Systematically Modulating Aptamer Affinity and Specificity by Guanosine-to-Inosine Substitution

Area of Science:

• Biotechnology
• Molecular Biology
• Biosensing

Background:

• Aptamers are crucial for small molecule detection due to their specificity, stability, and cost-effectiveness.
• A key challenge in aptamer-based sensing is matching binding affinity to the target analyte concentration range.
• Existing affinity modulation methods often cause significant, undesirable changes in aptamer folding.

Purpose of the Study:

• To introduce a novel, less perturbative method for modulating aptamer binding affinity.
• To explore the use of guanosine-to-inosine substitutions for tuning aptamer affinity and specificity.
• To generate aptamers with a range of binding affinities for diverse biosensing applications.

Main Methods:

• Systematic substitution of guanosine with inosine in the guanosine-to-inosine sequence of a known aptamer.
• Utilized the cocaine binding aptamer as a model system for these modifications.
• Assessed binding affinity, specificity, and folding stability (via melting temperature) of modified aptamers.

Main Results:

• Generated aptamer sequences with a broad range of binding affinities, from 230 nM to 80 μM.
• Demonstrated that guanosine-to-inosine substitutions can modulate aptamer specificity towards related analytes.
• Observed expected impacts on aptamer folding stability due to the nucleotide substitutions.

Conclusions:

• Guanosine-to-inosine substitution offers a convenient and reliable strategy for tuning aptamer binding affinity.
• This method allows for the rapid generation of aptamers with tailored affinity and specificity for biosensing.
• The approach provides a valuable tool for developing next-generation aptamer-based detection systems.