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Review
. 2007 Nov-Dec;1769(11-12):603-12.
doi: 10.1016/j.bbaexp.2007.09.001. Epub 2007 Sep 29.

Inhibition of gene expression in human cells using RNase P-derived ribozymes and external guide sequences

Kihoon Kim  1 , Fenyong Liu
Affiliations
Review

Inhibition of gene expression in human cells using RNase P-derived ribozymes and external guide sequences

Kihoon Kim et al. Biochim Biophys Acta. 2007 Nov-Dec.

Abstract

Ribonuclease P (RNase P) complexed with an external guide sequence (EGS) represents a novel nucleic acid-based gene interference approach to modulate gene expression. This enzyme is a ribonucleoprotein complex for tRNA processing. In Escherichia coli, RNase P contains a catalytic RNA subunit (M1 ribozyme) and a protein subunit (C5 cofactor). EGSs, which are RNAs derived from natural tRNAs, bind to a target mRNA and render the mRNA susceptible to hydrolysis by RNase P and M1 ribozyme. When covalently linked with a guide sequence, M1 can be engineered into a sequence-specific endonuclease, M1GS ribozyme, which cleaves any target RNAs that base pair with the guide sequence. Studies have demonstrated efficient cleavage of mRNAs by M1GS and RNase P complexed with EGSs in vitro. Moreover, highly active M1GS and EGSs were successfully engineered using in vitro selection procedures. EGSs and M1GS ribozymes are effective in blocking gene expression in both bacteria and human cells, and exhibit promising activity for antimicrobial, antiviral, and anticancer applications. In this review, we highlight some recent results using the RNase P-based technology, and offer new insights into the future of using EGS and M1GS RNA as tools for basic research and as gene-targeting agents for clinical applications.

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Figures

Figure 1
Figure 1
(A–B) Representation of natural substrates (pre-tRNA (A) and 4.5S RNA (B)). (C–E) A hybridized complex of a target RNA (e.g. mRNA) and an EGS that resembles a part of structure of a tRNA and can be cleaved by RNase P. (D) results from (C) by deleting the anticodon domain of the EGS, which is dispensable for EGS targeting activity, while (E) results from (D) by further deleting the D stem/loop and variable regions. Substrates in (C) and (D) can be cleaved by human RNase P and M1 ribozyme. In contrast, the stem structure in (E) can only serve as a substrate for M1 RNA and can not be cleaved by human RNase P. (F) A complex formed between an M1GS ribozyme and a target mRNA substrate. (G, H) Complexes between the HCMV capsid scaffolding protein (CSP) mRNA and EGS CSP1 and CSP2, respectively [82]. (I) Representation of an M1GS RNA construct to which a target RNA has hybridized. The arrow shows the site of the cleavage by RNase P and M1 RHA.
Figure 2
Figure 2
Schematic representation of the in vitro evolution procedure for the generation of highly active M1GS RNA ribozyme variants that specifically cleave a target mRNA.
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