DNA aptamers that bind to chitin
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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