Construction And Application Of The Drug Screening Model Based On PPARγ Assembled With DNA Origami

Drug screening model is one of the important methods to find and discover new drugs. In recent years, with the rapid development of molecular biology, pharmacy and modern chromatography technology, a large number of new drug screening models have emerged. Compared with traditional drug screening models, these new drug screening models have the advantages of simple screening methods, rapid screening processes and high-throughput.In recent years, the development and application of DNA origami technology becomes more mature, the main advantages of DNA origami include:addressable space, simple preparation process, good biocompatibility and easy to modify the surface. In this study, we used DNA origami as a biological carrier to load the nuclear receptor PPARy on its surface, and then the conjugates were bonded to the surface of inorganic materials silica by chemical bonding method. At last, the silica was filled in stainless steel column to construct a new type of drug screening model based on DNA origami chromatography.First of all, M13mp18 phage was propagated in E. coli JM109. The phage was purified by polyethylene glycol precipitation. The single-stranded DNA was then extracted with phenol and characterized by agarose gel. E. coli BL21 (DE3) cells transformed with pET-28a (+)-hPPARy-LBD recombinant plasmids were induced by IPTG. The recombinant protein was purified through a Ni-NTA-Sefinose column and analyzed by 10% SDS-PAGE electrophoresis. Short DNA staple strands and the long M13mp18 scaffold strand were mixed in 1×TAE-Mg buffer. The DNA origami combinatorial structures were formed with predefined annealing programs using a PCR. PPARy protein was assembled on DNA origami surface by the complementary base pairing between the "capture chain" on the surface of DNA origami structure and the DNA strand modified PPARy protein. The PPARy-DNA origami conjugates were characterized by agarose gel and AFM. In the presence of SMCC, PPARy-DNA origami conjugates were bonded to the silica gel surface to obtain PPARy assembled with DNA origami chromatography stationary phase. The stationary phase was packed in a stainless steel column to build PPARy assembled with DNA origami chromatography drug screening model.Secondly, the affinity and specificity of PPARy assembled with DNA origami chromatography drug screening model were evaluated. The affinity of the drug screening model was evaluated by observing the retention behavior of positive drug rosiglitazone on the unassembled with PPARy negative control column. The affinity of the drug screening model was studied by observing the retention behaviors of positive drug rosiglitazone and negative drug paracetamol on the PPARy assembled with DNA origami column. The results showed that PPARy assembled with DNA origami chromatography drug screening model can identify rosiglitazone specifically.Once again, the application of the PPARy assembled with DNA origami chromatography drug screening model in screening active components from Ginseng was studied. Total ginsenosides were analyzed on PPARy assembled with DNA origami chromatography column, and the reserved components and unreserved components were collected, respectively. Total ginsenosides, ginsenoside Re standards, the reserved components and unreserved components were analyzed by reverse phase chromatography. The results showed that ginsenoside Re can interact with with PPARy and had a strong interaction.Finally, the reproducibility and stability of the PPARy assembled with DNA origami chromatography drug screening model were investigated. Analysed ginsenosides on three batches of PPARy assembled with DNA origami chromatography column, in order to evaluate the reproducibility of PPARy assembled with DNA origami chromatography drug screening model; analysed ginsenosides on one of the column within one month, in order to evaluate the stability of the drug screening model. The results showed that the reproducibility of the drug screening model was well but the stability needed to be further optimized.