Designing Bcl-2 protein binding aptamers using an entropic fragment-based approach and in vitro validate the drug efficacy.

Abstract

Antiapoptotic Bcl 2 proteins are overexpressed in cancer cells. To regulate Bcl 2 protein functions we have designed a set of nucleic acid aptamers using a patented entropic fragment-based approach (EFBA), which integrates the concept of information processing with the seed-and-grow strategy to determine the probability distribution of the nucleotide sequences that most likely interact with target structures by ruling out the ones that do not satisfy the entropic criterion. Based on molecular dynamics (MD) simulations to understand the stability of the aptamer-protein complex we finalized eight aptamer sequences and ranked them. Our discovered aptamers appeared with 9-10 nucleotides in the sequences. We then tested these aptamers to quantify their in vitro Bcl 2 protein binding ability. In this experimental validation, we applied our patented technology (US9529006B1) helping us quantify the protein-bound aptamers in actual concentration ‘micromole’ directly at the protein site. This way we quantify protein-bound aptamers in proportion to the aptamer concentrations in the protein incubating solution. Our MD simulation-based best-ranked aptamers appeared with higher in vitro protein binding potency. We then performed fluorescence quenching experiments in the Bcl 2-aptamer complex using the best aptamer, as detected in both MD simulation and in vitro binding assay-based rankings. Here we observed substantial effects on fluorescence quenching of the protein as a result of the aptamer binding. We conclude that we have found a set of aptamer candidates that may qualify as therapeutic drugs capable of regulating Bcl 2 protein functions in cancer cells and would thus favor the induction of apoptosis.