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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
Aptamer clone sequences. (A) DNA template from which the initial RNA pool was generated. (B) Sequences of clones from the final aptamer pool. The codeine-binding properties of the sequences marked with an asterisk were characterized by the described direct coupling SPR assay. The number in parenthesis represents the frequency of a particular clone in the sequenced pool.
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fig1: Aptamer clone sequences. (A) DNA template from which the initial RNA pool was generated. (B) Sequences of clones from the final aptamer pool. The codeine-binding properties of the sequences marked with an asterisk were characterized by the described direct coupling SPR assay. The number in parenthesis represents the frequency of a particular clone in the sequenced pool.

Mentions: A random DNA library was generated through PCR using the following oligonucleotide sequences: a 59 nt DNA template 5′-GGGACAGGGCTAGC(N30)GAGGCAAAGCTTCCG-3′, primer1 5′-TTCTAATACGACTCACTATAGGGACAGGGCTAGC-3′ and primer2 5′-CGGAAGCTTTGCCTC-3′. All DNA synthesis was performed by Integrated DNA Technologies, Inc. The template contains a 30 nt randomized region flanked by two fixed primer-binding regions (Figure 1A). Primer1 contains a 17 nt T7 promoter sequence (italic). NheI and HindIII restriction endonuclease sites (underlined) were included in primer1 and primer2, respectively, for cloning of aptamer sequences.


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)

Aptamer clone sequences. (A) DNA template from which the initial RNA pool was generated. (B) Sequences of clones from the final aptamer pool. The codeine-binding properties of the sequences marked with an asterisk were characterized by the described direct coupling SPR assay. The number in parenthesis represents the frequency of a particular clone in the sequenced pool.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Aptamer clone sequences. (A) DNA template from which the initial RNA pool was generated. (B) Sequences of clones from the final aptamer pool. The codeine-binding properties of the sequences marked with an asterisk were characterized by the described direct coupling SPR assay. The number in parenthesis represents the frequency of a particular clone in the sequenced pool.
Mentions: A random DNA library was generated through PCR using the following oligonucleotide sequences: a 59 nt DNA template 5′-GGGACAGGGCTAGC(N30)GAGGCAAAGCTTCCG-3′, primer1 5′-TTCTAATACGACTCACTATAGGGACAGGGCTAGC-3′ and primer2 5′-CGGAAGCTTTGCCTC-3′. All DNA synthesis was performed by Integrated DNA Technologies, Inc. The template contains a 30 nt randomized region flanked by two fixed primer-binding regions (Figure 1A). Primer1 contains a 17 nt T7 promoter sequence (italic). NheI and HindIII restriction endonuclease sites (underlined) were included in primer1 and primer2, respectively, for cloning of aptamer sequences.

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