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Published November 2006 | Published
Journal Article Open

Codeine-binding RNA aptamers and rapid determination of their binding constants using a direct coupling surface plasmon resonance assay


RNA aptamers that bind the opium alkaloid codeine were generated using an iterative in vitro selection process. The binding properties of these aptamers, including equilibrium and kinetic rate constants, were determined through a rapid, high-throughput approach using surface plasmon resonance (SPR) analysis to measure real-time binding. The approach involves direct coupling of the target small molecule onto a sensor chip without utilization of a carrier protein. Two highest binding aptamer sequences, FC5 and FC45 with Kd values of 2.50 and 4.00 µM, respectively, were extensively studied. Corresponding mini-aptamers for FC5 and FC45 were subsequently identified through the described direct coupling Biacore assays. These assays were also employed to confirm the proposed secondary structures of the mini-aptamers. Both aptamers exhibit high specificity to codeine over morphine, which differs from codeine by a methyl group. Finally, the direct coupling method was demonstrated to eliminate potential non-specific interactions that may be associated with indirect coupling methods in which protein linkers are commonly employed. Therefore, in addition to presenting the first RNA aptamers to a subclass of benzylisoquinoline alkaloid molecules, this work highlights a method for characterizing small molecule aptamers that is more robust, precise, rapid and high-throughput than other commonly employed techniques.

Additional Information

© 2006 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Received May 30, 2006; Revised September 6, 2006; Accepted September 18, 2006. Nucleic Acids Research Advance Access originally published online on October 11, 2006 The authors thank Professor Pamela J. Bjorkman for generously allowing us the use of the Biacore 2000 instrument, which was used to develop the direct small molecule-aptamer characterization assays. The authors also gratefully acknowledge Dr Laure Jason-Moller and William Hunter (Biacore Life Sciences) and Katie Saliba (Caltech Chemistry Department) for their technical support and advice regarding codeine coupling chemistry to the sensor chip. The authors also thank Dr Kevin Hoff (Caltech Chemical Engineering Department) for his critical reading of the manuscript. This work was funded by Caltech startup funds, the Center for Biological Circuit Design at Caltech (fellowship support for M.N.W.), and the National Institutes of Health (training grant for J.S.K.). Conflict of interest statement. None declared.

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