Treatment of inflammatory or malignant disease using dnazymes
A DNAzyme and nucleotide technology, used in gene therapy, bone diseases, respiratory diseases, etc., can solve the meaningful treatment results of unsuccessful DNAzyme treatment, insufficient DNAzyme, and hinder the combination of DNAzymes. and cleavage of target mRNA
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Embodiment 1
[0152] Example 1 Design of DNAzyme construct
[0153] Two DNA constructs named ND1 and ND2 are designed according to the 10-23 catalytic motif (Santoro and Joyce, 1997). The catalytic motif is flanked by two substrate recognition domains, and each of the two substrate recognition domains has 9 Deoxynucleotides. Place an inverted thymidine at the end of the 3-primer of the oligodeoxynucleotide. This exposes the distinct 5-primer ends, thereby making the construct resistant to 3-primer exonuclease activity.
[0154] Construct ND1 is a RelA (p65) messenger RNA designed to cleave the AUG translation start site between A80 and U81. Construct ND2 is designed to cleave RelA (p65) messenger RNA at the next AU or GU site in the 3'direction, that is, between G91 and U92. Their respective controls, ND1c and ND2c contain random hybridization arms. In addition to a single base change at the 5'end of the catalytic motif, the control oligonucleotide also has a consensus sequence of 10-23 catalyt...
Embodiment 2
[0156] Example 2 In vitro cleavage of synthetic RNA targets by deoxyribozymes ND1 and ND2
[0157] Combine oligonucleotides ND1, ND1c, ND2 and ND2c with RelA (p65) RNA (61-110) in 10mM Mg 2+ Incubate at 37°C for the indicated time. RNA is used before incubation with deoxyribozyme 32 P End tag. For ND1 and ND2, cleavage into a single product of the desired molecular weight was observed (data not shown). The control oligonucleotides ND1c and ND2c were not cleaved. The cleavage of ND2 is more effective than ND1. Example 3 In the presence of liposomes and CellFectin, the effect of DNAzyme ND2 on NF-κB and AP-1-dependent luciferase reporter gene (Life Technoloqies)
Embodiment 3
[0158] HeLa cells were transfected with a plasmid containing a luciferase gene (Promega) transcribed from an artificial promoter that relies on 6 NF-κB binding sites and 3 AP-1 sites. Deoxyribozyme (Dz) was compounded with CellFectin at a ratio of 2.5ug / ml CellFectin 1μM deoxyribozyme. After Dz was administered to HeLa cells, luciferase was induced with 10ng / ml interleukin-1β. figure 1 It was shown that ND2 caused a concentration-dependent inhibition of NF-κB-dependent gene expression. The inhibition by ND2 was significantly higher than the control ND2c and the vector alone at all concentrations. Most importantly, ND2 or ND2c did not inhibit AP-1-dependent gene expression, indicating the specificity of ND2 for the transcription factor NF-κB compared to another inducible transcription factor.
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