Selection Of A BACE1-specific DNA Aptamer Which Inhibits BACE1 Activity

Alzheimer’s disease (AD) is a progressive degenerative disorder of central nervous system, manifested mainly by cognitive impairment, especially memory deterioration. Patients are not able to take care of themselves during advanced stage thus results in a huge economic problem to their family and the society. Due to an aging population and higher diagnosis rates, the morbidity of AD increased by years and AD has become a more serious healthcare problem than ever before. The actual cause of the disease has not been identified with certainty and there are no drugs for etiological treatment clinically. Currently the commonly used acetylcholinesterase and NMDA receptor antagonist can only be applied to symptomatic treatment. Amyloid plaques are a central pathological feature of AD and largely consist of amyloid beta (Aβ) peptides. Aβ acts as a crucial factor in AD pathogenesis. Aβ is formed through sequential proteolytic processing of amyloid precursor protein (APP), catalysed by the β-and γ-secretases. y-secretase cleaves APP at several different positions, generating a variety of Aβ peptides with different amino acid numbers, of which Aβ40 and Aβ42 are two dominating Aβ monomers. Various kinds Aβ monomers with their oligomeric and fibrous forms abnormally aggregate and then deposit in the brain, participate to neurotoxicity. Prevention and reduction of excessive Aβ production seems to be a dominant strategy to treat AD.Beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) also called β-secretase 1, is the main β-secretase and is the rate-limiting enzyme during the process of Aβ generation. Increased expression and activity of BACE1 in post-mortem brains of AD patients have been reported. BACE1 has been shown to be elevated in cerebrospinal fluid (CSF) of prodromal AD patients. In patients carrying the Swedish mutation in the APP gene (APPsw), there is an increased affinity of APP binding to BACE1 and consequently, increases Aβ production. A coding mutation (A673T) in the APP gene at a site proximal to the BACE1 proteolytic site reduces BACE1 cleavage of APP and is protective against AD, which provides further support for the idea that blocking BACE1 cleavage of APP may protect against AD. Several studies have convincingly demonstrated that reduction of BACE1 activity can alter amyloid burden in mice. Therefore inhibition of BACE1 activity appears an attractive target in the AD field. Until now, the development of BACE1 inhibitors has been challenging and there are no safe and effective BACE1 inhibitors used in clinic.Aptamers are oligonucleotides (single-stranded DNA or RNA) capable of binding to target molecules with high affinity and good specificity due to their tertiary structures. Compared to conventional antibodies, there are many attractive features for aptamers:low molecular weight, quick and reproducible synthesis in vitro, easy modification, good stability easy to long-term storage, low toxicity, low immunogenicity, rapid and nice tissue penetration. These advantages have made aptamer an excellent alternative as molecular probe for fundamental research and clinical applications. Aptamers are selected by a technology called systematic evolution of ligands by exponential enrichment (SELEX). SELEX technology is mainly proceeded as follows:(1) the in vitro constructed random oligonucleotide library with an amount of 1013-1015 molecules is incubated with target molecule; (2) the oligonucleotide complexes with the target are separated from non-bound oligonucleotides; (3) the bound sequences are amplified by PCR. After several or dozens of rounds of repetitive selection, highly efficient and specific oligonucleotide sequences to the target molecule can be enriched gradually. Purified proteins are the most common targets for SELEX selection. Aptamers selected directly against proteins are able to inhibit the activity of target proteins with high affinity and good specificity, and can be used for investigation of mechanisms of interaction between proteins and nucleic acids.In this study, we used purified human BACE1 extracellular domain as target, conducted SELEX process to obtain highly efficient and specific aptamer to BACE1 from an in vitro constructed ssDNA library containing a random region of 30 nucleotides (nt) in length. Taking advantage of biotin-streptavidin binding system, we applied indirect ELISA and pull-down assay to test the binding affinity and specificity of the selected aptamer to BACE1. Next we used fluorescence resonance energy transfer (FRET) assay to detect the inhibition effect of aptamer to BACE1 activity in vitro. After aptamer was incubated with M17-APPsw cells (an AD cell model), we used a double-antibody sandwich ELISA and western blot analysis to identify the inhibition effect of aptamer to BACE1 activity in AD cell model.Firstly, we constructed in vitro an ssDNA library with a full length of 76 nt, which comprises of oligonucleotide fixed sequence at both ends and a random sequence of 30 nt in length in the middle. Purified human BACE1 extracellular domain was used as target and the selection procedure was conducted on microplate. The main steps of each round included:incubation and binding of ssDNA library with BACE1, seperation of unbound sequences by washing, amplification of bound sequences by asymmetrical PCR, recovery of target ssDNA to obtain secondary library, incubation of the secondary library with BACE1 to enter into next round of selection. Since the fourth round, the secondary library was firstly incubated with a blank well for counter-selection, thus eliminating nonspecific sequences. Seven rounds of iterated selection was conducted.SsDNA obtained from the last round was amplified, purified, cloned by T vector and sequenced. Primer 5.0 software was used to analyse sequencing results, and sequences with the same fixed sequence at both ends as primary library and a random sequence of 30 nt in length in the middle were chosen as desired sequences. MEME online software was applied to analyse sequence homology. RNA STRUCTURE 5.5 software were employed to predict secondary structure of the sequences. Four repetitive sequences were singled out, and named aptamer A1, A2, A3, A4 respectively, of which frequency of Al was the highest. The common mortif of the four sequences was not found during sequence homology analysis. All sequences mainly had stem-loop structure as indicated by secondary structure analysis.In order to verify BACE1 was the target protein of these selected aptamers and their binding affinity, we performed an indirect ELISA based on the principle of classical ELISA and biotin-streptavidin binding system. Twice gradient concentrations of biotinylated aptamers were incubated with BACE1, then with horseradish peroxidase-conjugated strepavidin. Chromogenic reaction was developed by TMB solution and absorbance was measured at 450 nm. Apparent equilibrium dissociation constants (Kd) values for each aptamer were determined by nonlinear regression according to Michaelis Menten equation using SigmaPlot 12.0. Incubation of anti-BACE1 antibody with BACE1 was used as control for comparison of binding affinity. Incubation of unrelated aptamer U31 with BACE1 was used as control for comparison of binding specificity. Results showed the binding curves of A1 and A4 fitted well, similar to that of anti-BACE1, with R values all reaching 0.99 (P<0.01). On the contrary, the binding curve of U31 was not able to fit. The Kd values of A1 and A41 were 68.5655±8.1237 nM,15.3497±2.0262 nM respectively, close to that of anti-BACE1 (2.7498±0.2785 nM). These results indicated A1 and A4 can specifically bind to BACE1 with high affinity.Subsequently, we performed pull-down assay to observe whether aptamers can bind to BACE1 in AD cell model. A1 with the highest repetition frequency was chosen to do the experiment. After incubating with streptavidin magnetic beads, biotinylated Al was then incubated with M17-APPsw cell lysate. The proteins which interacted with aptamer were immobilized on the streptavidin bead-aptamer complex. After washing the complex was isolated by SDS-PAGE gel, transferred to PVDF membrane, incubated with anti-BACE1 antibody and developed by ECL. Biotinylated Gp30, biotinylated U31 and a no aptamer treated group served as control. Result revealed there was an obvious band at 70KDa corresponding to molecular weight of BACE1 in A1 group, the band at 70KDa was very thin in Gp30 group, no bands were detectable in U31 group and blank group, illustrating that Al can specifically bind to BACE1 protein in M17-APPsw cell.Next fluorescence resonance energy transfer assay was used to testify the inhibition effect of Al on BACE1 activity in vitro. BACE1 substrate is linked to a fluorescent donor on one end and to a quenching acceptor group on its other end. Due to intramolecular energy transfer to the quenching acceptor, fluorescence from the donor can be quenched by the acceptor. Upon substrate cleavage by BACE1 enzyme, there is a disturbance of the energy transfer resulting in the enhancement of the fluorescent signal. The fluorescence of the substrate is significantly reduced when substrate cleavage is blocked by an inhibitor. Based on the above principle, various concentrations of A1 was added into a reaction system containing BACE1 and its fluorescent substrate, then changes of fluorescence intensity were detected. A BACE1 specific inhibitor, Gp30 and U31 served as control. Following the increase of concentration, fluorescence intensity in standard inhibitor group declined gradually, with a significant decline at a concentration of 500 nM compared to positive control (P<0.01). Fluorescence intensity in A1 group decreased significantly at a concentration of 250 nM (P<0.01). No significant decline of fluorescence intensities occurred in Gp30 and U31 groups. The percent inhibition was calculated by the following expression:100-(IFi/IFo×100), where IFi and IFo were the fluorescence intensities obtained for BACE1 in the presence and in the absence of inhibitor, respectively. Inhibition curves were obtained by plotting the percent inhibition versus the logarithm of inhibitor concentration by linear regression. The IC50 value of A1 extrapolated was 139.81 nM, which was smaller than that of standard inhibitor (242.50 nM), demonstrating Al can act as a potent inhibitor to BACE1 activity in vitro.Various concentrations of A1 (200 nM-10 μM) were incubated with M17-APPsw cells for 24 h, then MTT experiment was performed to evaluate the effect of A1 on cell survival. At a final concentration of no more than 3 μM M17-APPsw cell viability was not affected. Thus A1, in a concentration range between 0.1 μM and 3 μM, were incubated with M17-APPsw cells for 24 h, cell lysate were collected and double-antibody sandwich ELISA was performed to test Aβ40 and Aβ42 production. When the final concentration of Al was 3 μM, both Aβ40 and Aβ42 concentration decreased significantly compared to blank group (without any treatment group) (P<0.01). Hence A1 with a concentration of 3 μM was applied to incubate with cells, the resulted cell lysates and media were resolved on double-antibody sandwich ELISA, respectively.3 μM Gp30 treated group,3 μM U31 treated group and a blank group served as control. Results showed the concentration of Aβ40 secreted by M17-APPsw cell intracellularly and in the culture media both decreased significantly compared to all control groups (P<0.01). The concentration of Aβ40 secreted by M17-APPwt cell displayed a similar phenomenon. The concentration of AP42 secreted by M17-APPsw cell also decreased significantly compared to control groups (P<0.01). To identify effect of A1 on sAPPβ protein expression in AD cell model, M17-APPsw cell lysates treated with A1, Gp30 and U31 were subjected to western blot ananlysis, respectively. The primary antibody used were anti-APP that specific to APP amino terminal and anti-BACE1. Results showed sAPPβ protein expression level in A1 group decreased significantly compared to all control groups (P<0.01, P<0.05). There were no significant differences in protein expression level of sAPPa and BACE1 among all groups. The above results prompted A1 can inhibit BACE1 activity in AD cell model.In this study, we have successfully selected an DNA aptamer able to bind BACE1 with high affinity using SELEX technology, and demonstrated the aptamer can inhibit BACE1 activity in AD cell model, which provides research foundation for the development of newly specific potent BACE1 inhibitor, and offers new way and ideas for the treatment of AD.