Specific repression of mutant K-RAS by 10-23 DNAzyme: Sensitizing cancer cell to anti-cancer therapies

Point mutations of the Ras family are frequently found in human cancers at a prevalence rate of 30%. The most common mutation K-Ras(G12V), required for tumor proliferation, survival, and metastasis due to its constitutively active GTPase activity, has provided an ideal target for cancer therapy. 10-23 DNAzyme, an oligodeoxyribonucleotide-based ribonuclease consisting of a 15-nucleotide catalytical domain flanked by two target-specific complementary arms, has been shown to effectively cleave the target mRNA at purine-pyrimidine dinucleotide. Taking advantage of this specific property, 10-23 DNAzyme was designed to cleave mRNA of K-Ras(G12V)(GGU → GUU) at the GU dinucleotide while left the wild-type (WT) K-Ras mRNA intact. The K-Ras(G12V)-specific 10-23 DNAzyme was able to reduce K-Ras(G12V) at both mRNA and protein levels in SW480 cell carrying homozygous K-Ras(G12V). No effect was observed on the WT K-Ras in HEK cells. Although K-Ras(G12V)-specific DNAzymes alone did not inhibit proliferation of SW480 or HEK cells, pre-treatment of this DNAzyme sensitized the K-Ras(G12V) mutant cells to anti-cancer agents such as doxorubicin and radiation. These results offer a potential of using allele-specific 10-23 DNAzyme in combination with other cancer therapies to achieve better effectiveness on cancer treatment.     

Optimisation of the 10-23 DNAzyme-substrate pairing interactions enhanced RNA cleavage activity at purine-cytosine target sites

The 10–23 RNA cleaving DNAzyme has been shown to cleave any purine–pyrimidine (RY) junction under simulated physiological conditions. In this study, we systematically examine the DNAzymes relative activity against different RY combinations in order to determine the hierarchy of substrate core dinucleotide sequence susceptibility. The reactivity of each substrate dinucleotide compared in the same background sequence with the appropriately matched DNAzyme was found to follow the scheme AU = GU ≥≥ GC >> AC. The relatively poor activity of the DNAzyme against AC and GC containing substrates was found to be improved substantially by modifications to the binding domain which subtly weaken its interaction with the substrate core. The most effective modification resulting in rate enhancement of up to 200-fold, was achieved by substitution of deoxyguanine with deoxyinosine such that the base pair interaction with the RNA substrates core C is reduced from three hydrogen bonds to two. The increased cleavage activity generated by this modification could be important for application of the 10–23 DNAzyme particularly when the target site core is an AC dinucleotide.     

An efficient RNA-cleaving DNA enzyme can specifically target the 5'-untranslated region of severe acute respiratory syndrome associated coronavirus (SARS-CoV)

BACKGROUND: The worldwide epidemic of severe acute respiratory syndrome (SARS) in 2003 was caused by a novel coronavirus called SARS-CoV. We report the use of DNAzyme (catalytic DNA) to target the 5'-untranslated region (5'UTR) of a highly conserved fragment in the SARS genome as an approach to suppression of SARS-CoV replication. A mono-DNA enzyme (Dz-104) possessing the 10-23 catalytic motif was synthesized and tested both in vitro and in cell culture. MATERIALS AND METHODS: SARS-CoV total RNA was isolated, extracted from the SARS-CoV-WHU strain and converted into cDNA. We designed a RNA-cleaving 10-23 DNAzyme targeting at the loop region of the 5'UTR of SARS-CoV. The designed DNAzyme, Dz-104, and its mutant version, Dz-104 (mut), as a control consist of 9 + 9 arm sequences with a 10-23 catalytic core. In vitro cleavage was performed using an in vitro transcribed 5'UTR RNA substrate. A vector containing a fused 5'UTR and enhanced green fluorescent protein (eGFP) was co-transfected with the DNAzyme into E6 cells and the cells expressing eGFP were visualized with fluorescence microscopy and analyzed by fluorescence-activated cell sorting (FACS). RESULTS AND CONCLUSIONS: Our results demonstrated that this DNAzyme could efficiently cleave the SARS-CoV RNA substrate in vitro and inhibit the expression of the SARS-CoV 5'UTR-eGFP fusion RNA in mammalian cells. This work presents a model system to rapidly screen effective DNAzymes targeting SARS and provides a basis for potential therapeutic use of DNA enzymes to combat the SARS infection.     

Suppression of platelet-type 12-lipoxygenase activity in human erythroleukemia cells by an RNA-cleaving DNAzyme.

Human platelet-type 12-lipoxygenase (12-LOX) and its metabolites play a crucial role in tumor angiogenesis. A "10-23" deoxyribozyme (DNAzyme) and its phosphorothioate-modified version were designed and synthesized against the 12-LOX mRNA. Both DNAzymes were able to cleave their substrate efficiently in a time- and concentration-dependent manner in vitro. Under a multiple turnover condition, both performed well at 37 degrees C, showing the k(cat) of 1 and 0.26 min(-1), respectively. The phosphorothioate modification of the DNAzyme significantly increased its stability in cells without a substantial loss of kinetic efficiency in vitro. In a cell culture system, transfection of the DNAzymes into HEL cells resulted in a significant down-regulation of the 12-LOX mRNA. Furthermore, the cell extracts from the DNAzyme-transfected cells exhibited a marked reduction in the 12-LOX enzyme activity. The present results indicated the potential use of DNAzyme technology for gene function study and cancer therapy.     

10-23型DNA酶作为鉴定mRNA靶点有效性的新工具

10-23DNA酶是能主动切割mRNA的一类反义寡核苷酸.利用10-23DNA酶的直接切割作用验证mRNA结构靶点的有效性.对筛选的绿色荧光蛋白(GFP)基因mRNA的4个靶点平行设计了4条反义寡核苷酸和4条10-23DNA酶,对照组反义寡核苷酸将最佳靶点——靶点2的反义寡核苷酸突变2个碱基,对照组10-23DNA酶将靶点2的10-23DNA酶结合臂中央突变2个碱基.体外4条10-23DNA酶切割mRNA的结果和相应的4条反义寡核苷酸依赖的RNaseH降解结果完全相似,细胞内4条10-23DNA酶对绿色荧光蛋白的表达抑制作用与相应的4条反义寡核苷酸相似,表明10-23DNA酶显示的最佳作用靶点同样是最佳作用效果的反义寡核苷酸结合靶.10-23DNA酶可以作为评价mRNA结构靶点有效性的新工具.     

Efficient silencing of gene expression by an ASON-bulge-DNAzyme complex

Background

DNAzymes are DNA molecules that can directly cleave cognate mRNA, and have been developed to silence gene expression for research and clinical purposes. The advantage of DNAzymes over ribozymes is that they are inexpensive to produce and exhibit good stability. The “10-23 DNA enzyme” is composed of a catalytic domain of 15 deoxynucleotides, flanked by two substrate-recognition domains of approximately eight nucleotides in each direction, which provides the complementary sequence required for specific binding to RNA substrates. However, these eight nucleotides might not afford sufficient binding energy to hold the RNA substrate along with the DNAzyme, which would interfere with the efficiency of the DNAzyme or cause side effects, such as the cleavage of non-cognate mRNAs.

Methodology

In this study, we inserted a nonpairing bulge at the 5′ end of the “10–23 DNA enzyme” to enhance its efficiency and specificity. Different sizes of bulges were inserted at different positions in the 5′ end of the DNAzyme. The non-matching bulge will avoid strong binding between the DNAzyme and target mRNA, which may interfere with the efficiency of the DNAzyme.

Conclusions

Our novel DNAzyme constructs could efficiently silence the expression of target genes, proving a powerful tool for gene silencing. The results showed that the six oligo bulge was the most effective when the six oligo bulge was 12–15 bp away from the core catalytic domain.     

Hairpin DNAzymes: a new tool for efficient cellular gene silencing

BACKGROUND: RNA-based gene silencing is potentially a powerful therapeutic strategy. Catalytic 10-23 DNAzymes bind to target RNA by complimentary sequence arms on a Watson-Crick basis and thus can be targeted to effectively cleave specific mRNA species. However, for in vivo applications it is necessary to stabilise DNAzymes against nucleolytic attack. Chemical modifications can be introduced into the binding arms to increase stability but these may alter catalytic activity and in some cases increase cell toxicity. METHODS: We designed novel 10-23 DNAzyme structures that incorporate stem-loop hairpins at either end on the DNAzyme binding arms. The catalytic activity of hairpin DNAzymes (hpDNAzyme) were tested in vitro against 32P-labelled cRNA encoding the muscle acetylcholine receptor (AChR) alpha-subunit. Resistance of hpDNAzymes to nucleolytic degradation was tested by incubation of the hpDNAzymes with Bal-31, DNase1 or HeLa cell extract. Gene silencing by hpDNAzymes was assessed by measuring reduced fluorescence from DsRed2 and EGFP reporters in cell culture systems, and reduced 125I-alpha-bungarotoxin binding in cells transfected with cDNA encoding the AChR. RESULTS: We show that hpDNAzymes show remarkable resistance to nucleolytic degradation, and demonstrate that in cell culture systems the hpDNAzymes are far more effective than standard 10-23 DNAzymes in down-regulating protein expression from target mRNA species. CONCLUSION: hpDNAzymes provide new molecular tools that, without chemical modification, give highly efficient gene silencing in cells, and may have potential therapeutic applications.     

Introduction of wild-type p53 in a human ovarian cancer cell line not expressing endogenous p53.

Utilizing a temperature sensitive p53 mutant (pLTRp53cGval135) which expresses mutant p53 at 37 degrees C and a wild-type like p53 at 32 degrees C, we transfected a human ovarian cancer cell line (SKOV3) which does not express endogenous p53. Among the different clones obtained, we selected three clones. Two were obtained from simultaneous transfection of p53 and neomycin resistance expression plasmids (SK23a and SK9), the other was obtained from transfection experiments utilizing the neomycin resistance gene only (SKN). Introduction of mutant p53 did not alter the morphology or growth characteristics of this ovarian cancer cell line. Upon shifting to the permissive temperature, a dramatic change in morphology and growth rate was observed in SK23a and SK9 cells that is associated with the presence of a wild-type like p53. SKN and SKOV3 cells maintained at 32 degrees C did not change morphology and only slightly reduced proliferation. Both SK23a and SK9 cells did not show evidence of apoptosis when measured up to 72 hours of maintenance at 32 degrees C. In contrast to what observed in other cell lines, SK23a and SK9 cells maintained at 32 degrees C were not blocked in G1, but they were accumulated in G2-M. This accumulation was transient and could be due either to a blockade or to a delay in the G2 progression. No down-regulation of c-myc was observed in p53 expressing clones when shifted to the permissive temperature. In these conditions gadd45 mRNA expression was highly stimulated in SK9 and SK23a cells but not in SKN cells. In both clones Gas1 mRNA was not detected either at 37 degrees C or 32 degrees C. This system represents a new and useful model for studying the effect of the absence of p53 (SKOV3 or SKN), presence of mutated p53 (SK23a and SK9 kept at 37 degrees C) or wild type p53 (SK23a and SK9 kept at 32 degrees C) on the mechanism of response of cancer cells to DNA damaging agents.     

DNAzyme-mediated silencing of ornithine decarboxylase

The value of reducing the activity of ornithine decarboxylase (ODC), a key enzyme in the biosynthesis of polyamines, is well-appreciated. Polyamines are necessary components for cell growth, and manipulation of polyamine homeostasis may be an effective strategy for the treatment of a number of disorders, including neoplastic diseases. An approach to develop an effective DNAzyme, using the 10-23 model, against ODC is described in these studies. DNAzymes able to cleave the target ODC RNA were identified in vitro and further characterized by the effect each had on ODC protein and activity levels using in vitro translated ODC RNA. ODC protein levels and activity correlated well with the RNA cleavage activity of the DNAzyme. One of the DNAzymes, DZ IV, which exhibited good activity, was optimized for use in cell culture studies. The DNAzyme hybridization arms were altered from equal length arms varying in length (8, 9, 10, or 11 nucleotides) or to unequal length arms (7/11 nucleotides), and kinetic analyses were performed to identify the most catalytically efficient configuration. DZ IV with equal arms nine nucleotides in length proved to be the most catalytically efficient. In HEK 293 cells, DZ IV was able to reduce the amount of translated ODC protein, resulting in approximately 80% reduction in ODC activity-a statistically significant enhancement over the apparent antisense effect of a catalytically inactive DNAzyme. These results indicate that this DNAzyme may be a useful tool to study the function of ODC and may have potential therapeutic uses.     

Deletion analysis in the catalytic region of the 10-23 DNA enzyme

In this study, the functional relevance of the core nucleotides of the RNA cleaving 10-23 DNA enzyme (DNAzyme) was investigated. Systematic deletion studies revealed that DNAzymes lacking thymine at position 8 (T8) retain catalytic activity comparable to that of the wild-type enzyme. Deletion of the adjacent cytosine at position 7 (C7) also resulted in a highly active enzyme and even the double deletion mutant C7/T8 displayed cleavage activity, although the catalytic rate under multiple turnover conditions was found to be reduced by one order of magnitude. The identification of non-essential nucleotides in the catalytic core might help to stabilize the DNAzyme against nucleolytic degradation and to overcome problems in elucidating its three-dimensional structure.     

Defective apoptosis and B-cell lymphomas in mice with p53 point mutation at Ser 23

Phosphorylation of the p53 tumor suppressor at Ser20 (murine Ser23) has been proposed to be critical for disrupting p53 interaction with its negative regulator, MDM2, and allowing p53 stabilization. To determine the importance of Ser23 for the function of p53 in vivo, we generated a mouse in which the endogenous p53 locus was targeted to replace Ser23 with alanine. We show that, in mouse embryonic fibroblasts generated from Ser23 mutant mice, Ser23 mutation did not dramatically reduce IR-induced p53 protein stabilization or p53-dependent cell cycle arrest. However, in Ser23 mutant thymocytes and in the developing cerebellum, p53 stabilization following IR was decreased and resistance to apoptosis was observed. Homozygous Ser23 mutant animals had a reduced lifespan, but did not develop thymic lymphomas or sarcomas that are characteristic of p53-/- mice. Instead, Ser23 mutant animals died between 1 and 2 years with tumors that were most commonly of B-cell lineage. These data support an important role for Ser20/23 phosphorylation in p53 stabilization, apoptosis and tumor suppression.     

Codon 249 of the human TP53 tumor suppressor gene is no hot spot for aflatoxin B1 in a heterologous background

Mutations in the TP53 tumor suppressor gene are the most common alteration in cancer, and human primary liver cancers related to previous dietary exposure to the mycotoxin aflatoxin B1 (AFB1) exhibit a specific hot spot mutation at TP53 codon 249. We have asked whether the 249 hot spot is related to a particular susceptibility to AFB1 of this TP53 region or whether it is related to a phenotype of the 249S p53 mutant protein. This was addressed by constructing a metabolically competent variant of Saccharomyces cerevisiae strain yIG397 expressing human cytochrome P450 1A2 and P450-reductase and isolating AFB1-induced mutants that failed to express the genomic ADE2 reporter gene. Molecular analysis revealed that only 8/40 mutants had a mutation in the TP53 target gene, whereas 32/40 mutants were due to a recombination event eliminating the ADE2 reporter gene. None of 19 mutations identified in the eight mutant TP53 plasmids altered codon 249, thus this codon was no hot spot if the TP53 gene was in the heterologous background yeast. The genotoxic action of AFB1 was completely different from that of the alkylating agent ethyl-methane-sulfonate, where 28/30 induced mutations were linked to the TP53 target gene.