744. Optimizing Conditions for Aptamer Folding Using a High-Throughput Aptamer Fluorescence Binding and Internalization (AFBI) Assay

Aptamers are small structured RNA or DNA oligonucleotides that bind target molecules with high avidity and affinity. Aptamer selection begins with a hugely complex library of aptamer sequences that are enriched for a specific target using the SELEX process, Systematic Evolution of Ligands by EXponentional enrichment. An important consideration in the SELEX process is the protocol used to fold aptamers into their active conformations. The folding conditions include multiple variables, such as temperature, buffer components, incubation time and aptamer concentration. Aptamer folding protocols vary widely across the aptamer field, and most published folding conditions primarily describe only temperature and folding time. To understand how variations in folding conditions impact aptamer function, we developed a novel high-throughput assay to interrogate the optimal folding conditions of several published aptamers. The Aptamer Fluorescence Binding and Internalization (AFBI) assay is a cell-based platform that uses a 96-well plate format to rapidly and efficiently screen multiple fluorescent-labelled aptamers against hundreds of conditions. The AFBI assay can be applied to rapidly determine aptamer binding constants (Kd) on cells, time course of aptamer internalization and cross reactivity of aptamers against different cell types. Using the AFBI assay, we screened several different folding parameters against published aptamers. We found that the buffer components contributed significantly more to an aptamer's function than any other examined factor. The concentration of an aptamer during folding was important for some aptamers but not others, including aptamers that originated from the same selection. Most surprising was that most temperature protocols had little impact on aptamer function after folding, the exception being that high temperature (95°C) often attenuated aptamer function. In summary, our data using the AFBI assay revealed that aptamer folding is more dependent on buffer components than the temperature protocol. Furthermore, aptamers derived from the same selection may have different optimal folding conditions. These data demonstrate that optimal aptamer folding protocols need to be more carefully interrogated on a per aptamer basis and reported in detail to allow for efficacious and reproducible results.