DNA aptamers that bind to chitin

doi:10.1016/S0960-894X(00)00013-5

Bioorganic & Medicinal Chemistry Letters

Volume 10, Issue 5, 6 March 2000, Pages 423-425

https://doi.org/10.1016/S0960-894X(00)00013-5 Get rights and content

Abstract

We have succeeded in the acquisition of DNA aptamers that recognize chitin using in vitro selection. The obtained DNA aptamers have the stem-loop or bulge loop structures with guanine rich loop clusters and the clockwise B-form stems.

Introduction

Oligosaccharide antigens play essential biological roles in cellular adhesion, molecular recognition, and so on. However, little is known about the interaction concerning oligosaccharide from the viewpoints of its molecular basis. The ability to discriminate the presence of different saccharide types with relatively subtle structural differences would be of benefit in both experimental applications and diagnostics. High-affinity nucleic acid ligands, termed ‘aptamers’, can be selected from a random pool of nucleic acid sequences.1, 2, 3 The isolation methodology of the ligands is one of combinatorial chemistry techniques, termed ‘in vitro selection’ or ‘systematic evolution of ligands by exponential enrichment (SELEX)’. Such selection produces high-affinity ligands that can be propagated indefinitely with little risk of losing the stock in significantly lower cost than antibodies, and this method completely obviates the use of animals. In addition to possible ethical considerations, this also provides an opportunity to generate ligands to antigens that are toxic to humans and animals. This selection procedure involves the iterative isolation of ligands out of the random sequence pool with affinity for a defined target molecule together with PCR-based amplification of the selected oligonucleotides after each round of isolation. Using the above methodology, various RNA ligands specific to various target molecules such as amino acids, bases, nucleotides and enzymatic cofactors have been identified.4, 5 Recently, single-strand(ss)DNA ligands that bind thrombin, organic dye, ATP have been isolated via SELEX screenings.3, 6

In the present study, DNA ligands that selectively bind to ‘chitin’, poly-beta-1,4-N-acetylglucosamine, was isolated using SELEX.

Section snippets

Results and Discussions

In vitro selection of the DNA oligomers specific for chitin was performed according to the procedures described in notes.⁷ For the initial selection cycle, synthetic 103-mer oligonucleotides with a random region of 59 nucleotides was amplified using the forward primer (P1) and the reverse primer (P2). After 8 rounds of selections, 20 clones were sequenced⁸ from the pool of selected DNAs to give seven unique sequences. Several sequences among the 20 clones were identical, suggesting multiple

Acknowledgements

This research was supported in part by a grant-in-aid (No. 11132242) for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.

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Compared to the pH = 6 condition, at pH = 6.96, the kinetic parameter ka is slightly smaller, kd is slightly larger, and KA is smaller. Stems and loops on the aptamer structure can affect the affinity of the aptamer for binding to the analyte, which has been widely demonstrated [36,43–51]. Many studies have used a method of intercepting the stem-loop structure to determine which part of the aptamer plays a key role.
Glycated hemoglobin (HbA1c) has been widely explored as an important marker for monitoring and diagnosing diabetes. Due to the advantages of high selectivity, easy preparation, and convenient preservation of aptamers, research on glycated hemoglobin detection utilizing aptasensors has received much attention in recent years. However, factors such as the pH and the salt concentration of the solution and the structure of the aptamer could influence the interactions between HbA1c and the aptamer. In this study, the factors were evaluated using surface plasmon resonance (SPR). The results show that the pH and the salt concentration can greatly affect the formation of a complex between the aptamer and HbA1c. In the stereostructure of the aptamer, loop L1 may be an important motif for recognizing glycated hemoglobin. In addition, the best condition for detecting HbA1c was at pH 6, with a high sensitivity and a low limit of detection(LOD) (1.06 × 10⁻³ $\frac{RU}{nM}$ /2.55 nM). The results also demonstrated that the use of an SPR aptamer biosensor can be a sensitive technique to improve the accuracy and correctness of HbA1c measurement.
Generation and screening of histamine-specific aptamers for application in a novel impedimetric aptamer-based sensor
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Thymine was found at a significantly greater (37 ± 7%) abundance within the Round 11 pool – than those of either adenine, t(96) = 12.8, p > .05 (21 ± 6%); guanine, t(96) = 12.0, p > .05 (22 ± 6%); or cytosine, t(96) = 13.8, p > .05 (20 ± 5%) nucleotide residues. Nucleotide base favouring has been observed following SELEX, e.g. guanine [63,87,90]. Distinct nucleotide base favouring may indicate that thymine could potentially be important to histamine affinity as differing nucleotides and tertiary structure complexity of DNA molecules contribute towards the DNA-histamine interaction [43].
Histamine is an important biomarker in both biomedical and food quality assurance sectors. Current methods of monitoring this compound via fluorescent, electrochemical, and enzymatic means have several drawbacks, preventing routine detection. This work reports on the isolation of single-stranded DNA-based, histamine-targeting aptamers generated by the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) and the characterisation of these candidates via bioinformatics analysis. Aptamer binding affinity was determined by magnetic bead-based enzyme linked oligonucleotide assays, followed by the detection of unmodified histamine at a physiological pH via electrochemical impedance spectroscopy (EIS). Aptamer H47 demonstrated the lowest apparent binding affinity (72.8 ± 13.9 nmol L⁻¹) towards bead immobilised histamine. When immobilised to a gold surface, H47 demonstrated the largest biosensor response (ΔR_ct = 6.83 ± 2.00) compared to other single-stranded DNA sequences in the presence of dissolved histamine. The H47 EIS aptasensor also displayed a highly selective, concentration-dependent response towards histamine (linear range = 1 μmol L⁻¹ - 5 mmol L⁻¹), compared to other similar small molecules. Possessing an apparent binding affinity, limit of detection and limit of quantification of 7.80 ± 1.70 mmol L⁻¹, 4.83 mmol L⁻¹ and 16.08 mmol L⁻¹, respectively, the H47 EIS aptasensor demonstrates promise towards the development of aptasensors in applications which require the rapid detection of histamine in solution.
Screening and evaluation of aptamers against somatostatin, and sandwich-like monitoring of somatostatin based on atomic force microscopy
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A gold-coated silicon cantilever (Olympus Co. Ltd., Tokyo, Japan, BL-RC150VB-C1, spring constant: 0.006 N/m) was used in all experiments. The aptamers against SST were selected based on previously reported methods [25,26]. A double-stranded DNA (dsDNA) library was prepared as follows.
A sensing system was constructed to monitor the target peptide via two aptamer-based sensors pinching. First, aptamers against somatostatin (SST) were selected via the systematic evolution of ligands by exponential enrichment, and four aptamers were selected from a single-stranded DNA library. Their specificities to SST were evaluated via surface plasmon resonance and atomic force microscopy (AFM). Next, two aptamers with higher specificities to SST were used as aptamer-based sensors; one aptamer was modified with a chip, and the other was modified with a probe. Based on AFM system, the probe was surveyed on the chip in SST solutions, simultaneously measuring an interactive force. The label-free SST could be detected, and then the change in its concentration could be monitored at levels that ranged from 2 to 2000 nM. The interactive force of a single pair was approximately 45 pN, and the molecule number was associated with the interactive force. Therefore, we could firstly select the aptamers against somatostatin, and the sandwich-like monitoring system can be used to promote peptide sensor or monitoring system using an aptamer-based sensor.
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Carbohydrates consist of only hydroxyl groups, which are able to form noncovalent bonds with significant stability; therefore, the carbohydrate “antigens” lack both the charged interactions and aromatic ring structures to have strong nucleic acid interactions. Currently, there are less than ten reports in the literature that have screened DNA aptamers for binding to carbohydrates such as cellulose [14], sialyllactose [15], cellobiose [16], (1→3)-β-d-glucans [17] and chitin [18]. These aptamers recognize their targets with Kd values from 10−5 to 10−7 M.
We proposed to use a novel stepwise sequence-constructive SELEX method to develop DNA aptamers that can recognize Globo H which is a tumor-associated carbohydrate antigen. A combinatorial synthetic library that consisted of DNA molecules with randomized regions of 15-bases was used as the starting library for the first SELEX procedure. The input DNA library for the second round of SELEX consisted of the extension of the 5′ and 3′-ends with 7-bases that were randomized from four selected aptamers. The third round of SELEX was performed following the same procedures as described for the second round of SELEX. The experimental results indicate that the binding affinity of DNA aptamers to Globo H was enhanced when using the sequence-constructive SELEX approach. The selectivity of the DNA aptamers for related disaccharides, mannose derivatives, and Globo H analogs demonstrated the ability of the DNA aptamers to discriminate the presence of various glycans with different structures.
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Double-stranded DNAs (dsDNA) were then purified using the MinElute kit (Qiagen) and used as templates to prepare ssDNA oligonucleotides. In this case, an asymmetric PCR was performed only with the primer P1 [22]. After seven rounds of selection, selected ssDNA were amplified with the Pfu polymerase (Promega).
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DNA aptamers that bind to chitin

Abstract

Introduction

Section snippets

Results and Discussions

Acknowledgements

Nature

Science

Chem. Rev.

Annu. Rev. Biochem.

Annu. Rev. Biochem.