The Isolation Of An RNA Aptamer Targeting To P53Protein With A Single Amino Acid Mutation

p53protein, also known as tumor suppressive protein, is involved in cell cycle regulation, activation of DNA repairing, maintaining genome stability and promoting cell apoptosis. p53loss of function is found in almost50%of the human cancers, including single site mutation, multisite mutagenesis and structural distortion. An increasing number of evidence shows that once p53is mutated, its conformation is alterd, resulting in lots of critical biological functions such as dysfunction of cell cycle regulation and promotion of cell apoptosis. Meanwhile, p53mutation also leads to an effect of Gain-of-Function (GOF) in cells like enhanced cell migration and invasiveness, changing from tumor inhibitor to tumor protein. Up to date, more and more research groups are focusing the p53mutants, from which reactivation of p53mutants and inhibition of their Gain-of-Function effects are the major points of the research, which makes it biologically and pharmacologically meaningful for the cancer targeted therapy.Aptamers are short single stranded nucleic acid molecules which can bind a variety of target molecules in a structure-dependent manner. Its targets can be proteins, small molecules, sugar, lipids etc.. It is screened out by a evolutionary procedure called SELEX (Systematic Evolution of Ligand Exponential enrichment). SELEX techonology was invented by three different research groups in1990and is widely used in many biological and pharmocological applications like riboswitch, targeted therapy. Thus we are confident that through this method we could screened out an aptamer that habors high affinity and specificity to the p53mutants.Up to date, there is no report about isolation of any p53mutant aptamers with high affinity. In this work, our primary goal is to screen out p53R175H aptamer. Since there is only one amino acid difference compared with its wild type, we then modified the conventional SELEX procedures as what we call Contrast Screening in which p53wild type and mutant were respectively coupled to different solid phase and were subject to competitive binding of their potential targets. This competitive system enhances, at a very high degree, the specificity of screening and the differences of the two proteins. After five rounds of selection, we obtained21aptamers that were believed to have much higher affinity towards p53R175H. Of them, p53R175H-APT was found to much higher affinity towards p53mutant over p53wildtype. In gel shift assay, we finally confirmed that p53R175H-APT and p53R175H have direct binding in vitro. We then set out to investigate the following functional study of p53R175H-APTAlthough p53R175H-APT exhibited higher affinity to p53R175H, its function in cell culture is yet to be investigated. In this work, we tried to illustrate how p53R175H-APT functioned in cells. Firstly we have to make sure that this kind of p53mutant aptamer has stringent specificity towards its targets rather than other molecules. We transfected p53R175H-APT to HEK293T and HeLa cells which habor p53wild type. No matter from phenotype nor cell growth and cell death, no significant difference was observed compared with sramble control. At the same time, in p53R273H expressing H1299cells, we also got no significant difference. After completion of the above experimets and making sure that p53R175H-APT has no significant function towards neither p53wild type and other p53mutant, We then established p53R175H-HI299stable cell line for our following experiments. We found that cell growth is significantly suppressed and cell death is increased after treatment of p53R175H-APT, which means p53R175H-APT specifically binds to p53R175H, inhibits cell growth and induce cell death. In order to make sure that so significant difference is due to the specific binding of p53R175H-APT and p53R175H, we carried out p53RIP assay and confirmed that in cell culture p53R175H-APT binds more to the p53mutant rather than IgG or scramble control. Meanwhile, clonogenic assay and soft agar assay indicated decreased colony formation ability. Since many p53mutants are GOF mutation, the ability of migration and invasiveness is enhanced after mutation, transwell assay and wound healing assay demonstrated that after treatment of p53R175H-APT, the migration and invasiveness are significantly decreased. Additionally in molecular level, p53R175H-APT treated cells could transactivate the p53target genes, which means rescue of p53mutant. In order to make it more direct, we performed immunostaining with p53wild type antibody PAbl620. The results indicated that structually p53R175H-APT treated p53mutant is more like with p53wild type which means a successful rescue of p53mutant.In order to verify the amazing effects of p53R175H-APT at a higher level in vivo, we established a nude mice xenograft model. Through intradermal injection and intravenous injection, we both found significantly smaller tumors in p53R175H-APT treated mice compared with scramble control. In TUNEL apoptosis staining, we found much more apoptotic cells in p53R175H-APT group, which indicated that p53R175H-APT functioned in vivo by inducing apoptosis of the cells. Above all, all the results leads to the conclusion that p53R175H-APT can promote tumor cell apoptosis, decrease the cell growth and clonogenesis, inhibit cell migration and transactivate p53target genes in cell culture and in vivo.Above all, this work illustrated a novel SELEX screening strategy by which we could successfully screen out an aptamer p53R175H-APT towards p53R175H, which only has one amino acid difference. In vitro experiments demonstrated that it could inhibit tumor cell growth and induce cell death, in vivo experiments further verified its potential clinical value. It offers a new insight to the cancer targeted therapy.