title: Evaluation of LNA-modified DNAzymes targeting a single nucleotide polymorphism in the large subunit of RNA polymerase II.
abstract: Allele-specific inhibition (ASI) is a new strategy to treat cancer through a vulnerability created by the loss of large segments of chromosomal material by loss of heterozygosity (LOH). Using antisense approaches, it is possible to target single nucleotide polymorphisms (SNP) in the remaining allele of an essential gene in the tumor, thus killing the tumor while the heterozygous patient survives at the expense of the other nontargeted allele lost by the tumor. In this study, the feasibility of using locked nucleic acid (LNA)-modified DNAzymes (LNAzymes) of the 10-23 motif as allele-specific drugs was investigated. We demonstrate that incorporation of LNA into 10-23 motif DNAzymes increases their efficacy in mRNA degradation and that, in a cell-free system, the 10-23 motif LNAzyme can adequately discriminate and recognize an SNP in the large subunit of RNA polymerase II (POLR2A), an essential gene frequently involved in LOH in cancer cells. However, the LNAzymes, optimized under in vitro conditions, are not always efficient in cleaving their RNA target in cell culture, and the efficiency of RNA cleavage in cell culture is cell type dependent. The cleavage rate of the LNAzyme is also much slower than RNase H-recruiting DNA phosphorothioate antisense oligonucleotides. Moreover, compared with DNA phosphorothioates, the ability of the LNAzymes to differentially knock down two POLR2A alleles in cultured cancer cells is limited.
authors.affiliation: Department of Neurogenetics, Academic Medical Center, 1105 AZ Amsterdam, The Netherlands. k.fluiter@amc.uva.nl
authors.forename: Kees
authors.initials: K
authors.lastname: Fluiter
authors.forename: Miriam
authors.initials: M
authors.lastname: Frieden
authors.forename: Jeroen
authors.initials: J
authors.lastname: Vreijling
authors.forename: Troels
authors.initials: T
authors.lastname: Koch
authors.forename: Frank
authors.initials: F
authors.lastname: Baas
mesh_terms: D001483:Base Sequence; D045744:Cell Line, Tumor; D021881:DNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008297:Male; D009690:Nucleic Acid Conformation; D009696:Nucleic Acids / Q000737:chemistry*; D020641:Polymorphism, Single Nucleotide; D012319:RNA Polymerase II / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics
chemical_list: D021881:DNA, Catalytic; D009696:Nucleic Acids; D012333:RNA, Messenger; D012319:RNA Polymerase II