Allosteric trans-splicing group i ribozyme whose activity of target-specific rna replacement is controlled by theophylline
A technology of trans-splicing and theophylline, which is applied in the determination/inspection of enzymes, transferases, and microorganisms, and can solve the problem that the therapeutic effect will not be maximized.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment
[0075] Hereinafter, exemplary embodiments of the present invention will be described with reference to the accompanying drawings, but the present invention is not particularly limited thereto.
[0076] Reference Example 1: Preparation of Substrate (hTERT) RNA
[0077] To prepare target RNA, primers described in SEQ ID NO: 9 (5'-GGGGAATTCTAATACGACTCACTATAGGGCAGGCAGCGCTGCGTCCT-3') and primers described in SEQ ID NO: 10 (5'-CGGGATCCCTGGCGGAAGGAGGGGGCGGCGGG-3') were paired with hTERT-containing hTERT-1 The pCl-neo vector (exon 1-2) of the DNA sequence to +218th was subjected to PCR amplification to obtain a DNA fragment encoding hTERT RNA. The DNA fragments thus prepared are transcribed into RNA in vitro. Add DNA template (3 μ g), 10x transcription buffer, 10 mM DTT (Sigma), 0.5 mM ATP, GTP, CTP, and UTP (Roche), 80 U of RNase inhibitor (Cosco (Kosco)), 200U of T7 RNA polymerase (Ambion), and diethylpyrocarbonate water (DEPC-H 2 O) to a final volume of 100 μl and then mixed. T...
Embodiment 7
[0091] Reference Example 7: Cell Culture
[0092] Incubate hTERT-positive cell lines in 5% CO 2 293 (human kidney / normal), HT-29 (colon / colon adenocarcinoma), Capan-1 (pancreas / adenocarcinoma) and HepG2 (liver / hepatocellular carcinoma) were used as reference, and place hTERT-negative cell lines in 5% CO 2 Cultured at 37°C in an incubator with IMR-90 (lung / fibroblast / normal) and SK-LU1 (lung / adenocarcinoma) American Type Collection (ATCC) as reference.
[0093] Reference Example 8: Confirmation of Specificity and Efficiency of Trans-splicing Aptamase in Cell Lines
[0094] 1) The test of the optimum concentration of theophylline
[0095] 293 cells in a 35mm dish in 3×10 5 The concentrations were inoculated and grown to approximately 80% confluency. In this case, growing 293 cells were transfected with 1 μg of Mu P9 6t8t with liposomal LipofectAMINE (Invitrogen). The transfected 293 cells were cultured in increasing concentrations of theophylline or caffeine (0.1 mM, 0.3 m...
Embodiment 1
[0111] Example 1: Preparation of a trans-splicing ribozyme with theophylline aptamer attached and specifically targeting hTERT RNA
[0112] As the basic trans-splicing ribozyme backbone for the development of allosteric ribozymes, a type I intronic ribozyme that specifically recognizes the +21nt site of hTERT and has a target The antisense sequence of 300nt of RNA is annealed P1, P10 and extended IGS ( figure 2 ). This ribozyme was observed to induce specific apoptosis in hTERT-expressing cancer cells by specifically expressing hTERT RNA in cells and animal models (Mol. Ther. 2005; 12:824, Mol Ther .2008;16:74)
[0113] To prepare theophylline-dependent allosteric ribozymes, theophylline RNA aptamer (Science 1994; 263:1425) used as the acceptor region of theophylline was simultaneously attached to either of the P6 or / and P8 regions or Both, this plays an important role in RNA folding for the catalytic function of the hTERT-specific T / S ribozyme developed by the applicant's...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More