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1.
Methods Mol Biol ; 2564: 47-52, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36107336

RESUMO

Many fluorescent proteins are available nowadays suitable for in vivo imaging experiments. Each fluorescent protein has unique biophysical properties, such as emission and excitation spectra, quantum yield, oligomerization state, pH sensitivity, fluorescence lifetime, and stability within the cellular environment. Even a small variation in fluorescent protein properties might result in significant differences in the experimental outcomes. This chapter discusses the aspects that need to be considered while selecting the fluorescent proteins for in vivo imaging experiment.


Assuntos
Diagnóstico por Imagem , Proteínas , Biofísica , Corantes , Diagnóstico por Imagem/métodos , Fluorescência
2.
Methods Mol Biol ; 2564: 53-74, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36107337

RESUMO

Fluorescent proteins are standard tools for addressing biological questions in a cell biology laboratory. The genetic tagging of protein of interest with fluorescent proteins opens the opportunity to follow them in vivo and to understand their interactions and dynamics. In addition, the latest advances in optical microscopy image acquisition and processing allow us to study many cellular processes in vivo. Techniques such as fluorescence lifetime microscopy and hyperspectral imaging provide valuable tools for understanding fluorescent protein interactions and their photophysics. Finally, fluorescence fluctuation analysis opens the possibility to address questions of molecular diffusion, protein-protein interactions, and oligomerization, among others, yielding quantitative information on the subject of study. This chapter will cover some of the more important advances in cutting-edge technologies and methods that, combined with fluorescent proteins, open new frontiers for biological studies.


Assuntos
Corantes , Proteínas , Fenômenos Fisiológicos Celulares , Microscopia de Fluorescência/métodos
3.
Methods Mol Biol ; 2564: 99-119, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36107339

RESUMO

Fluorescent proteins have revolutionized cell biology and cell imaging through their use as genetically encoded tags. Structural biology has been pivotal in understanding how their unique fluorescent properties manifest through the formation of the chromophore and how the spectral properties are tuned through interaction networks. This knowledge has in turn led to the construction of novel variants with new and improved properties. Here we describe the process by which fluorescent protein structures are determined, starting from recombinant protein production to structure determination by molecular replacement. We also describe how to incorporate and determine the structures of proteins containing non-natural amino acids. Recent advances in protein engineering have led to reprogramming of the genetic code to allow incorporation of new chemistry at designed residue positions, with fluorescent proteins being at the forefront of structural studies in this area. The impact of such new chemistry on protein structure is still limited; the accumulation of more protein structures will undoubtedly improve our understanding and ability to engineer proteins with new chemical functionality.


Assuntos
Aminoácidos , Código Genético , Aminoácidos/química , Corantes , Cristalização , Engenharia de Proteínas/métodos , Proteínas Recombinantes/genética
4.
Methods Mol Biol ; 2564: 223-246, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36107345

RESUMO

DNA binding fluorescent proteins are a powerful tool for single-molecule visualization. In this chapter, we discuss a protocol for the synthesis of DNA binding fluorescent proteins and visualization of single DNA molecules. This chapter includes stepwise methods for molecular cloning, reversible staining, two-color staining, sequence-specific staining, and microscopic visualization of single DNA molecules in a microfluidic device. This content will be useful for DNA characterization using DNA binding fluorescent proteins and its visualization at the single-molecule level.


Assuntos
Proteínas de Ligação a DNA , DNA , DNA/química , Proteínas de Ligação a DNA/metabolismo , Corantes Fluorescentes , Microscopia de Fluorescência/métodos , Coloração e Rotulagem
5.
Methods Mol Biol ; 2564: 247-258, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36107346

RESUMO

Citrate is a central intracellular metabolite with roles in a variety of normal and aberrant biological processes. The methods for quantifying citrate concentration in cells can enable the study of the molecular mechanisms of citrate-related biological processes and diseases. Compared to existing analytical methods such as enzymatic assays and mass spectrometry, genetically encoded biosensors based on fluorescent proteins (FPs) offer the advantage of noninvasively tracking intracellular ion and small molecule dynamics with high spatial-temporal resolution. These biosensors are less toxic than chemosensors and can be targeted to specific organelles for subcellular imaging. Here we present a protocol for quantification of cytosolic and mitochondrial citrate in mammalian cells with recently reported genetically encoded biosensors for citrate.


Assuntos
Técnicas Biossensoriais , Ácido Cítrico , Animais , Técnicas Biossensoriais/métodos , Citratos , Mamíferos , Proteínas
6.
Methods Mol Biol ; 2570: 85-102, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36156776

RESUMO

Although SELEX can identify high-affinity aptamers, Doped-SELEX is often performed post-selection for the identification of better variants. Starting from a partially randomized (doped) library derived from an already identified aptamer, this method can screen rapidly several thousand substitutions in order to identify those that can improve the binding of the aptamers. It can also highlight the positions that do not tolerate substitutions, which suggest they are crucial for the interaction of the aptamer with its target. High-throughput sequencing (HTS), also named next-generation sequencing (NGS), can dramatically improve this method by studying millions of sequences. This high number of sequences ensures a statistically robust analysis of variants even for those with a low frequency in the library. It can reduce the number of selection rounds and provide a more in-depth analysis of the positions that are crucial for the aptamer affinity. In this chapter, we provide a protocol to simultaneously study and improve an aptamer using Doped-SELEX and HTS analysis, including the design of the doped library, the selection, HTS, and analysis. This protocol could be useful to improve the affinity of an aptamer and to reduce its size as well as to improve ribozyme.


Assuntos
Aptâmeros de Nucleotídeos , RNA Catalítico , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Técnica de Seleção de Aptâmeros/métodos
7.
Methods Mol Biol ; 2570: 105-118, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36156777

RESUMO

Isothermal titration calorimetry (ITC) is a technique where the heat given off, or absorbed, during a binding event is measured and used to determine the binding thermodynamics and affinity associated with binding. This protocol focuses on ITC applications for studying aptamer interactions with small molecule ligands where ITC has the advantage of being a label-free solution-based technique. The limitation of ITC using a relatively large amount of material compared to other analytical techniques is not applicable here as large amounts of nucleic acids, especially DNA, can be readily obtained. In this chapter we describe how to use ITC methods to measure the thermodynamics and affinity of binding using the interaction of quinine with a DNA cocaine-binding aptamer as an example.


Assuntos
Aptâmeros de Nucleotídeos , Cocaína , Ácidos Nucleicos , Aptâmeros de Nucleotídeos/química , Calorimetria/métodos , Cocaína/química , Ligantes , Ácidos Nucleicos/metabolismo , Ligação Proteica , Quinina/química , Termodinâmica
8.
Methods Mol Biol ; 2570: 141-153, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36156780

RESUMO

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.


Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Aptâmeros de Nucleotídeos/química , Fenômenos Biofísicos , Técnicas Biossensoriais/métodos , Humanos , Cinética , Ressonância de Plasmônio de Superfície/métodos , Ativador de Plasminogênio Tipo Uroquinase
9.
Methods Mol Biol ; 2570: 177-185, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36156782

RESUMO

Super-resolution microscopy methods enable the visualization of biological processes on the level of a few nanometers. However, the application of these techniques in biological systems is limited by the availability of small affinity reagents. Slow off-rate-modified aptamers as nucleic acid analogues to antibodies have been successfully applied to improve the resolution and quantification of DNA-PAINT. In this chapter, we describe a protocol for using SOMAmers as labeling reagents for super-resolution microscopy.


Assuntos
Ácidos Nucleicos , Oligonucleotídeos , DNA , Microscopia de Fluorescência/métodos
10.
Methods Mol Biol ; 2570: 187-196, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36156783

RESUMO

Aptamer-functionalized field-effect transistor (FET) biosensors enable detection of small-molecule targets in complex environments such as tissue and blood. Conventional FET-based platforms suffer from Debye screening in high ionic strength physiological environments where the effective sensing distance is limited to less than a nanometer from the surface of the sensor. Aptamers that undergo significant conformational rearrangement of negatively charged backbones upon target recognition within or in close proximity to the Debye length, facilitate the transduction of electronic signals through the semiconducting channel. Herein, the fabrication of high-performance, ultrathin-film FETs and subsequent aptamer functionalization are described. Moreover, electronic sensing measurement protocols alongside calibration methods to minimize device-to-device variations are covered.


Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Técnicas Biossensoriais/métodos , Transistores Eletrônicos
11.
Methods Mol Biol ; 2570: 197-204, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36156784

RESUMO

The impedimetric detection of a protein, lysozyme (LYS), was carried out herein by aptamer-immobilized single-use pencil graphite electrodes (PGEs). The aptamer was immobilized onto electrochemically activated surface of electrode without using any chemical agents, or any types of nanomaterials. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques were applied to analyze the electrochemical behavior of unmodified PGE and aptamer immobilized PGE. The interaction of aptamer with its target protein, LYS, was then investigated by EIS. The limit of detection for LYS was found to be 1.44 µg/mL (equals to 100.65 nM). The developed aptasensor specific to LYS presented high selectivity against to bovine serum albumin and thrombin.


Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Grafite , Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Eletrodos , Ouro , Grafite/química , Muramidase/química , Prostaglandinas E , Soroalbumina Bovina , Trombina
12.
Methods Mol Biol ; 2570: 235-242, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36156787

RESUMO

Aptamers are single-stranded oligonucleotides able to recognize a target with high affinity and specificity. Aptamers are used in different diagnostics applications, highlighting, among all, variations of the traditional enzyme-linked immunosorbent assay (ELISA). In this chapter, we show the procedures for the development of two types of indirect ELONA: a sandwich ELONA and a direct ELONA coupled to either real-time quantitative PCR as a direct and sensitive readout.


Assuntos
Aptâmeros de Nucleotídeos , Ensaios Enzimáticos , Ensaio de Imunoadsorção Enzimática , Reação em Cadeia da Polimerase em Tempo Real , Técnica de Seleção de Aptâmeros/métodos
13.
Methods Mol Biol ; 2570: 243-269, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36156788

RESUMO

Small-molecule sensing is a major issue as they can serve both in fundamental science and as makers of various diseases, contaminations, or even environment pollution. RNA aptamers are single-stranded nucleic acids that can adopt different conformations and specifically recognize a wide range of ligands, making them good candidates to develop biosensors of small molecules. Recently, light-up RNA aptamers have been introduced and used as starting building blocks of RNA-based fluorogenic biosensors. They are typically made of three domains: a reporter domain (a light-up aptamer), connected to a sensor domain (another aptamer) via a communication module. The latter is instrumental as being in charge of information transmission between the sensor and the reporting domains. Here we present an ultrahigh-throughput screening procedure to develop RNA-based fluorogenic biosensors by selecting optimized communication modules through an exhaustive functional exploration of every possible sequence permutation using droplet-based microfluidics and next-generation sequencing.


Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Aptâmeros de Nucleotídeos/genética , Técnicas Biossensoriais/métodos , Ligantes , Microfluídica/métodos , RNA/genética
14.
Methods Mol Biol ; 2570: 271-280, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36156789

RESUMO

Electrochemical aptamer-based (E-AB) sensors using conformational change-induced electron transfer kinetics are sensitive, reagent-less, and cost-effective tools for molecular sensing. Current advances in this technology can allow continuous drug pharmacokinetic monitoring in living animals (Dauphin-Ducharme et al., ACS Sens 4(10):2832-2837, 2019; Idili et al., Chem Sci 10(35):8164-8170, 2019), as well as automated analysis of hormone pulsatility (Liang et al., Nat Commun 10(1):852, 2019). In this chapter, we provide the methodology for an automated E-AB conformational change-based robotic sensing platform. By using an open-source programmable robotic system, this method can be adapted to a wide range of experimental scenarios.


Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Procedimentos Cirúrgicos Robóticos , Animais , Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Eletrodos , Ouro/química , Hormônios
15.
Food Chem ; 400: 134032, 2023 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-36055145

RESUMO

The formation mechanism of heat-induced egg yolk granules (EYGs)/sodium alginate (SA) emulsion gel was studied under pH 6.2 and 7.5. Particle size, water holding capacity, LF NMR, and protein solubility revealed that pH 7.5 increased the surface charge of EYGs and enhanced non-covalent interaction with SA, and hydrogen bonding dominated of the gel formation process. Microscopy and rheological analysis indicated that samples with 0.75% SA had the smallest particle size and highest G', with chain-like oil droplets. Excess SA (1%) led to depletion flocculation due to SA structural rearrangements around oil droplets caused by the increase in negatively charged, causing uneven network structure. The in vitro release property and storage stability of ß-carotene loaded in the EYGs/SA emulsion gel showed that SA increased storage stability and decreased bioaccessibility of ß-carotene with delayed digestion rate. These results provide a theoretical basis for the nutrient delivery system in gel foods.


Assuntos
Alginatos/química , Gema de Ovo/química , beta Caroteno/química , Emulsões/química , Géis/química , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Imageamento por Ressonância Magnética/métodos , Tamanho da Partícula , Proteínas/química , Reologia , Solubilidade , Água/química
16.
Med Gas Res ; 13(1): 15-22, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-35946218

RESUMO

Postherpetic neuralgia (PHN) is a devastating disease with extraordinarily poor treatment outcomes. Both pulsed radiofrequency (PRF) and ozone have good effects on the treatment of the disease. However, whether PRF and ozone have a synergistic effect on PHN remains unclear. Therefore, this study aimed to assess the therapeutic effects of ozone alone and in combination with PRF in the treatment of PHN. Ninety-one patients with PHN were assigned into two groups: PRF combined with ozone (PRF + ozone group, n = 44) and ozone therapy alone (ozone group, n = 47). In PRF + ozone group, the high-voltage, long-duration PRF was applied to the target dorsal root ganglions. Then ozonated water (11.5 µg/mL) was injected through the inner cannula. In the ozone group, all other processes were the same as those in the PRF + ozone group apart from the electrical stimulation. The therapeutic efficacy was evaluated by visual analog scale and tactile sensation at pre-treatment and post-treatment 3, 6, and 12 months. Compared with pre-treatment data, the visual analog scale score was significantly decreased in both groups after treatment. Compared with the ozone group, the visual analog scale score was significantly decreased in the PRF + ozone group at 3, 6, and 12 months. Similarly, the tactile sensation was also significantly decreased at post-treatment when compared to pre-treatment. However, there were no statistical differences between the two groups. Regression analysis results showed that the history of diabetes mellitus and age had significant negative and positive effects, respectively, on the treatment results. To conclude, the administration of PRF + ozone and ozone therapy alone could both improve pain symptoms. Moreover, treatment effects and total efficacy rates tended to be higher for the combination of PRF and ozone than ozone alone. This conclusion was especially true for long-term therapeutic effects.


Assuntos
Neuralgia Pós-Herpética , Ozônio , Tratamento por Radiofrequência Pulsada , Gânglios Espinais , Humanos , Neuralgia Pós-Herpética/tratamento farmacológico , Ozônio/uso terapêutico , Tratamento por Radiofrequência Pulsada/métodos , Estudos Retrospectivos
17.
Methods Mol Biol ; 2578: 27-39, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36152278

RESUMO

The analytical performance of the microarray technique in screening the affinity and reactivity of molecules toward a specific target is highly affected by the coupling chemistry adopted to bind probes to the surface. However, the surface functionality limits the biomolecules that can be attached to the surface to a single type of molecule, thus forcing the execution of separate analyses to compare the performance of different species in recognizing their targets. Here, we introduce a new N,N-dimethylacrylamide-based polymeric coating, bearing simultaneously different functionalities (N-acryloyloxysuccinimide and azide groups) to allow an easy and straightforward method to co-immobilize proteins and oriented peptides on the same substrate. The bifunctional copolymer has been obtained by partial post-polymerization modification of the functional groups of a common precursor. This strategy represents a convenient method to reduce the number of analyses, therefore possible systematic or random errors, besides offering a drastic shortage in time, reagents, and costs.


Assuntos
Azidas , Polímeros , Azidas/química , Análise em Microsséries/métodos , Peptídeos/química , Polimerização , Polímeros/química
18.
Methods Mol Biol ; 2578: 17-25, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36152277

RESUMO

Antibody-mediated neurological diseases constitute an emerging clinical entity that remains to be fully explored. Recent studies identified autoantibodies that directly confer pathogenicity, and it was shown that in these cases immunotherapies can result in profound positive patient responses. These advances highlight the urgent need for improved means to effectively screen patient samples for novel autoantibodies (aAbs) and their subsequent characterization. Here, we discuss challenges and opportunities for peptide microarrays to contribute to the identification, mapping, and characterization of the underlying monospecific disease-defining binding surfaces. We outline control experiments, workflow modifications and bioinformatic filtering methods that enhance the predictive power of array-based studies. Further, we highlight experimental and computer-based display approaches that have the potential to expand the use of synthetic microarrays over the detection of discontinuous epitopes. Knowledge over the autoantibody epitopes in neurological disease will enhance our understanding of the pathological mechanisms and thereby potentially contribute to novel diagnostic approaches or even innovative antigen-specific treatments that avoid the serious adverse effects seen with currently used immunosuppressive therapies.


Assuntos
Autoanticorpos , Doenças do Sistema Nervoso , Biologia Computacional , Mapeamento de Epitopos/métodos , Epitopos , Humanos , Análise em Microsséries , Doenças do Sistema Nervoso/diagnóstico , Peptídeos/química
19.
Methods Mol Biol ; 2578: 63-81, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36152281

RESUMO

Understanding antibody specificity and defining response profiles to antigens continue to be essential to both vaccine research and therapeutic antibody development. Peptide scanning assays enable mapping of continuous epitopes in order to delineate antibody-antigen interactions beyond traditional immunoassay formats. We have developed a relatively low-cost method to generate peptide microarray slides for antibody binding studies that allow for interrogation of up to 1536 overlapping peptides derived from the target antigens on a single microslide. Using an IntavisAG MultiPep RS peptide synthesizer and a Digilab MicroGrid II 600 microarray printer robot, each peptide is tagged with a polyethylene glycol aminooxy terminus to improve peptide solubility, orientation, and conjugation efficiency to the slide surface. Interrogation of the surface can then be performed using polyclonal immune sera or monoclonal antibodies, and sensitive detection using an InnoScan 1100 AL scanner with fluorescent-conjugated secondary reagents maximizes conservation of reagents.


Assuntos
Análise Serial de Proteínas , Vacinas , Anticorpos Monoclonais , Mapeamento de Epitopos/métodos , Epitopos , Soros Imunes , Peptídeos , Polietilenoglicóis , Análise Serial de Proteínas/métodos
20.
Methods Mol Biol ; 2578: 103-120, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36152283

RESUMO

This chapter describes the principles for selection of antigenic peptides for the development of anti-peptide antibodies suitable for microarray-based multiplex affinity assays and optional mass spectrometry detection. The methods described here are mostly applicable to small- and medium-scale multiplex affinity assay and microarrays. Although the same principles of peptide selection may also be applied to larger-scale arrays (with 100+ features), informatics software and printing methods may well differ. Due to the sheer number of proteins/peptides to be processed and analyzed, dedicated software with high processing capacity and enterprise-level array robotics may be required for larger-scale efforts. This report aims to provide practical advice to those seeking to develop or use arrays with up to ~100 different peptide or protein features.


Assuntos
Peptídeos , Análise Serial de Proteínas , Antígenos , Espectrometria de Massas/métodos , Peptídeos/química , Análise Serial de Proteínas/métodos , Proteínas
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