Bio-nanocapsules for oriented immobilization of DNA aptamers on aptasensors

The Analyst ◽  
2022 ◽  
Author(s):  
Masumi Iijima ◽  
Yuki Yamada ◽  
Hideo Nakano ◽  
Tsutomu Nakayama ◽  
Shun'ichi Kuroda
Keyword(s):  
Binding Capacity ◽  
Dna Aptamers ◽  
Oriented Immobilization ◽  
Target Binding

The oriented immobilization of sensing molecules (e.g., IgGs, receptors, lectins, and DNA aptamers) on sensor chips is particularly important for maximizing the sensitivity and target-binding capacity of biosensors.

scholarly journals A Combined ELONA-(RT)qPCR Approach for Characterizing DNA and RNA Aptamers Selected against PCBP-2

Molecules ◽  
2019 ◽  
Vol 24 (7) ◽  
pp. 1213 ◽  
Author(s):  
Miguel Moreno ◽  
María Fernández-Algar ◽  
Javier Fernández-Chamorro ◽  
Jorge Ramajo ◽  
Encarnación Martínez-Salas ◽  
...  
Keyword(s):  
Binding Capacity ◽  
Rna Aptamers ◽  
Dna Aptamers ◽  
General Strategy ◽  
Label Free ◽  
Mobility Shift ◽  
Real Time Quantitative Pcr ◽  
High Affinity ◽  
Dna And Rna ◽  
Electrophoretic Mobility Shift Assays

Improvements in Systematic Evolution of Ligands by EXponential enrichment (SELEX) technology and DNA sequencing methods have led to the identification of a large number of active nucleic acid molecules after any aptamer selection experiment. As a result, the search for the fittest aptamers has become a laborious and time-consuming task. Herein, we present an optimized approach for the label-free characterization of DNA and RNA aptamers in parallel. The developed method consists in an Enzyme-Linked OligoNucleotide Assay (ELONA) coupled to either real-time quantitative PCR (qPCR, for DNA aptamers) or reverse transcription qPCR (RTqPCR, for RNA aptamers), which allows the detection of aptamer-target interactions in the high femtomolar range. We have applied this methodology to the affinity analysis of DNA and RNA aptamers selected against the poly(C)-binding protein 2 (PCBP-2). In addition, we have used ELONA-(RT)qPCR to quantify the dissociation constant (Kd) and maximum binding capacity (Bmax) of 16 high affinity DNA and RNA aptamers. The Kd values of the high affinity DNA aptamers were compared to those derived from colorimetric ELONA performed in parallel. Additionally, Electrophoretic Mobility Shift Assays (EMSA) were used to confirm the binding of representative PCBP-2-specific RNA aptamers in solution. We propose this ELONA-(RT)qPCR approach as a general strategy for aptamer characterization, with a broad applicability in biotechnology and biomedicine.

scholarly journals Exploring the Antihyperglycemic Chemical Composition and Mechanisms of Tea Using Molecular Docking

2020 ◽  
Vol 2020 ◽  
pp. 1-12
Author(s):  
Yue Sun ◽  
Lufei Wang ◽  
Lily K. Shaughnessy ◽  
Yan Lin ◽  
Qingliang Xu ◽  
...  
Keyword(s):  
Molecular Docking ◽  
Molecular Mechanisms ◽  
Binding Capacity ◽  
Close Correlation ◽  
Tea Plant ◽  
Protein Targets ◽  
Docking Approach ◽  
Bioactive Ingredients ◽  
Target Binding ◽  
Potential Interactions

Tea, a widely consumed beverage, has long been utilized for promoting human health with a close correlation to hyperglycemia. The Tea Metabolome Database (TMDB), the most complete and comprehensive curated collection of tea compounds data containing 1271 identified small molecule compounds from the tea plant (Camellia sinensis), was established previously by our research team. More recently, our studies have found that various tea types possess an antihyperglycemic effect in mice. However, the bioactive ingredients from tea have potential antihyperglycemic activity and their underlying molecular mechanisms remain unclear. In this study, we used a molecular docking approach to investigate the potential interactions between a selected 747 constituents contained in tea and 11 key protein targets of clinical antihyperglycemic drugs. According to our results, the main antihyperglycemic targets of tea composition were consistent with those of the drug rosiglitazone. The screening results showed that GCG, ECG3’Me, TMDB-01443, and CG had great target binding capacity. The results indicated that these chemicals of tea might affect hyperglycemia by acting on protein targets of rosiglitazone.

Efficacy of Base-Modification on Target Binding of Small Molecule DNA Aptamers

2013 ◽  
Vol 135 (25) ◽  
pp. 9412-9419 ◽  
Author(s):  
Yuri Imaizumi ◽  
Yuuya Kasahara ◽  
Hiroto Fujita ◽  
Shunsuke Kitadume ◽  
Hiroaki Ozaki ◽  
...  
Keyword(s):  
Small Molecule ◽  
Dna Aptamers ◽  
Base Modification ◽  
Target Binding

scholarly journals Preactivation Crosslinking—An Efficient Method for the Oriented Immobilization of Antibodies

2019 ◽  
Vol 2 (2) ◽  
pp. 35 ◽  
Author(s):  
Barbara Schroeder ◽  
Hoa Le Xuan ◽  
Jule L. Völzke ◽  
Michael G. Weller
Keyword(s):  
Magnetic Particles ◽  
Binding Capacity ◽  
Protein A ◽  
Covalent Immobilization ◽  
High Yield ◽  
Binding Partner ◽  
Oriented Immobilization ◽  
Novel Method ◽  
Antibody Preparations ◽  
Culture Supernatants

Crosslinking of proteins for their irreversible immobilization on surfaces is a proven and popular method. However, many protocols lead to random orientation and the formation of undefined or even inactive by-products. Most concepts to obtain a more targeted conjugation or immobilization requires the recombinant modification of at least one binding partner, which is often impractical or prohibitively expensive. Here a novel method is presented, which is based on the chemical preactivation of Protein A or G with selected conventional crosslinkers. In a second step, the antibody is added, which is subsequently crosslinked in the Fc part. This leads to an oriented and covalent immobilization of the immunoglobulin with a very high yield. Protocols for Protein A and Protein G with murine and human IgG are presented. This method may be useful for the preparation of columns for affinity chromatography, immunoprecipitation, antibodies conjugated to magnetic particles, permanent and oriented immobilization of antibodies in biosensor systems, microarrays, microtitration plates or any other system, where the loss of antibodies needs to be avoided, and maximum binding capacity is desired. This method is directly applicable even to antibodies in crude cell culture supernatants, raw sera or protein-stabilized antibody preparations without any purification nor enrichment of the IgG. This new method delivered much higher signals as a traditional method and, hence, seems to be preferable in many applications.

Development of an RNA aptamer that acquires binding capacity against HIV-1 Tat protein via G-quadruplex formation in response to potassium ions

2017 ◽  
Vol 53 (52) ◽  
pp. 7056-7059 ◽  
Author(s):  
Yudai Yamaoki ◽  
Takashi Nagata ◽  
Tsukasa Mashima ◽  
Masato Katahira
Keyword(s):  
Binding Capacity ◽  
Binding Activity ◽  
Potassium Ions ◽  
Rna Aptamer ◽  
Tat Protein ◽  
G Quadruplex ◽  
Quadruplex Formation ◽  
Target Binding ◽  
Hiv 1

The development of the first K+-responsive RNA aptamer demonstrating ON/OFF switching of its target-binding activity by sensing the addition/removal of K+ is reported.

DEVELOPMENT OF THE METHOD OF ORIENTED IMMOBILIZATION OF RECOMBINANT FRAGMENT OF PROTEIN А (А-Z2) DIMER

2020 ◽  
pp. 36-38
Author(s):  
A. Ischenko ◽  
S. Rodin
Keyword(s):  
Recombinant Protein ◽  
Binding Capacity ◽  
Recombinant Fragment ◽  
Oriented Immobilization ◽  
Conventional Methods

The method of oriented immobilization of modified recombinant protein А-Z2 on sepharose matrix has been developed. In comparison with conventional methods of immobilization, the developed method allows to increase binding capacity of a sorbent.

scholarly journals Preactivation Crosslinking – An Efficient Method for the Oriented Immobilization of Antibodies

2019 ◽  
Author(s):  
Barbara Schroeder ◽  
Hoa Le Xuan ◽  
Jule L. Völzke ◽  
Michael G. Weller
Keyword(s):  
Magnetic Particles ◽  
Binding Capacity ◽  
Protein A ◽  
Covalent Immobilization ◽  
High Yield ◽  
Binding Partner ◽  
Oriented Immobilization ◽  
Novel Method ◽  
Antibody Preparations ◽  
Culture Supernatants

Crosslinking of proteins for their irreversible immobilization on surfaces is a proven and popular method. However, many protocols lead to random orientation and the formation of undefined or even inactive by-products. Most concepts to obtain a more targeted conjugation or immobilization requires the recombinant modification of at least one binding partner, which is often impractical or prohibitively expensive. Here a novel method is presented, which is based on the chemical preactivation of Protein A or G with selected conventional crosslinkers. In a second step, the antibody is added, which is subsequently crosslinked in the Fc part. This leads to an oriented and covalent immobilization of the immunoglobulin with a very high yield. Protocols for Protein A and Protein G with murine and human IgG are presented. This method may be useful for the preparation of columns for affinity chromatography, immunoprecipitation, antibodies conjugated to magnetic particles, permanent and oriented immobilization of antibodies in biosensor systems, microarrays, microtitration plates or any other system, where the loss of antibodies needs to be avoided, and maximum binding capacity is desired. This method is directly applicable even to antibodies in crude cell culture supernatants, raw sera or protein-stabilized antibody preparations without any purification nor enrichment of the IgG. This new method delivered much higher signals as a traditional method and, hence, seems to be preferable in many applications.

scholarly journals Selection of DNA Aptamers for Ovarian Cancer Biomarker CA125 Using One-Pot SELEX and High-Throughput Sequencing

2017 ◽  
Vol 2017 ◽  
pp. 1-9 ◽  
Author(s):  
Delia J. Scoville ◽  
Tae Kyu Brian Uhm ◽  
Jamie A. Shallcross ◽  
Rebecca J. Whelan
Keyword(s):  
Ovarian Cancer ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Selection Process ◽  
Response To Therapy ◽  
Diagnostic Tools ◽  
Dna Aptamers ◽  
One Pot ◽  
Protein Target ◽  
Target Binding

CA125 is a mucin glycoprotein whose concentration in serum correlates with a woman’s risk of developing ovarian cancer and also indicates response to therapy in diagnosed patients. Accurate detection of this large, complex protein in patient samples is of great clinical relevance. We suggest that powerful new diagnostic tools may be enabled by the development of nucleic acid aptamers with affinity for CA125. Here, we report on our use of One-Pot SELEX to isolate single-stranded DNA aptamers with affinity for CA125, followed by high-throughput sequencing of the selected oligonucleotides. This data-rich approach, combined with bioinformatics tools, enabled the entire selection process to be characterized. Using fluorescence anisotropy and affinity probe capillary electrophoresis, the binding affinities of four aptamer candidates were evaluated. Two aptamers, CA125_1 and CA125_12, both without primers, were found to bind to clinically relevant concentrations of the protein target. Binding was differently influenced by the presence of Mg2+ions, being required for binding of CA125_1 and abrogating binding of CA125_12. In conclusion, One-Pot SELEX was found to be a promising selection method that yielded DNA aptamers to a clinically important protein target.

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