presented, followed by investigations on the binder morphology versus surface energy and cadaverine capture. We demonstrated a method for hindering binder crystallization on functionalized surfaces, leading to reproducible target capture. The results show that cantilever surface treatment is a promising method for achieving a high degree of functionalization reproducibility for industrial cantilever sensors, by controlling binder morphology and uniformity.

Proteins interact with one another across a broad spectrum of affinities. Our understanding of the low end of this spectrum, as characterized by millimolar dissociation constants, relies on a handful of cases in which weak encounters have experimentally been identified. These weak interactions away from the specific target binding site can lead toward a higher-affinity complex. Recently, we detected weak encounters between two paralogous phosphotransferase pathways of Escherichia coli, which regulate various metabolic processes and stress responses. In addition to encounters that are known to occur between cognate proteins, i.e., those that can exchange phosphate groups with each other, surprisingly, encounters involving noncognates were also observed. It is not clear whether these “futile” encounters have a cooperative or competitive role. Using agent-based simulations, we find that the encounter complexes can be cooperative or competitive so as to increase or lower the effective binding affinity of the specific complex under different circumstances. This finding invites further questions into how organisms might exploit such low affinities to connect their signaling components.

Split aptamers (SPAs) are a pair of oligonucleotide fragments generated by cleaving a long parent aptamer. SPAs have many compelling advantages over the parent aptamer such as sandwich target binding, optimized concise structure, and low cost. However, only a limited number of SPAs have been developed so far because the traditional theory restricts the splitting to the functionally dispensable site that many parent aptamers do not possess. In this work, the traditional mechanism and hypothesis that SPAs can also be generated by splitting the parent aptamer at the functionally essential site while still preserving the biorecognition capability are challenged. To prove the hypothesis, three SPAs with Broken initial small-molecule binding Pockets (BPSPAs) are discovered and their binding capabilities are validated both in the wet lab and in silico. An allosteric binding mechanism of BPSPAs, in which a new binding pocket is formed upon the target binding, is revealed by all-atom microsecond-scale molecular dynamics simulations. Our work highlights the important role of MD simulations in predicting the ligand binding potency with functional nucleic acids at the molecular level. The findings will greatly promote discovery of new SPAs and their applications in molecular sensing in many fields. Accepted version The authors acknowledge Prof. Ai-Qun Liu from Nanyang Technological University and Prof. Xinhui Lou from Capital Normal University for valuable discussions. This research was funded by National Nature Science Foundation of China (Grant 21677082) and the Shanghai Sailing Program (Grant 18YF1406600). We acknowledge the Tsinghua University Branch of China National Center for Protein Sciences for providing the facility support. We also thank the ECNU Multifunctional Platform for Innovation (001) for computational resources.

The detection of Aflatoxin B1 (AFB1) has attracted extensive attention for food safety is a worldwide public health problem. Herein, a novel, simple and label-free fluorescent aptasensor, based on quaternized tetraphenylethene salt (TPE-Z), graphene oxide (GO) and AFB1 aptamer, has been constructed to detect AFB1. In the presence of AFB1, AFB1 aptamer undergoes a conformational switch from single stranded structure to the AFB1/AFB1 aptamer complex upon target binding, which induces the release of TPE-Z/AFB1 aptamer from the surface of GO. Thus, the fluorescence of TPE-Z/AFB1 aptamer is recovered. The assay can be performed by simply mixing TPE-Z, AFB1 aptamer, the GO and the AFB1 samples with a detection limit of 0.25 ng/mL. It is highly selective against other aflatoxins in foods and its performance has been verified in food samples (corn, milk and rice) with known concentration AFB1.

Complex mechanical changes in response to an external trigger are pervasive in natural soft materials and often sought for applications. Be it the reversible stiffening of sea cucumber, the failure of a polymeric or colloidal gel under load, or the dissolution of a biosensing hydrogel upon target binding, mechanical transitions are typically enabled, and critically affected, by heterogeneous structures and reversible bonds. New possibilities to monitor evolving properties and to gain access to stress propagation with temporal and spatial resolution are being disclosed by mechanochromic molecules and molecular complexes, which transduce a mechanical stress into a light signal and act as built-in stress reporters. I will review recent strategies and identify future directions for the design of mechanically responsive soft networks and for their optical mapping, focusing particular attention on the emerging class of hydrogels based on DNA self-assembly.

Photo-controlled affinity reagents seek to provide modular spatiotemporal control of bioactivity by conferring photo-switchability of function on an affinity reagent scaffold. Here we used Rosetta-based computational methods to screen for sites on the Fynomer affinity reagent structure for attachment of photoswitchable cross-linkers. Both established UV-based cross-linkers (azobenzene-iodoacetamide (IAC)) and an azonium-based efficient red light switchable cross-linker, piperazino-tetra-ortho-methoxy azobenzene (PIP), were then tested experimentally. Several sites compatible with Fynomer function were identified, including sites showing rapid (

To explore thermofluorimetric analysis (TFA) in detail, we compared two related aptamers. The first, LINN2, is a DNA aptamer previously selected against EGFR recombinant protein. In this work we selected a second aptamer, KM4, against EGFR-overexpressing A549 cells. The two aptamers were derived from the same pool and bind the same target but behave differently in TFA. Our results suggest four overall conclusions about TFA of aptamers: 1. Some aptamers show reduced fluorescence upon target binding suggesting that target-bound aptamer is not always fluorescent. 2. Many aptamers do not obey the intuitive assumptions that aptamer–target interactions stabilize a folded conformation. 3. TFA may be most appropriate for aptamers with significant double-stranded structure. 4. Kinetic effects may be significant and the order of operations in preparing samples should be carefully optimized., Graphical Abstract

The current work focuses on optimizing aptamer scaffolds that are tailored to allow for the formation of binding pockets for both a redox active signaling molecule and the target miR-92a. These newly designed allosteric nucleic acid systems are studied for efficacy to undergo a target based conformational switch. Two hairpin scaffolds were designed with differing stem stabilities and were explored using fluorescence quenching measurements. The dose dependent data for the detection of miR-92a shows the importance of scaffold design where the stability of the intra-molecular hairpin structure has to be optimized for target binding. Additional experiments explored the selectivity of the aptamer scaffolds in the presence of competing miR’s and mismatched sequences. These results provide an important precursor to constructing nucleic acid scaffolds for the detection of miR’s using label-free redox signaling.

Accessible and adaptable nucleic acid diagnostics remains a critical challenge in managing the evolving COVID‐19 pandemic. Here, an integrated molecular nanotechnology that enables direct and programmable detection of SARS‐CoV‐2 RNA targets in native patient specimens is reported. Termed synergistic coupling of responsive equilibrium in enzymatic network (SCREEN), the technology leverages tunable, catalytic molecular nanostructures to establish an interconnected, collaborative architecture. SCREEN mimics the extraordinary organization and functionality of cellular signaling cascades. Through programmable enzyme–DNA nanostructures, SCREEN activates upon interaction with different RNA targets to initiate multi‐enzyme catalysis; through system‐wide favorable equilibrium shifting, SCREEN directly transduces a single target binding into an amplified electrical signal. To establish collaborative equilibrium coupling in the architecture, a computational model that simulates all reactions to predict overall performance and optimize assay configuration is developed. The developed platform achieves direct and sensitive RNA detection (approaching single‐copy detection), fast response (assay reaction is completed within 30 min at room temperature), and robust programmability (across different genetic loci of SARS‐CoV‐2). When clinically evaluated, the technology demonstrates robust and direct detection in clinical swab lysates to accurately diagnose COVID‐19 patients., A molecular nanotechnology is developed utilizing interconnected enzyme–DNA nanostructures to achieve cooperative multi‐enzyme catalysis. Through programmable nanostructures and favorable equilibrium coupling in the system architecture, the technology achieves sensitive and versatile detection for the direct measurement of SARS‐CoV‐2 RNA targets in native patient specimens (30 min at room temperature).

Nanobodies are becoming increasingly popular as tools for manipulating and visualising proteins in vivo. The ability to control nanobody/antigen interactions using light could provide precise spatiotemporal control over protein function. We develop a general approach to engineer photo-activatable nanobodies using photocaged amino acids that are introduced into the target binding interface by genetic code expansion. Guided by computational alanine scanning and molecular-dynamics simulations, we tune nanobody/target binding affinity to eliminate binding before uncaging. Upon photo-activation, binding is restored. We use this approach to generate improved photocaged variants of two anti-GFP nanobodies. These variants exhibit photo-activatable binding triggered by illumination with 365nm light. We demonstrate that the photocaged nanobodies we have created are highly robust and function in a complex cellular environment. We apply them to control subcellular protein localisation in the nematode worm C. elegans. Our approach provides a rare example of computationally designed proteins being directly applied in living animals and demonstrates the importance of accounting for in vivo effects on protein-protein interactions.

In this work, hydrazone ligation assisted DNAzyme walking nanomachine is explored to couple with CRISPR-Cas12a trans-cleavage. Hydrazone ligation with high efficiency can mediate signal input which can be induced by target binding, thereby regulating the performance of DNAzyme walking nanomachine. The product strand from DNAzyme walking nanomachine can further activate the trans-cleavage of Cas12a. So, cascade signal amplification can be achieved to enhance the sensitivity for target detection. Subsequently, hydrazone ligation assisted DNAzyme walking nanomachine coupled with CRISPR-Cas12a has been further developed as a biosensor to analyze lipopolysaccharides. The developed biosensor exhibits a linear range from 0.05 ng/mL to 106 ng/mL and a lowest limit of detection of 7.31 fg/mL. This research provides a new mode for the signal output of DNAzyme walking nanomachine, so as to sensitively analyze different biomolecules.

The ever-increasing use of magnetic particle bioconjugates (MPB) in biosensors calls for methods of comprehensive characterization of their interaction with targets. Label-free optical sensors commonly used for studying inter-molecular interactions have limited potential for MPB because of their large size and multi-component non-transparent structure. We present an easy-to-use method that requires only three 20-min express measurements to determine the key parameters for selection of optimal MPB for a biosensor: kinetic and equilibrium characteristics, and a fraction of biomolecules on the MPB surface that are capable of active targeting. The method also provides a prognostic dependence of MPB targeting efficiency upon interaction duration and sample volume. These features are possible due to joining a magnetic lateral flow assay, a highly sensitive sensor for MPB detection by the magnetic particle quantification technique, and a novel mathematical model that explicitly describes the MPB-target interactions and does not comprise parameters to be fitted additionally. The method was demonstrated by experiments on MPB targeting of cardiac troponin I and staphylococcal enterotoxin B. The validation by an independent label-free technique of spectral-correlation interferometry showed good correlation between the results obtained by both methods. The presented method can be applied to other targets for faster development and selection of MPB for affinity sensors, analytical technologies, and realization of novel concepts of MPB-based biosensing in vivo.

Bispecific antibodies (BsAbs) are engineered to simultaneously bind two different antigens, and offer promising clinical outcomes for various diseases. The dual binding properties of BsAbs may enable superior efficacies and/or potencies compared to standard monoclonal antibodies (mAbs) or combination mAb therapies. Characterizing BsAb binding properties is critical during biotherapeutic development, where data is leveraged to predict efficacy and potency, assess critical quality attributes and improve antibody design. Traditional single-target, single-readout approaches (e.g., ELISA) have limited usefulness for interpreting complex bispecific binding, and double the benchwork. To address these deficiencies, we developed and implemented a new dual-target/readout binding assay that accurately dissects the affinities of both BsAb binding domains directly and simultaneously. This new assay uses AlphaPlex® technology, which eliminates traditional ELISA wash steps and can be miniaturized for automated workflows. The optimized BsAb AlphaPlex assay demonstrates 99-107% accuracy within a 50-150% linear range, and detected >50% binding degradation from photo- and thermal stress conditions. To the best of our knowledge, this is the first instance of a dual-target/readout BsAb AlphaPlex assay with GMP-suitable linear range, accuracy, specificity, and stability-indicating properties. As a highly customizable and efficient assay, BsAb AlphaPlex may be applicable to numerous bispecific formats and/or co-formulations against a variety of antigens beyond the clinical therapeutic space.

Botulinum neurotoxin (BoNT) serotypes A, B and E cause the vast majority of human botulism cases and pose the greatest bioterrorism threats. We previously identified multiple camelid single-domain antibodies (VHHs) that each neutralize BoNT/A, BoNT/B or BoNT/E. We also demonstrated that heterodimers of linked toxin-neutralizing VHHs are much more potent than VHH monomer pools in preventing BoNT intoxication. In this study, we expressed two different heterohexamer proteins (VNA1-ABE and VNA2-ABE) of ~100 kDa secreted from mammalian host cells, each containing the same six linked anti-BoNT VHH components ordered in two different combinations. Each heterohexamer contained two VHHs that neutralize BoNT/A, BoNT/B or BoNT/E. Both heterohexameric antitoxins displayed similar strong binding properties for the three targeted BoNT serotypes by ELISA. One ug of each heterohexameric antitoxin fully protected groups of mice co-administered with 100 LD50 of BoNT/A, BoNT/B or BoNT/E, or a pool containing 100 LD50 of each of the three toxins. The results demonstrate that long chains of at least six different linked VHHs can be expressed such that all component VHHs in the multimer retain their target binding activities. These findings make more feasible the development of a BoNT antitoxin product consisting of a small pool of proteins that, in combination, neutralize all known BoNT serotypes and subtypes.Key ContributionHeteromultimeric proteins consisting of six linked, VHH antibodies, and including VHHs that neutralize BoNT/A, BoNT/B and BoNT/E, retain high potency to protect mice challenged with high doses of all three of these BoNT serotypes.