Functional Selection Of SsDNA Aptamers That Specifically Bind Hepatitis C Virus Envelope Glycoprotein E2

Objective.To Obtain ssDNA aptamers with high affinity and specificity for Hepatitis C Virus (HCV) envelope glycoproteins (HCV-E2), which could become early diagnostic and protective reagent for HCV infection.Methods.In the present study, a 97nt single stranded DNA(ssDNA) random library was subjected to 14 rounds of selection against HCV envelope glycoproteins E2 expressed on the mammalian cell CT26 by CS-SELEX method. In this model system, we carried out CS-SELEX (cell surface-systematic evolution of ligands by exponential enrichment) by establishing a stable cell line that ectopically expressed HCV E2 glycoprotein on the cell surface and we examined the binding affinities and functions of selected ssDNA aptamers. The Binding rates of the 14 pools aptamers with E2-CT26 cells were detected by flow cytometer (FCM). We selected 13th pool aptamers which had the highest binding affinities with the positive cells and were cloned and sequenced. The primary sequences and structure of the aptamers were analyzed by DNAMAN sofeware and the affinities of aptamers to HCV E2 protein were determinated. Then, the structural analysis of aptamer and the dissociation constant (Kd) between aptamers and target selecting CT26 cell ectopically and stabilitily expressing HCV E2 on the cell surface were carried out by FCM or by ELISA.Furthermore we observed a dose dependent binding property between the single aptamer from 13 pools, ZE18, and HCV-E2 protein which was confirmed by CONFOCAL and by CEMSA, the values of IC50 were determined to estimate aptamers’ cellular toxicity by MTT method. We detected HCVcc with our aptamers using ZE18 sandwich ELISA. The inhibitory function of the ZE18 on HCVcc in human Huh7.5.1 cells was evaluated by FCM and by real-time QRT-PCR. Also we measured binding rates between CT26 cells and the aptamers by FCM in the presence of CD81 protein which is one of HCV receptors. Last we have observed the binding rate between cut and mutative single aptamer that is mZE18 and ZE18-N30 and HCV-E2 protein cut to small peptide (from P1 to P15) so that to understand the binding sites the aptamer with HCVcc or HCV of patients’ serum by ELISA.Last we deteced the bingding affinities of the single aptamer ZE18 with the different HCV genotypes by ELISA.Results.1. In this study, we firstly applied a cell surface SELEX to have obtained successfully ssDNA aptamer pools of HCV envelope protein E2 stably expreesed on CT26 cells’ surface. The 13th pool of the 14 E2 aptamer pools had the strongest binding rate to CT26-E2 cells.2.The aptamers of the 13th pool have cloned into pU19 to get 30 single aptamer named from ZE1 to ZE30, in which ZE18 had appeared to have the most affinity for its ligand by FCM. The dissociation constant(Kd) of ZE18 with its target molecule E2 is 1.05±0.4 nM-63.1±9.3 nM. The sequence of binding affinitieswas as follows:ZE18 >13th P>ZE25>ZE16>12th P>ZE21>10th P>4th P>14th P.The mutated aptamer of ZE18, mZE18,can also bind to E2 protein in a dose dependent manner same as ZE18, but the binding rate of mZE18 is lower than ZE18 and can penetrate karyotheca into cell nucleus. The selected aptamers have very low or no toxicity for hepatic cells:IC50 is 10.47mM。3.ZE18, mZE18 and 13pool aptamers can bind to HCVcc in a dose dependent manner, in which ZE18 has the strongest binding rate. The results of ZE18 for the serum of patients infected with HCV is in consistency with results obtained by the methods of HCV antibody or QRT-PCR for HCV RNA in clinic and at the same time the detected values for ZE18 was inversely correlated with serum dilutions.4.The E2 aptamers can inhibit HCV E2 protein to bind with target cellular receptors; Furthore more, aptamers could competitatively block CD81 receptor protein binding to target cells. E2 aptamers could also significantly block HCVcc infection to Huh7.5.1 in vitro. 5.The binding rate between ZE18 and mZE18 with the P7 peptide segment of HCV-E2 protein is higher than other segments, furthermore there were lower binding affinity after cutting into three segments for P7.ZE18-N30 and mZE18-N30, only had random N30 segment, could also bind with E2-HepG2 target cells、ZE18 or ZE18-N30 has higher binding rate than mZE18 or mZE18-N30 toward HCVcc and the serum of HCV patients(sHCV).6.HCV genotypes 1a,1b, and 2a of E2 could be significantly captured by aptamer ZE18,and the binding affinities of aptamer ZE18 with genotypes la, lb, and 2a of E2 were two-fold higher than those of genotypes 3,4,5,and 6.Conclusions.In summary, this study shows that the ssDNA aptamers that bind specifically to HCV-E2 have been successfully generated by CS-SELEX for the first time. It can be used to antagonize HCV early in infection and as a potential new drug against HCV infections in both prevention of infection and in therapeutic treatment. It can also hold great promise in developing a new reagent for early diagnosis for HCV and can also serve as a tool for analysis of the HCV-host cell interactions.