This project developed a split aptamer that achieves enhanced target-binding affinity through cooperative binding.
The project produced a split cocaine-binding aptamer that incorporates two binding domains, such that target binding at one domain greatly increases the affinity of the second domain. It experimentally demonstrated that the resulting cooperative-binding split aptamer (CBSA) exhibits higher target binding affinity and is far more responsive in terms of target-induced aptamer assembly compared to the single-domain parent split aptamer (PSA) from which it was derived. Researchers further confirmed that the target-binding affinity of the CBSA can be affected by the cooperativity of its binding domains and the intrinsic affinity of its PSA. The researchers believe that CBSA-5335 has the highest cocaine affinity of any split aptamer described to date. The CBSA-based assay also demonstrated excellent performance in target detection in complex samples. Using this CBSA, the project achieved specific, ultra-sensitive, one-step fluorescence detection of cocaine within fifteen minutes at concentrations as low as 50 nM in 10-percent saliva without signal amplification. This limit of detection meets the standards recommended by the European Union's Driving under the Influence of Drugs, Alcohol and Medicines program. The assay also demonstrated excellent reproducibility of results, confirming that this CBSA-platform represents a robust and sensitive means for cocaine detection in actual clinical samples. (publisher abstract modified)