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

Win MN, Klein JS, Smolke CD - Nucleic Acids Res. (2006)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Chemistry and Chemical Engineering, 1200 E. California Boulevard, MC 210-41 California Institute of Technology, Pasadena, CA 91125, USA.

ABSTRACT
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 K(d) values of 2.50 and 4.00 microM, 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.

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Equilibrium codeine-binding curves of (A) FC5 and (B) FC45.
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fig4: Equilibrium codeine-binding curves of (A) FC5 and (B) FC45.

Mentions: The binding data from the SPR assay supports the qualitative binding data obtained from the chromatography-based assay. The data indicate that there was little to no detectable binding (Figure 3A) between the initial pool and codeine, whereas the final pool bound codeine with significant binding responses (Figure 3B). The overall Kd value of the final pool was evaluated to be ∼15 μM, whereas that of the initial pool was estimated to be in the high millimolar range. This latter value is only an estimate as no binding curve could be established for the initial pool due to its insufficient binding response. Therefore, codeine-binding affinity of the final pool was enhanced over 1000-fold from that of the initial pool. The Kd values of the analyzed aptamer clones are listed in Table 1. Several of the aptamer sequences have Kd values that are much lower than that of the enriched final pool. Two of the highest binding aptamers FC45 and FC5, with Kd values of 2.50 ± 0.06 μM and 4.00 ± 0.13 μM, respectively, were subject to further characterization studies (Figure 4). Despite their similar affinities for codeine, FC5 and FC45 may form different binding pockets since their corresponding mini-aptamers adopt different predicted secondary structures supported by structural studies described in a later section. In addition, FC5 and FC45 exhibit fairly different binding kinetics (Table 2), where the latter has faster kinetics (both binding and dissociation) than that of the former. Some clones, such as FC3, FC13, FC34 and C9 have observed dissociation constants on the same order as that of FC5, while other clones, such as FC23, A3, A20, B11, C15 and C23 exhibit similar dissociation kinetics to FC45 (data not shown). The kinetic data of the modified FC5 and FC45 sequences discussed in later sections are also reported in Table 2.


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

Win MN, Klein JS, Smolke CD - Nucleic Acids Res. (2006)

Equilibrium codeine-binding curves of (A) FC5 and (B) FC45.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1636496&req=5

fig4: Equilibrium codeine-binding curves of (A) FC5 and (B) FC45.
Mentions: The binding data from the SPR assay supports the qualitative binding data obtained from the chromatography-based assay. The data indicate that there was little to no detectable binding (Figure 3A) between the initial pool and codeine, whereas the final pool bound codeine with significant binding responses (Figure 3B). The overall Kd value of the final pool was evaluated to be ∼15 μM, whereas that of the initial pool was estimated to be in the high millimolar range. This latter value is only an estimate as no binding curve could be established for the initial pool due to its insufficient binding response. Therefore, codeine-binding affinity of the final pool was enhanced over 1000-fold from that of the initial pool. The Kd values of the analyzed aptamer clones are listed in Table 1. Several of the aptamer sequences have Kd values that are much lower than that of the enriched final pool. Two of the highest binding aptamers FC45 and FC5, with Kd values of 2.50 ± 0.06 μM and 4.00 ± 0.13 μM, respectively, were subject to further characterization studies (Figure 4). Despite their similar affinities for codeine, FC5 and FC45 may form different binding pockets since their corresponding mini-aptamers adopt different predicted secondary structures supported by structural studies described in a later section. In addition, FC5 and FC45 exhibit fairly different binding kinetics (Table 2), where the latter has faster kinetics (both binding and dissociation) than that of the former. Some clones, such as FC3, FC13, FC34 and C9 have observed dissociation constants on the same order as that of FC5, while other clones, such as FC23, A3, A20, B11, C15 and C23 exhibit similar dissociation kinetics to FC45 (data not shown). The kinetic data of the modified FC5 and FC45 sequences discussed in later sections are also reported in Table 2.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Chemistry and Chemical Engineering, 1200 E. California Boulevard, MC 210-41 California Institute of Technology, Pasadena, CA 91125, USA.

ABSTRACT
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 K(d) values of 2.50 and 4.00 microM, 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.

Show MeSH