Targeting mRNAs by Engineered Sequence-Specific RNase P Ribozymes

Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, CA, USA.
Methods in molecular biology (Clifton, N.J.) (Impact Factor: 1.29). 01/2012; 848:357-68. DOI: 10.1007/978-1-61779-545-9_22
Source: PubMed


The methods of using engineered RNase P catalytic RNA (termed as M1GS RNA) for in vitro and in vivo in trans-cleavage of target viral mRNA are described in this chapter. Detailed information is focused on (1) mapping accessible regions of target viral mRNA in infected cells, (2) generation and in vitro cleavage assay of the customized M1GS ribozyme, (3) stable expression of M1GS RNAs and evaluation of its antiviral activity in cultured cells. Using these methods, we have constructed functional M1GS ribozyme that can cleave an overlapping region of the mRNAs coding for the human cytomegalovirus (HCMV) capsid scaffolding protein (CSP) and assemblin in vitro. Further study has demonstrated that, in cultured human cells expressing the functional M1GS ribozyme and infected with HCMV, more than 85% reduction in the expression of CSP and assemblin and a 4,000-fold reduction in viral growth were achieved. Our study provided the direct evidence that the customized M1GS ribozyme can be used as an effective gene-targeting agent for in trans-cleavage of viral genes and inhibition of viral growth in cultured cells.

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    • "Moreover, M1 RNA is able to cleave a target RNA sequence efficiently if an additional small RNA is covalently linked to the 3′ end of M1 RNA. The new sequence-specific ribozyme is named M1GS ribozyme (Figure 1A) [7,8], and any RNA could in principle be targeted by a custom-designed M1GS for specific cleavage. When introduced into human cells, M1GS ribozyme can function independently from the endogenous human RNase P to cleave a targeting sequence that base pairs with the guide sequence [9]. "
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