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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.

Show MeSH
Codeine-binding mini-aptamer characterization. Proposed secondary structures from mfold of (A) the FC5 mini-aptamer (FC5L) and (B) the FC45 mini-aptamer (FC45L), and the corresponding equilibrium codeine-binding curves of (C) FC5L and (D) FC45L.
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fig6: Codeine-binding mini-aptamer characterization. Proposed secondary structures from mfold of (A) the FC5 mini-aptamer (FC5L) and (B) the FC45 mini-aptamer (FC45L), and the corresponding equilibrium codeine-binding curves of (C) FC5L and (D) FC45L.

Mentions: These truncation experiments support the importance of the formation of the correct binding pocket for aptamer molecular recognition capabilities. In addition, both FC5 and FC45 mini-aptamers lack the 3′ constant terminal sequence, indicating that the 3′ terminus is not involved in binding codeine. Secondary structure predictions from mfold and RNAstructure indicate that the 3′ terminus forms a small hairpin (Supplementary Figure 3C), isolating itself from the remaining sequences of FC5 and FC45. The proposed secondary structures of the FC5 and FC45 mini-aptamers (Figure 6) are supported by the structural modification and structural probing experiments described in the next section.


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)

Codeine-binding mini-aptamer characterization. Proposed secondary structures from mfold of (A) the FC5 mini-aptamer (FC5L) and (B) the FC45 mini-aptamer (FC45L), and the corresponding equilibrium codeine-binding curves of (C) FC5L and (D) FC45L.
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Related In: Results  -  Collection

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

fig6: Codeine-binding mini-aptamer characterization. Proposed secondary structures from mfold of (A) the FC5 mini-aptamer (FC5L) and (B) the FC45 mini-aptamer (FC45L), and the corresponding equilibrium codeine-binding curves of (C) FC5L and (D) FC45L.
Mentions: These truncation experiments support the importance of the formation of the correct binding pocket for aptamer molecular recognition capabilities. In addition, both FC5 and FC45 mini-aptamers lack the 3′ constant terminal sequence, indicating that the 3′ terminus is not involved in binding codeine. Secondary structure predictions from mfold and RNAstructure indicate that the 3′ terminus forms a small hairpin (Supplementary Figure 3C), isolating itself from the remaining sequences of FC5 and FC45. The proposed secondary structures of the FC5 and FC45 mini-aptamers (Figure 6) are supported by the structural modification and structural probing experiments described in the next section.

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