Selection Of P-glycoprotein Aptamers And Interaction Study With Dragon's Blood Molecules

P-gp?Permeablity Glycoprotein?is an efflux protein located in the cytoplasmic membrane which is encoded by an MDR1?multidrug resistance 1?gene.It is mainly distributed in the brain capillary endothelial cells,intestinal columnar epithelial cells,etc and can transport the substrate outside of the cells from the cytoplasm.P-gp highly expressed on the brain capillary endothelial cells plays an important role in restricting exogenous substances into the brain and maintaining the stability of the brain environment.Rare Dai medicine Dragon's Blood is a kind of red resin which is extracted from the dragon blood?Dracaena cochinchinensis?Lour.?S.C.Chen?tree.It can promote blood circulation and protect the nervous system under weightlessness condition,and it is used clinically in the treatment of ischemic stroke.The concentration of Dragon's Blood in rat brain is very low,so that the treatment dose is high,This may be related to its difficulty in passing through the blood-brain barrier.Dragon's Blood is rich in flavonoids and other compounds,Among them,Loureirin A?LA?,Loureirin B?LB?,and Loureirin C?LC?are the main active ingredients of Dragon's Blood,which play the role of promoting blood circulation and removing blood stasis.Previous studies have demonstrated that LA,LB and LC are substrates of P-gp,and the efflux action of P-gp in the blood-brain barrier limits the brain concentration of these active components.Thus improving the concentration of LA,LB and LC become the bottleneck problem of Dragon's Blood for protective effect in nervous system,Aptamer is a new recognition molecule emerging in recent years.With the the characteristics of specificity,high affinity and wide range of targets,aptamer has been widely applied to the quantitative detection of biological target protein,imaging analysis,drug-targeted transportation and other fields.Based on the difficult problems with Dragon's Blood research,the aim of this study was to screen the specific aptP-gp of P-gp using Systematic Evolution of Ligands by Exponential Enrichment?SELEX?and to construct the interaction system of Apt-drug,Apt-P-gp,indicating the total affinity in the interaction system by capillaryzone electrophoresis and calculating the binding constants of drug LA-Apt,LB-Apt and Apt-P-gp.In this paper,the following research results are obtained:1)Establishment of screening method of Apt basing on magnetic beads and SELEX.In the study,we obtain 14 P-gp-specific aptamers with a length of 81 bp were selected,respectively,named Apt1 to Apt14 number.Then,were compared the relative affinities of Apt with P-gp,ovalbumin,BSA,that the binding between the aptamer and P-gp is far higher than the aptamer with ovalbumin and BSA,indicating that the specificity of Apt on P-gp.Sequence analysis of these aptamers shows that there were some similar base sequences,such as TTTTCC,CCCCC,TGGG and so on.The secondary structure shows that the aptamers were rich in convex ring domain,These specific base sequences and convex ring domains,may be closely related to the ability of the specifically of aptamers binding to the target protein P-gp.2)Determination of the affinity and the dissociation constants of Apt with P-gpThe dissociation constants?Kd?of 14 aptamers with P-gp were determined by ELISA method.From Apt1 to Apt14 were 28.56,60.87,75.11,380.5,49.82,17.46,318.2,694.3,50.52,60.01,25.15,116.7,71.50,55.92 nM.The dissociation constant of Apt6 was 17.46 nM,and the lowest dissociation constant shows the highest affinities with P-gp.The high affinity with P-gp of Apt6 and Apt1 is rich in G and C bases,indicating that the GC content in the base sequence significantly affects the affinity of P-gp to aptamers.Apt6 with highest specificity and affinity was selected to study the interaction between drug,proteins and aptamers.3)Characterization the interaction between Apt-drug and Apt-P-gpThe affinity of Apt6-Pgp,Apt6-LA,Apt6-LB and Apt6-LC were detected by capillary zone electrophoresis.The binding constant?nK?between Apt6 and P-gp was3.07×10⁶ M^-1.The binding constants of Apt6 to LB and LC were 2.90×10⁵ M^-1 and3.19×10⁵ M^-1,respectively.The binding constant between aptamer and protein was significantly higher than that between aptamer and LC and LB,indicating that the affinity of aptamer and P-gp protein was much higher than that of aptamer with effect molecules of Dragon's Blood.This paper first screened the aptamer of P-gp,which can be used as a specific recognition target for P-gp.The strong binding ability between aptamers and drug molecules indicates that aptamers can be used as target molecules for drug delivery.Aptamers are easy to label and can be used as imaging molecules for drug distribution.These results may provide molecular basis for molecular target transport,target recognition,in vivo imaging studies.