Molecular Specificity Research Articles

Label-Free Determination of the Kinetic Parameters of Protein-Aptamer Interaction by Surface Plasmon Resonance.

Due to their high specificity and affinity to target molecules, aptamers can be used as powerful tools in diagnostics, therapeutics, and environmental or food analytics. For the use in various applications, the detailed characterization of their binding behavior is an important step after selection to determine the interaction strength between the aptamer and its target and to find the best kinetics depending on the field of application. The surface plasmon resonance (SPR) spectroscopy is a powerful technology to investigate important parameters in molecular interaction, for example, kinetics, affinity, and specificity. The most-used system is the Biacore™ SPR system which comprises an optical biosensor for label-free monitoring of binding events in real time based on SPR. This biophysical phenomenon describes the changes in refractive index on a sensor surface which can be used to measure binding events and to determine kinetic constants. In this chapter, a detailed protocol for the determination of kinetic constants for protein-aptamer interaction is provided. An 82-nt long ssDNA aptamer which are targeted against human urokinase is used as a model system for determination of binding and dissociation constants using Biacore™ SPR technology. A detailed note section provides useful tips and pitfalls at the end of this chapter.

Chemigenetic indicators based on synthetic chelators and green fluorescent protein.

Molecular fluorescent indicators are versatile tools for dynamic imaging of biological systems. We now report a class of indicators that are based on the chemigenetic combination of a synthetic ion-recognition motif and a protein-based fluorophore. Specifically, we have developed a calcium ion (Ca2+) indicator that is based on genetic insertion of circularly permuted green fluorescent protein into HaloTag protein self-labeled with a ligand containing the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. We have demonstrated the versatility of this design by also developing a sodium ion (Na+) indicator using a crown-ether-containing ligand. This approach affords bright and sensitive ion indicators that can be applicable to cell imaging. This design can enable the development of chemigenetic indicators with ion or molecular specificities that have not been realized with fully protein-based indicators.

The binding model of adenosine-specific DNA aptamer: Umbrella sampling study

We report a novel model of the selective binding mechanism of adenosine-specific DNA aptamer. Our theoretical investigations of AMP (Adenosine monophosphate) dissociation from aptamer-AMP complexes reveals new details of aptamer molecular specificity and stabilisation factors. Umbrella sampling MD calculations using parmbsc1 force field shows that the disordered structure of the internal loop of the unbound aptamer hairpin has a characteristic packing of guanines, which prevents barrier-free penetration of ligands into the site cavity. Also, this disordered structure of the unbound aptamer has a network of hydrogen bonds stabilising the cavity near the target guanines within the binding sites during the whole binding process. We suggested that the first AMP molecule binds to the disordered structure of the site closest to the aptamer hairpin stem and spends some free energy on ordering of the internal loop. Then the second AMP molecule binds to the ordered site closest to the aptamer hairpin loop with a lower energy gain. As a result, the induced-fit binding model is the most applicable for this aptamer and does not contradict the modern experimental NMR and calorimetry data.

Rapid and robust analysis of aristolochic acid I in Chinese medicinal herbal preparations by surface-enhanced Raman spectroscopy

The use of Chinese herbs containing aristolochic acid can induce the exchange of adenine and thymine in gene mutations and even cause liver cancer. To eliminate the harm of aristolochic acids (AAs) to humans, a rapid and robust method of AAs screening is a prerequisite. In this work, a facile and robust Surface-enhanced Raman spectroscopy (SERS) method was used for the qualitative and quantitative detection of AAs in Chinese medicinal herbal preparations based on the mandelic acid modified Ag nanoparticles SERS substrate. Qualitative and quantitative SERS detection of Aristolochic acid I (AAI) was achieved with a good linear relationship ranging from 0.2 − 120.0 μM and a limit of detection (LOD) of 0.06 μM. The proposed method demonstrates a refined strategy for sensitivity analysis of AAs with the advantages of easy operation, time-saving, high sensitivity, and molecular specificity, making it a preferred platform for the screening of AAI in regular inspections of herbal products and regulatory supervision of the supply chain.

Molecular Characteristics, Receptor Specificity, and Pathogenicity of Avian Influenza Viruses Isolated from Wild Ducks in Russia.

Avian influenza viruses (AIV) of wild ducks are known to be able to sporadically infect domestic birds and spread along poultry. Regular surveillance of AIV in the wild is needed to prepare for potential outbreaks. During long-year monitoring, 46 strains of AIV were isolated from gulls and mallards in Moscow ponds and completely sequenced. Amino acid positions that affect the pathogenicity of influenza viruses in different hosts were tested. The binding affinity of the virus for receptors analogs typical for different hosts and the pathogenicity of viruses for mice and chickens were investigated. Moscow isolates did not contain well-known markers of pathogenicity and/or adaptation to mammals, so as a polybasic cleavage site in HA, substitutions of 226Q and 228G amino acids in the receptor-binding region of HA, and substitutions of 627E and 701D amino acids in the PB2. The PDZ-domain ligand in the NS protein of all studied viruses contains the ESEV or ESEI sequence. Although several viruses had the N66S substitution in the PB1-F2 protein, all Moscow isolates were apathogenic for both mice and chickens. This demonstrates that the phenotypic manifestation of pathogenicity factors is not absolute but depends on the genome context.

Self-supervised classification of subcellular morphometric phenotypes reveals extracellular matrix-specific morphological responses

Cell morphology is profoundly influenced by cellular interactions with microenvironmental factors such as the extracellular matrix (ECM). Upon adhesion to specific ECM, various cell types are known to exhibit different but distinctive morphologies, suggesting that ECM-dependent cell morphological responses may harbour rich information on cellular signalling states. However, the inherent morphological complexity of cellular and subcellular structures has posed an ongoing challenge for automated quantitative analysis. Since multi-channel fluorescence microscopy provides robust molecular specificity important for the biological interpretations of observed cellular architecture, here we develop a deep learning-based analysis pipeline for the classification of cell morphometric phenotypes from multi-channel fluorescence micrographs, termed SE-RNN (residual neural network with squeeze-and-excite blocks). We demonstrate SERNN-based classification of distinct morphological signatures observed when fibroblasts or epithelial cells are presented with different ECM. Our results underscore how cell shapes are non-random and established the framework for classifying cell shapes into distinct morphological signature in a cell-type and ECM-specific manner.

CNN-based two-branch multi-scale feature extraction network for retrosynthesis prediction

BackgroundRetrosynthesis prediction is the task of deducing reactants from reaction products, which is of great importance for designing the synthesis routes of the target products. The product molecules are generally represented with some descriptors such as simplified molecular input line entry specification (SMILES) or molecular fingerprints in order to build the prediction models. However, most of the existing models utilize only one molecular descriptor and simply consider the molecular descriptors in a whole rather than further mining multi-scale features, which cannot fully and finely utilizes molecules and molecular descriptors features.ResultsWe propose a novel model to address the above concerns. Firstly, we build a new convolutional neural network (CNN) based feature extraction network to extract multi-scale features from the molecular descriptors by utilizing several filters with different sizes. Then, we utilize a two-branch feature extraction layer to fusion the multi-scale features of several molecular descriptors to perform the retrosynthesis prediction without expert knowledge. The comparing result with other models on the benchmark USPTO-50k chemical dataset shows that our model surpasses the state-of-the-art model by 7.4%, 10.8%, 11.7% and 12.2% in terms of the top-1, top-3, top-5 and top-10 accuracies. Since there is no related work in the field of bioretrosynthesis prediction due to the fact that compounds in metabolic reactions are much more difficult to be featured than those in chemical reactions, we further test the feasibility of our model in task of bioretrosynthesis prediction by using the well-known MetaNetX metabolic dataset, and achieve top-1, top-3, top-5 and top-10 accuracies of 45.2%, 67.0%, 73.6% and 82.2%, respectively.ConclusionThe comparison result on USPTO-50k indicates that our proposed model surpasses the existing state-of-the-art model. The evaluation result on MetaNetX dataset indicates that the models used for retrosynthesis prediction can also be used for bioretrosynthesis prediction.

In Vitro Infant Fecal Fermentation Characteristics of Human Milk Oligosaccharides Were Controlled by Initial Microbiota Composition More than Chemical Structure.

Human milk oligosaccharides (HMOs), multifunctional glycans naturally present in human milk, are known to contribute to the infant's microbiota and immune system development. However, the molecular specificity of HMOs on microbiota and associated fermentation is not yet fully understood, and is important for the development of infant formula optimum functionality. In vitro fermentation is carried out on structurally different HMOs with infant fecal inocula dominated by Bifidobacterium longum, Bifidobacterium breve, and Bacteroides. The gas, metabolite (SCFA, lactate, and succinate) profiles, and microbiota responses differ between individual microbiota inocula patterns regardless of HMO structure. In terms of HMO pairs with same sugar composition but different glycosidic bonds, gas and metabolite profiles are similar with the B. longum- and B. breve-dominated inocula. However, large individual variations are observed with the Bacteroides-dominated inocula. The microbial communities at the end of fermentation are closely related to the initial microbiota composition. The findings demonstrate that short-term in vitro fermentation outcomes largely depend on the initial gut microbiota composition more than the impact of HMO molecular specificity. These results advance the current understanding for the design of personalized infant nutritional solutions and therapies in future.

Selective TnsC recruitment enhances the fidelity of RNA-guided transposition.

Bacterial transposonsare pervasive mobile genetic elements that use distinct DNA-binding proteins for horizontal transmission. For example, Escherichia coli Tn7 homes to a specific attachment site using TnsD1, whereas CRISPR-associated transposons use type I or type V Cas effectors to insert downstream of target sites specified by guide RNAs2,3. Despite this targeting diversity, transposition invariably requires TnsB, a DDE-family transposase that catalyses DNA excision and insertion, and TnsC, a AAA+ ATPase that is thought to communicate between transposase and targeting proteins4. How TnsC mediates this communication and thereby regulates transposition fidelity has remained unclear. Here we use chromatin immunoprecipitation with sequencing to monitor in vivo formation of the type I-F RNA-guided transpososome, enabling us to resolve distinct protein recruitment events before integration. DNA targeting by the TniQ-Cascade complex is surprisingly promiscuous-hundreds of genomic off-target sites are sampled, but only a subset of those sites is licensed for TnsC and TnsB recruitment, revealing a crucial proofreading checkpoint. To advance the mechanistic understanding of interactions responsible for transpososome assembly, we determined structures of TnsC using cryogenic electron microscopy and found that ATP binding drives the formation of heptameric rings that thread DNA through the central pore, thereby positioning the substrate for downstream integration. Collectively, our results highlight the molecular specificity imparted by consecutive factor binding to genomic target sites during RNA-guided transposition, and provide a structural roadmap to guide future engineering efforts.

Label-Free Sensing with Metal Nanostructure-Based Surface-Enhanced Raman Spectroscopy for Cancer Diagnosis.

Surface-Enhanced Raman Spectroscopy (SERS) is a powerful analytical technique for the detection of small analytes with great potential for medical diagnostic applications. Its high sensitivity and excellent molecular specificity, which stems from the unique fingerprint of molecular species, have been applied toward the detection of different types of cancer. The noninvasive and rapid detection offered by SERS highlights its applicability for point-of-care (PoC) deployment for cancer diagnosis, screening, and staging, as well as for predicting tumor recurrence and treatment monitoring. This review provides an overview of the progress in label-free (direct) SERS-based chemical detection for cancer diagnosis with the main focus on the advances in the design and preparation of SERS substrates on the basis of metal nanoparticle structures formed via bottom-up strategies. It begins by introducing a synopsis of the working principles of SERS, including key chemometric approaches for spectroscopic data analysis. Then it introduces the advances of label-free sensing with SERS in cancer diagnosis using biofluids (blood, urine, saliva, sweat) and breath as the detection media. In the end, an outlook of the advances and challenges in cancer diagnosis via SERS is provided.

Clinical and Technical Aspects in Free Cortisol Measurement.

Accurate measurement of cortisol is critical in adrenal insufficiency as it reduces the risk associated with misdiagnosis and supports the optimization of stress dose. Comprehensive assays have been developed to determine the levels of bioactive free cortisol and their clinical and analytical efficacies have been extensively discussed because the level of total cortisol is affected by changes in the structure or circulating levels of corticoid-binding globulin and albumin, which are the main reservoirs of cortisol in the human body. Antibody-based immunoassays are routinely used in clinical laboratories; however, the lack of molecular specificity in cortisol assessment limits their applicability to characterize adrenocortical function. Improved specificity and sensitivity can be achieved by mass spectrometry coupled with chromatographic separation methods, which is a cutting-edge technology to measure individual as well as a panel of steroids in a single analytical run. The purpose of this review is to introduce recent advances in free cortisol measurement from the perspectives of clinical specimens and issues associated with prospective analytical technologies.

Highly multiplexed, label-free proteoform imaging of tissues by individual ion mass spectrometry.

Imaging of proteoforms in human tissues is hindered by low molecular specificity and limited proteome coverage. Here, we introduce proteoform imaging mass spectrometry (PiMS), which increases the size limit for proteoform detection and identification by fourfold compared to reported methods and reveals tissue localization of proteoforms at <80-μm spatial resolution. PiMS advances proteoform imaging by combining ambient nanospray desorption electrospray ionization with ion detection using individual ion mass spectrometry. We demonstrate highly multiplexed proteoform imaging of human kidney, annotating 169 of 400 proteoforms of <70 kDa using top-down MS and a database lookup of ~1000 kidney candidate proteoforms, including dozens of key enzymes in primary metabolism. PiMS images reveal distinct spatial localizations of proteoforms to both anatomical structures and cellular neighborhoods in the vasculature, medulla, and cortex regions of the human kidney. The benefits of PiMS are poised to increase proteome coverage for label-free protein imaging of tissues.

The Significance of Cell Surface N-Glycosylation for Internalization and Potency of Cytotoxic Conjugates Targeting Receptor Tyrosine Kinases.

Precise anticancer therapies employing cytotoxic conjugates constitute a side-effect-limited, highly attractive alternative to commonly used cancer treatment modalities, such as conventional chemotherapy, radiotherapy or surgical interventions. Receptor tyrosine kinases are a large family of N-glycoproteins intensively studied as molecular targets for cytotoxic conjugates in various cancers. At the cell surface, these receptors are embedded in a dense carbohydrate layer formed by numerous plasma membrane glycoproteins. The complexity of the cell surface architecture is further increased by galectins, secreted lectins capable of recognizing and clustering glycoconjugates, affecting their motility and activity. Cell surface N-glycosylation is intensively remodeled by cancer cells; however, the contribution of this phenomenon to the efficiency of treatment with cytotoxic conjugates is largely unknown. Here, we evaluated the significance of N-glycosylation for the internalization and toxicity of conjugates targeting two model receptor tyrosine kinases strongly implicated in cancer: HER2 and FGFR1. We employed three conjugates of distinct molecular architecture and specificity: AffibodyHER2-vcMMAE (targeting HER2), vcMMAE-KCK-FGF1.E and T-Fc-vcMMAE (recognizing different epitopes within FGFR1). We demonstrated that inhibition of N-glycosylation reduced the cellular uptake of all conjugates tested and provided evidence for a role of the galectin network in conjugate internalization. In vitro binding studies revealed that the reduced uptake of conjugates is not due to impaired HER2 and FGFR1 binding. Importantly, we demonstrated that alteration of N-glycosylation can affect the cytotoxic potential of conjugates. Our data implicate a key role for cell surface N-glycosylation in the delivery of cytotoxic conjugates into cancer cells.

Orientation Polarization Spectroscopy-Toward an Atomistic Understanding of Dielectric Relaxation Processes.

The theory of orientation polarization and dielectric relaxation was developed by P. Debye more than 100 years ago. It is based on approximating a molecule by a sphere having one or more dipole moments. By that the detailed intra- and intermolecular interactions are explicitly not taken into consideration. In this article, the principal limitations of the Debye approximation are discussed. Taking advantage of the molecular specificity of the infrared (IR) spectral range, measurements of the specific IR absorption of the stretching vibration υ(OH) (at 3370 cm−1) and the asymmetric υas(CH2) (at 2862.9 cm−1) are performed in dependence on the frequency and the strength of external electric fields and at varying temperature. The observed effects are interpreted as caused by orientation polarization of the OH and the adjacent CH2 moieties.

A viral toolbox for conditional and transneuronal gene expression in zebrafish.

The zebrafish is an important model in systems neuroscience but viral tools to dissect the structure and function of neuronal circuitry are not established. We developed methods for efficient gene transfer and retrograde tracing in adult and larval zebrafish by herpes simplex viruses (HSV1). HSV1 was combined with the Gal4/UAS system to target cell types with high spatial, temporal, and molecular specificity. We also established methods for efficient transneuronal tracing by modified rabies viruses in zebrafish. We demonstrate that HSV1 and rabies viruses can be used to visualize and manipulate genetically or anatomically identified neurons within and across different brain areas of adult and larval zebrafish. An expandable library of viruses is provided to express fluorescent proteins, calcium indicators, optogenetic probes, toxins and other molecular tools. This toolbox creates new opportunities to interrogate neuronal circuits in zebrafish through combinations of genetic and viral approaches.

Polarization-Dependent Heterodyne-Detected Sum-Frequency Generation Spectroscopy as a Tool to Explore Surface Molecular Orientation and Ångström-Scale Depth Profiling.

Sum-frequency generation (SFG) spectroscopy provides a unique optical probe for interfacial molecules with interface-specificity and molecular specificity. SFG measurements can be further carried out at different polarization combinations, but the target of the polarization-dependent SFG is conventionally limited to investigating the molecular orientation. Here, we explore the possibility of polarization-dependent SFG (PD-SFG) measurements with heterodyne detection (HD-PD-SFG). We stress that HD-PD-SFG enables accurate determination of the peak amplitude, a key factor of the PD-SFG data. Subsequently, we outline that HD-PD-SFG can be used not only for estimating the molecular orientation but also for investigating the interfacial dielectric profile and studying the depth profile of molecules. We further illustrate the variety of combined simulation and PD-SFG studies.

Antifungal Effect of Penicillamine Due to the Selective Targeting of L-Homoserine O-Acetyltransferase.

Due to the apparent similarity of fungal and mammalian metabolic pathways, the number of established antifungal targets is low, and the identification of novel ones is highly desirable. The results of our studies, presented in this work, indicate that the fungal biosynthetic pathway of L-methionine, an amino acid essential for humans, seems to be an attractive perspective. The MET2 gene from Candida albicans encoding L-homoserine O-acetyltransferase (CaMet2p), an enzyme catalyzing the first step in that pathway, was cloned and expressed as the native or the oligo-His-tagged fusion protein in Escherichia coli. The recombinant enzymes were purified and characterized for their basic molecular properties and substrate specificities. The purified MET2 gene product revealed the appropriate activity, catalyzed the conversion of L-homoserine (L-Hom) to O-acetyl-L-homoserine (OALH), and exhibited differential sensitivity to several L-Hom or OALH analogues, including penicillamine. Surprisingly, both penicillamine enantiomers (L- and D-Pen) displayed comparable inhibitory effects. The results of the docking of L- and D-Pen to the model of CaMet2p confirmed that both enantiomeric forms of the inhibitor are able to bind to the catalytic site of the enzyme with similar affinities and a similar binding mode. The sensitivity of some fungal cells to L-Pen, depending on the presence or absence of L-Met in the medium, clearly indicate Met2p targeting. Moreover, C. glabrata clinical strains that are resistant to fluconazole displayed a similar susceptibility to L-Pen as the wild-type strains. Our results prove the potential usefulness of Met2p as a molecular target for antifungal chemotherapy.

Multiscale optical and optoacoustic imaging of amyloid-β deposits in mice.

Deposits of amyloid-β (Aβ) in the brains of rodents can be analysed by invasive intravital microscopy on a submillimetre scale, or via whole-brain images from modalities lacking the resolution or molecular specificity to accurately characterize Aβ pathologies. Here we show that large-field multifocal illumination fluorescence microscopy and panoramic volumetric multispectral optoacoustic tomography can be combined to longitudinally assess Aβ deposits in transgenic mouse models of Alzheimer's disease. We used fluorescent Aβ-targeted probes (the luminescent conjugated oligothiophene HS-169 and the oxazine-derivative AOI987) to transcranially detect Aβ deposits in the cortex of APP/PS1 and arcAβ mice with single-plaque resolution (8 μm) and across the whole brain (including the hippocampus and the thalamus, which are inaccessible by conventional intravital microscopy) at sub-150 μm resolutions. Two-photon microscopy, light-sheet microscopy and immunohistochemistry of brain-tissue sections confirmed the specificity and regional distributions of the deposits. High-resolution multiscale optical and optoacoustic imaging of Aβ deposits across the entire brain in rodents thus facilitates the in vivo study of Aβ accumulation by brain region and by animal age and strain.

Fractionation by flash chromatography and molecular characterization of bio-oil by ultra-high-resolution mass spectrometry and NMR spectroscopy

Bio-oils obtained from the pyrolysis of lignocellulosic biomass are highly complex matrices with tens of thousands of organic compounds covering a wide polarity and mass range. Their upgrading into greener fuel or value-added chemicals requires an in-depth knowledge of their molecular composition prior and after valorization treatment, which can be obtained by different analytical techniques. Here, flash chromatography was employed as an efficient way to fractionate a bio-oil vacuum residue into four distinct fractions. This process was repeatable at both gravimetric and chromatographic levels and provided information about the weight contribution of each fraction to bio-oil composition. Achieved fractions were then characterized by ultra-high-resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and nuclear magnetic resonance spectroscopy. These two techniques ensured the good repeatability of bio-oil fractionation at the molecular level and also highlighted molecular specificities of each fraction. Thus, flash chromatography provided quantitative information that was complementary to FT-ICR MS results. It also simplifies the bio-oil compositional analysis, making it possible to detect more compounds. Eventually, this fractionation process can also be used to target fraction or compounds of interest for bio-oil upgrading.

Molecular Mapping of a Recessive Gene for Stripe Rust Resistance at the YrCf75 Locus Using Bulked Segregant Analysis Combined with Single Nucleotide Polymorphism Genotyping Arrays and Bulked Segregant RNA-Sequencing.

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most important diseases in wheat worldwide. Planting resistant varieties is the most economical, effective, and environment-friendly measure to control wheat stripe rust. Changfeng 75, a Chinese winter wheat variety, shows high stripe rust resistance in both seedling and adult-plant stages. The seedling tests of F1, F2, and F2:3 populations derived from Mingxian 169/Changfeng 75 inoculated with Chinese predominant Pst race CYR34 showed that the stripe rust resistance of Changfeng 75 was controlled by a single recessive gene. The locus was temporarily designated as YrCf75. Bulked segregant analysis (BSA) combined with the wheat 660K single-nucleotide polymorphism (SNP) array and bulked segregant RNA-sequencing indicated that the proportion of polymorphic SNPs on wheat chromosome 2A was the highest, which suggested that YrCf75 was likely located on chromosome 2A. Two hundred and twenty-five Kompetitive allele-specific PCR (KASP) and 75 simple sequence repeat (SSR) markers on chromosome 2A were used to map YrCf75 using the BSA approach. Linkage analysis indicated that 31 KASP markers and one SSR marker were linked to YrCf75, and the genetic distances of the two closest flanking KASP markers, AX-1110060462 and AX-111004763, were 1.2 and 2.7 cM, respectively. YrCf75 was located on wheat chromosome 2AL. The molecular detection, resistance specificity, and chromosome location showed that YrCf75 is likely a new gene that is different from the known stripe rust resistance genes (Yr1 and Yr32) on wheat chromosome 2AL.