title: Maxizyme-mediated specific inhibition on mutant-type p53 in vitro.
abstract: OBJECTIVE
  To evaluate the specific inhibition of maxizyme directing against mutant-type p53 gene (mtp53) at codon 249 in exon 7 (AGG-AGT) in vitro.
  
  
  METHODS
  Two different monomers of anti-mtp53 maxizyme (maxizyme right MzR, maxizyme left MzL) and control mutant maxizyme (G(5)-A(5)) were designed by computer and cloned into vector pBSKU6 (pBSKU6MzR, pBSKU6MzL). After being sequenced, the restrictive endonuclease site in pBSKU6MzR was changed by PCR and then U6MzR was inserted into pBSKU6MzL, the recombinant vector was named pU6Mz and pU6asMz (mutant maxizyme). Mtp53 and wild-type p53 (wtp53) gene fragments were cloned into pGEM-T vector under the T7 promoter control. The (32)p-labeled mtp53 transcript was the target mRNA. Cold maxizyme transcripts were incubated with (32)p-labeled target RNA in vitro and radioautographed after denaturing polyacrylamide gel electrophoresis.
  
  
  RESULTS
  In cell-free systems, pU6Mz showed a specific cleavage activity against target mRNA at 37 degrees and 25 mM MgCL(2). The cleavage efficiency of pU6Mz was 42 %, while pU6asMz had no inhibitory effect. Wtp53 was not cleaved by pU6Mz either.
  
  
  CONCLUSIONS
  pU6Mz had a specific catalytic activity against mtp53 in cell-free system. These lay a good foundation for studying the effects of anti-mtp53 maxizyme in HCC cell lines. The results suggest that maxizyme may be a promising alternative approach for treating hepatocellular carcinoma containing mtp53.
authors.affiliation: Institute of Liver Disease, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, China.
authors.forename: Xin-Juan
authors.initials: XJ
authors.lastname: Kong
authors.forename: Yu-Hu
authors.initials: YH
authors.lastname: Song
authors.forename: Ju-Sheng
authors.initials: JS
authors.lastname: Lin
authors.forename: Huan-Jun
authors.initials: HJ
authors.lastname: Huang
authors.forename: Nan-Xia
authors.initials: NX
authors.lastname: Wang
authors.forename: Nan-Zhi
authors.initials: NZ
authors.lastname: Liu
authors.forename: Bin
authors.initials: B
authors.lastname: Li
authors.forename: You-Xin
authors.initials: YX
authors.lastname: Jin
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000628:therapy; D002474:Cell-Free System; D003001:Cloning, Molecular; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D006801:Humans; D066298:In Vitro Techniques; D008113:Liver Neoplasms / Q000628:therapy; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
chemical_list: D016337:RNA, Catalytic; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53