An Experimental Research On Activity Of Deoxyribozymes Site-specifically Against Hepatitis C Virus

With the rapid development of modern molecular biology and correlated science, several kinds of short special DNA molecules with biocatalytic activity have been found in recent years. These catalytic DNAs are named as deoxyribozymes, and have different catalytic activity for the difference in their structures. Among the family of catalytic DNAs, those with the highly-conserved motif of 5'-GGCTAGCTACAACGA-3', which acts as the active center and contributes to the ability of cleaving target RNA site-specifically, have caught the great attention of scientists for their potential usage as the tools of gene modulation or therapy. Compared with antisense oligodeoxynucleotides (ASODN), these deoxyribozymes may not only have antisense inhibition effect on target RNAs, but also own the ability to break special RNAs by distorting their configurations, and one deoxyribozyme molecule can cleave several substrate RNAs. In comparison with ribozymes, the deoxyribozymes can recognize all the sequences characterized with 5'…Y↓R…3' (Y=A/G, R=U/C), thus have more target sites to be selected. Furthermore, they are more stable than ribozymes and have relatively simple structures. So it is considered that the deoxyribozymes may have both advantages of ASODN and ribozymes. Now the deoxyribozymes have been proved to be effective to certain extent in some incipient researches, such as cleaving RNAs of human immunodeficiency virus-1, human papilloma virus-16, and mRNAs of mutant genes in malignant tumors; but articles on their ability to cleave hepatitis virus are rarely seen.As is well known, the lack of ideally effective antiviral drugs is still an major problem in the treatment of viral hepatitis, and medical scientists have been trying their best to explore every new possible strategy to eradicate hepatitis virus in patients. The find of deoxyribozymes may bring a new hope for this purpose. In this study, we designed four groups of specific deoxyribozymes targeting at 5'-noncoding region (5'-NCR) of hepatitis C virus (HCV) according to its sequence and secondary structure, and investigated their possibility for using as antiviral measures by systematically evaluating the activity of cleavage in vitro at molecular level, inhibition or/and cleavage effect in HCV 5'-NCR-C transgeneic hepatoma cells and HCV-infected fetal hepatocytes.1. Selection of target sites and design of specific deoxyribozymesSelection of target sites is the essential steps for design of deoxyribozymes, and should be done according to several factors. HCV 5'-NCR is highly conserved, and containing important functional domains, such as internal ribosome entry sites (IRES). Chemical probing, nuclease digestion test, thermadynamics (free energy) calculation and computer simulation all demonstrate that the secondary structure of HCV 5'-NCR can be divided into domainⅠ～Ⅳ. Domain Ⅰ might have the ability to impede the start of HCV translation, but domain Ⅱ could counteract this effect by stabilizing the configuration of 5'-NCR and its binding to host ribosomes. Domain Ⅲ and Ⅳ are the main spanning regions of IRES, thus act as the most important functional regions in 5'-NCR for controlling initiation and modulation of HCV translation. Based on these findings, it is wise to locate the target sites in domain Ⅲ and Ⅳ preferably, or domain Ⅱ to some extent, but avoid domain Ⅰ. 5'-NCR may have some mutant bases among different clinical HCV separate strains, and sequences containing those mutant bases should not be selected as target sites in order to keep the designed deoxyribozymes have universal cleavage or/and inhibition effect on every HCV variants. On the other hand, though all sequences characterized with 5'…R↓Y…3' might be the potential target sites, the 5'…A↓U…3' or 5'…G↓U…3' had been proved by Santoro et al to be more easily cleaved than other sites, thus should be selected preferably.After confirmation of sequence of HCV 5'-NCR-C from pHCV-neo in a DNA sequencer, the secondary structure of 5'-NCR was figured and all the possib...