Optimized Targeting Transforming Growth Factor-β Receptor Ⅱ Extracellular Domain Aptamer Selection And Binding Sites Prediction Studies

Background:Glaucoma filtration surgery is the important therapeutic method currently, but filteringbleb scarring fibrosis after surgery is the main failing factor.Therefore there are often5-fluorouracil (5-FU) and mitomycin (MMC) drugs to inhibit scar formation clinically, butit cannot avoid complications such as filtering bleb leakage, low intraocular pressure,intraocular inflammation, corneal injury et al.So, to seek a more safe, effective and lesstoxic drugs is still the current research direction. TGF-β is believed to play an importantpart in both normal wound healing and tissue fibrosis. The TGF-β isoform TGF-β2has beenfound to be the predominant isoform related to ocular scarring diseases. The binding of aTGF-β ligand to TGF-β receptor Ⅱ(TβRⅡ) triggers the transdifferentiation of fibroblastsinto myofibroblasts (MFs) with high contractility at wound sites, which is the key processin scar formation. Herefore, to antagonism or block TGF-β combination of TβRⅡ, can beused as a new research approach of anti-scarring after glaucoma surgery treatment.In our previous study, a dominant sequence called aptamer S58, which was selected bySELEX, antagonised TGF-β-induced myofibroblast transdifferentiation in human Tenon’scapsule fibroblasts through sealing the targeting extracellular domain of TβRⅡ. Forerunneraptamer need optimize its structure to find the best sequence. Most ends of sequence areflanking sequence,which have weak influence on its function. The aptamer S58composesof60nucleic acid bases, the cost of synthesis is much high.So we optimaized the aptamerS58and shortened the sequence,then we verified their biological activity in vitro to find thebest one.This can provide new insight about experiment in vivo and treatment of scar inclinical for the future.Aptamer, which rely on identifying molecular targets, can closely integrated with thetarget molecules to form stable space structure through their adaptive structure change. So aptamer has similar to antibodies of high affinity, high specificity. the mechanism ofaction of the aptamer focus on studying the spatial structure and the binding sites of thetarget molecules. With the development of molecular biology and information science,bioinformatics has become a guidance in the field of biological science, the study ofprotein and nucleic acid interactions becomes more efficient. So to establish the bestthree-dimensional model of the nucleic acid aptamer,and study the binding sites of thereceptor protein is a necessary and feasible by bioinformatics method.Purpose:To find the best aptamer by optimaizing aptamer S58and confirming biologicalactivity in vitro, and establish the3D model and study the binding sites of the receptorprotein by bioinformatics method. The preliminary study on the mechanism of aptamer canprovide new insight treatment of scar in clinical for the future.Methods:First, we observe the combination of S58and fibroblasts by Laser confocalmicroscope. according to the sequence of S58,we shear gradually S58,5bp cut off eachtime, to get seven new shorter aptamer.Secondly,We tested the affinity and biologicalactivity of them by biosensor technology、flow cytometry、cell constriction experiment andwestern blot respectively to select the best one. Finally,we created3D structure by utilizingssDNA aptamer sequences, the crystal structure of the TβRII is searched by Protein DataBank (PDB)database,and the molecular docking was applied.Results:We can observe that the fluorescent of the FITC-S58displayed consistent with HTFsarrangement. Under the same concentration, the response peak was at350arc/s(S58)、200arc/s(S583’-5bp)、160arc/s(S583’-10bp)、130arc/s(S583’-15bp),80~120arc/s(S585’cut-off). Under the same concentration, the binding rate of S58and HTFs was72.9%, andthey were respectively:39.0%,27.6%,23.5%,27.6%(S58-1~4and HTFs),41.4%,38.7%,23.9%(S58-5~7and HTFs).The expression of α-SMA S58was reduced significantly (P=0.000), it of other groups had no difference (P>0.05).Similarly, cells contraction experiments displayed thatthe area rate of S58group was63.2%, they were respectively9.7%,10.4%(S585’-5bpgroup、S583’-5bp group) with no statistical difference (P>0.05).TRβII extracellular domain is composed of122amino acids,,and exists in the dimerstructure. the1PLO-3is most likely three-dimensional crystal structure in solution of10kinds of natural state. S58are oligonucleotides with60bases. The binding sites of S58andTRβII extracellular protein, respectively, including site I (T4, T5, G6, C7), site II (G13,A14, T15, C16, G17, C18), site III (T31, G32, T33, C34) and site IV (G40, A41, T42, T43,T44, G45, G46).Conclusions:The affinity of S58and TβRⅡwas highest in all, the combination rate of S58andHTFs was the best. The affinity of all the new ssDNA with TβRⅡwere lower thanS58,and the more shearing bases, the more lower affinity. In vitro S58can inhibit TGF-β2induced HTFs transdifferentiation, contraction,but all shortened ssDNA had no significanteffect, therefore biological activity of S58is still the best.The binding sites of S58and TβRⅡ extracellular domain was closely, once thebinding sites were cutted off, the biological activity of S58was significantly receded.SoS58with high structural stability, would still affect itself effect even if the binding siteswere not cutted off in vitro. Molecular docking is a kind of convenient and efficient methodto explore binding between DNAs and proteins, and provides a good theoretical basis formedical basic research.