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| DNAzymes target design for Ezrin mRNA antisense technologyPAN Huilong1 ZHANG Guoru2▲ WANG Jin3 WU Xingyuan2 |
| PAN Huilong1 ZHANG Guoru2▲ WANG Jin3 WU Xingyuan2 |
1.Guangzhou Medical University Graduate School, Guangdong Province, Guangzhou 510080, China;
2.Department of Osteoarthritis, the Third People′s Hospital of Hainan, Hainan Province, Sanya 572000, China;
3.Department of Bone and Soft Tissue, Cancer Hospital Affiliated to Zhongshan University, Guangdong Province, Guangzhou 510080, China |
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Abstract Objective To analyze the structure of distant metastasis associated protein (Ezrin mRNA) and find and verify the best target of DNAzymes. Methods RNAstructure and RNAdraw programs were used to analyze the structure of Ezrin mRNA and calculate its first or secondary structure. If RNAstructure and RNAdraw simultaneously calculate 4 or more unpaired single chain ring forming regions, the single chain ring forming region would be set as the target area of antisense technology for the design of the target of the DNAzymes. Then, according to the principle of minimum free energy, the OligoWalk program was used for screening, and the target of each antisense technology was screened by screening. The experimental method was used to verify the prediction results. Results There were 42 common single chain regions predicted by the two software, of which 21 were single matched, and 27 were coding regions. AU1655, AU1751, AU1766, AU1789 and GU2623 were located in the single chain region over 10 consecutive unpaired bases. Only AU1655, AU1751, AU1766 and AU1789 meet the requirements. The results of enzyme digestion showed that DNAzymes could cut Ezrin mRNA most ideally at AU1751 site. Conclusion Compared with the traditional experiment to find the target, the combined thermal dynamic parameters of the two stage structure of nucleic acid could deal with the design and selection of the target more accurately and quickly. The DNAzymes corresponding to the AU1751 locus is less likely to form stable self heterozygotes, which is beneficial to DNAzymes binding to RNA.
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