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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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Structural stabilization and sequence requirements of the FC5 mini-aptamer stems. Proposed secondary structures from mfold of (A) the original FC5 mini-aptamer (FC5L), (B) the FC5 mini-aptamer with a stabilized base stem (FC5L-S1), (C) the FC5 mini-aptamer with a stabilized base stem composed of randomly-selected nucleotides (FC5L-S2).
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fig7: Structural stabilization and sequence requirements of the FC5 mini-aptamer stems. Proposed secondary structures from mfold of (A) the original FC5 mini-aptamer (FC5L), (B) the FC5 mini-aptamer with a stabilized base stem (FC5L-S1), (C) the FC5 mini-aptamer with a stabilized base stem composed of randomly-selected nucleotides (FC5L-S2).

Mentions: The base stems of the mini-aptamers were modified with an extension of GC base pairs to stabilize the proposed structures of these mini-aptamers. The FC5 mini-aptamer (FC5L) was stabilized by extending the existing 3 bp stem with two GC base pairs (Figure 7B), based on the assumption that a few nucleotides present on each end of the original mini-aptamer are unessential for codeine-binding. Similarly, the FC45 mini-aptamer (FC45L) was stabilized by extending the base stem formed by the 5′-CUU and 3′-GGG pairing with two GC base pairs (Figure 8B), excluding several nucleotides from the 5′ end. Following the modification, the structures of these stabilized mini-aptamers were further analyzed in mfold using the DotPlot Partition Function, which confirms these structures to be the most favorable ones to adopt among others. The codeine-binding properties of the resulting mini-aptamers, referred to as FC5L-S1 and FC45L-S1, respectively, were determined using the described SPR assay. FC5L-S1 and FC45L-S1 were determined to bind codeine with Kd values of 5.51 ± 0.23 μM and 4.18 ± 0.48 μM, respectively (Supplementary Figure 2A and B). These results indicate that the modified mini-aptamers bind the target molecule codeine with affinities similar to the corresponding unmodified mini-aptamers. Therefore, these results support the proposed secondary structures of the FC5 and FC45 mini-aptamers (Figure 6) and that their codeine-binding affinities were minimally affected by extending the original base stems. Structural probing studies were performed on FC5 and FC45 full-length aptamers using a standard lead-based cleavage assay to confirm the structures predicted through the SPR analysis. Lead-induced and RNase T1 cleavage patterns were observed to be in agreement with the corresponding proposed structures (Supplementary Figure 4).


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)

Structural stabilization and sequence requirements of the FC5 mini-aptamer stems. Proposed secondary structures from mfold of (A) the original FC5 mini-aptamer (FC5L), (B) the FC5 mini-aptamer with a stabilized base stem (FC5L-S1), (C) the FC5 mini-aptamer with a stabilized base stem composed of randomly-selected nucleotides (FC5L-S2).
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Related In: Results  -  Collection

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fig7: Structural stabilization and sequence requirements of the FC5 mini-aptamer stems. Proposed secondary structures from mfold of (A) the original FC5 mini-aptamer (FC5L), (B) the FC5 mini-aptamer with a stabilized base stem (FC5L-S1), (C) the FC5 mini-aptamer with a stabilized base stem composed of randomly-selected nucleotides (FC5L-S2).
Mentions: The base stems of the mini-aptamers were modified with an extension of GC base pairs to stabilize the proposed structures of these mini-aptamers. The FC5 mini-aptamer (FC5L) was stabilized by extending the existing 3 bp stem with two GC base pairs (Figure 7B), based on the assumption that a few nucleotides present on each end of the original mini-aptamer are unessential for codeine-binding. Similarly, the FC45 mini-aptamer (FC45L) was stabilized by extending the base stem formed by the 5′-CUU and 3′-GGG pairing with two GC base pairs (Figure 8B), excluding several nucleotides from the 5′ end. Following the modification, the structures of these stabilized mini-aptamers were further analyzed in mfold using the DotPlot Partition Function, which confirms these structures to be the most favorable ones to adopt among others. The codeine-binding properties of the resulting mini-aptamers, referred to as FC5L-S1 and FC45L-S1, respectively, were determined using the described SPR assay. FC5L-S1 and FC45L-S1 were determined to bind codeine with Kd values of 5.51 ± 0.23 μM and 4.18 ± 0.48 μM, respectively (Supplementary Figure 2A and B). These results indicate that the modified mini-aptamers bind the target molecule codeine with affinities similar to the corresponding unmodified mini-aptamers. Therefore, these results support the proposed secondary structures of the FC5 and FC45 mini-aptamers (Figure 6) and that their codeine-binding affinities were minimally affected by extending the original base stems. Structural probing studies were performed on FC5 and FC45 full-length aptamers using a standard lead-based cleavage assay to confirm the structures predicted through the SPR analysis. Lead-induced and RNase T1 cleavage patterns were observed to be in agreement with the corresponding proposed structures (Supplementary Figure 4).

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