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  • Title: Engineered RNase P ribozymes inhibit gene expression and growth of cytomegalovirus by increasing rate of cleavage and substrate binding.
    Author: Trang P, Hsu A, Zhou T, Lee J, Kilani AF, Nepomuceno E, Liu F.
    Journal: J Mol Biol; 2002 Jan 25; 315(4):573-86. PubMed ID: 11812131.
    Abstract:
    We have previously employed an in vitro (genetic) selection procedure to select RNase P ribozyme variants for their activity in cleaving a mRNA substrate from a pool of ribozymes containing randomized sequences. In this study, one of the variants was used to target the overlapping region of the mRNAs encoding the major transcription regulatory proteins, IE1 and IE2, of human cytomegalovirus (HCMV). The ribozyme variant exhibited an enhanced substrate binding and rate of chemical cleavage, and was at least 25 times more efficient in cleaving the target mRNA in vitro than the ribozyme derived from the wild-type sequence. Our results provide the first direct evidence that a point mutation at nucleotide 86 of RNase P catalytic RNA from Escherichia coli (A(86)-->C(86)) increases the rate of chemical cleavage while another mutation at nucleotide 205 (G(205)-->C(205)) enhances substrate binding of the ribozyme. Moreover, the variant was also more effective in inhibiting IE1 and IE2 expression and HCMV growth in cultured cells. A reduction of more than 97% in IE1 and IE2 expression and a reduction of 3000-fold in viral growth were observed in cells expressing the variant. Thus, RNase P ribozyme variant is highly effective in inhibiting HCMV gene expression and growth. Our results provide the direct evidence that increasing the rate of chemical cleavage and substrate-binding affinity of the ribozymes should lead to an improvement of their anti-HCMV efficacy. Moreover, our data also suggest that highly effective anti-HCMV ribozyme variants can be developed using genetic engineering approaches including in vitro selection.
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