Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.26.5 (RNase P)
1,348 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ribozymes are promising gene-targeting agents for regulation of gene expression. In our recent studies, RnaseP (M1GS) ribozymes were constructed to target the overlapping region (IE mRNA) of IE1 and IE2 mRNAs of human cytomegalovirus (HCMV) and the mRNA (TK mRNA) coding for thymidine kinase (TK) of herpes simplex virus 1 (HSV-1). Our results indicate that RNase P ribozymes efficiently cleaved the IE mRNA and TK mRNA sequences in vitro. Significant inhibitions (approximately 75-85%) of HCMV IE1/IE2 and HSV-1 TK expression were observed in cells that expressed these ribozymes while a reduction of less than 10% was found in cells that did not express the ribozymes or expressed a disabled one that contained mutations abolishing catalytic activity. Ribozyme variants, which cleaved a TK mRNA sequence in vitro more efficiently than the ribozyme derived from the wildtype RNase P sequence, were selected by an in vitro selection system. When the selected ribozymes were expressed in cultured cells, they were more effective in inhibiting viral IE1/IE2 and TK expression and viral growth than the wildtype ribozyme sequence. Our results provide the first direct evidence that RNase P ribozymes are highly effective in inhibiting HCMV gene expression and growth. Moreover, a selection system was developed for generating novel ribozyme variants that cleave a mRNA substrate efficiently in vitro. These results suggest that M1GS ribozyme-mediated inhibition of expression of viral genes can be used as a new approach for the studies of HCMV gene function and the treatment of HCMV infection.
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PMID:RNase P ribozymes for the studies and treatment of human cytomegalovirus infections. 1236 58

Ribonuclease P (RNase P) is a ubiquitous ribonucleoprotein complex responsible for the biosynthesis of tRNA. This enzyme from Escherichia coli contains a catalytic RNA subunit (M1 ribozyme) and a protein subunit (C5 cofactor). M1 ribozyme cleaves an RNA helix that resembles the acceptor stem and T-stem structure of its natural tRNA substrate. When covalently linked with a guide sequence, M1 RNA can be engineered into a sequence-specific endonuclease, M1GS ribozyme, which can cleave any target RNA sequences that base pair with the guide sequence. Recent studies indicate that M1GS ribozymes efficiently cleave the mRNAs of herpes simplex virus 1, human cytomegalovirus, and cancer causing BCR-ABL proteins in vitro and effectively inhibit the expression of these mRNAs in cultured cells. Moreover, RNase P ribozyme variants that are more active than the wild type M1 RNA can be generated using in vitro selection procedures and the selected variants are also more effective in inhibiting gene expression in cultured cells. These results demonstrate that engineered RNase P ribozymes represent a novel class of promising gene-targeting agents for applications in both basic research and clinical therapy. This review discusses the principle underlying M1GS-mediated gene inactivation and methodologies involved in effective M1GS construction, expression in vivo and emerging prospects of this technology for gene therapy.
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PMID:Engineering of RNase P ribozyme for gene-targeting applications. 1295 77

RNase P ribozyme, such as M1 RNA, the catalytic RNA subunit of RNase P from Escherichia coli, cleaves an RNA helix that resembles the acceptor stem and T-stem structure of its natural ptRNA substrate. When covalently linked with a guide sequence, the M1 ribozyme can function as a sequence-specific endonuclease, M1GS RNA, and cleave any target RNA sequences that basepair with the guide sequence. Using the mRNA coding for the major transcription regulatory protein ICP4 of herpes simplex virus 1 (HSV-1) as the model target, we describe in this chapter the general design and construction of M1GS ribozymes for gene-targeting applications. Specifically, methods are described in detail to determine ideal target regions of an mRNA for M1GS ribozymes and to construct highly active RNase P ribozymes that target these regions. Extensive protocols for in vitro synthesis of the ribozymes and for the cleavage assay of the ribozyme activity are also included. These methods are intended to provide general guidelines for the design and construction of M1GS ribozymes for gene-targeting applications.
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PMID:General design and construction of RNase P ribozymes for gene-targeting applications. 1501 65

An in vitro selection procedure for identifying highly efficient RNase P ribozyme (M1GS RNA) variants is presented as a model system for engineering ribozymes to improve their catalytic efficiency. Detailed protocols as well as the rationale for setting up such a system are included, using the mRNA sequence that encodes the thymidine kinase (TK) of herpes simplex virus 1 (HSV 1) as a target substrate of choice. Using the selection system, we have successfully generated M1GS RNA variants that more efficiently cleave the TK mRNA in vitro and more effectively inhibit the TK expression in cultured cells than the ribozyme derived from the wild-type RNase P ribozyme sequence. The in vitro selection system represents a novel and effective approach for engineering highly active RNase P ribozymes that can be used in both basic research and clinical therapeutic settings.
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PMID:In vitro selection of RNase P ribozymes that efficiently cleave a target mRNA. 1501 66

RNase P, a tRNA processing enzyme, contains both RNA and protein subunits. M1 RNA, the catalytic RNA subunit of RNase P from Escherichia coli, recognizes its target RNA substrate mainly on the basis of its structure and cleaves a double stranded RNA helix at the 5' end that resembles the acceptor stem and T-stem structure of its natural tRNA substrate. Accordingly, a guide sequence (GS) can be covalently attached to the M1 RNA to generate a sequence specific ribozyme, M1GS RNA. M1GS ribozyme can target any mRNA sequence of choice that is complementary to its guide sequence. Recent studies have shown that M1GS ribozymes efficiently cleave the mRNAs of herpes simplex virus 1 and human cytomegalovirus, and the BCR-ABL oncogenic mRNA in vitro and effectively reduce the expression of these mRNAs in cultured cells. Moreover, an in vitro selection scheme has been developed to select for M1 GS ribozyme variants with more efficient catalytic activity in cleaving mRNAs. When expressed in cultured cells, these selected ribozymes also show an enhance ability to inhibit viral gene expression and growth. These recent results demonstrate the feasibility of developing the M1GS ribozyme-based technology as a promising gene targeting approach for basic research and clinical therapeutic application.
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PMID:Developing RNase P ribozymes for gene-targeting and antiviral therapy. 1510 92

Engineered RNase P ribozymes are promising gene-targeting agents that can be used in both basic research and clinical applications. We have previously selected ribozyme variants for their activity in cleaving an mRNA substrate from a pool of ribozymes containing randomized sequences. In this study, one of the variants was used to target the mRNA encoding thymidine kinase (TK) of herpes simplex virus 1 (HSV-1). The variant exhibited enhanced cleavage and substrate binding and was at least 30 times more efficient in cleaving TK mRNA in vitro than the ribozyme derived from the wild type sequence. Our results provide the first direct evidence to suggest that a point mutation at nucleotide 95 of RNase P catalytic RNA from Escherichia coli (G(95) --> U(95)) increases the rate of cleavage, whereas another mutation at nucleotide 200 (A(200) --> C(200)) enhances substrate binding of the ribozyme. A reduction of about 99% in TK expression was observed in cells expressing the variant, whereas a 70% reduction was found in cells expressing the ribozyme derived from the wild type sequence. Thus, the RNase P ribozyme variant is highly effective in inhibiting HSV-1 gene expression. Our study demonstrates that ribozyme variants increase their cleavage activity and efficacy in blocking gene expression in cells through enhanced substrate binding and rate of cleavage. These results also provide insights into the mechanism of how RNase P ribozymes efficiently cleave an mRNA substrate and, furthermore, facilitate the development of highly active RNase P ribozymes for gene-targeting applications.
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PMID:Engineered RNase P ribozymes increase their cleavage activities and efficacies in inhibiting viral gene expression in cells by enhancing the rate of cleavage and binding of the target mRNA. 1516 70

An external guide sequence (EGS) is a RNA sequence which can interact with a target mRNA to form a tertiary structure like a pre-tRNA and recruit intracellular ribonuclease P (RNase P), a tRNA processing enzyme, to degrade target mRNA. Previously, an in vitro selection procedure has been used by us to engineer new EGSs that are more robust in inducing human RNase P to cleave their targeted mRNAs. In this study, we constructed EGSs from a variant to target the mRNA encoding herpes simplex virus 1 (HSV-1) major transcription regulator ICP4, which is essential for the expression of viral early and late genes and viral growth. The EGS variant induced human RNase P cleavage of ICP4 mRNA sequence 60 times better than the EGS generated from a natural pre-tRNA. A decrease of about 97% and 75% in the level of ICP4 gene expression and an inhibition of about 7,000- and 500-fold in viral growth were observed in HSV infected cells expressing the variant and the pre-tRNA-derived EGS, respectively. This study shows that engineered EGSs can inhibit HSV-1 gene expression and viral growth. Furthermore, these results demonstrate the potential for engineered EGS RNAs to be developed and used as anti-HSV therapeutics.
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PMID:Inhibition of herpes simplex virus 1 gene expression and replication by RNase P-associated external guide sequences. 2727 82

Infections of the central nervous system (CNS) are often acute, with significant morbidity and mortality. Routine diagnosis of such infections is limited in developing countries and requires modern equipment in advanced laboratories that may be unavailable to a number of patients in sub-Saharan Africa. We developed a TaqMan array card (TAC) that detects multiple pathogens simultaneously from cerebrospinal fluid. The 21-pathogen CNS multiple-pathogen TAC (CNS-TAC) assay includes two parasites (Balamuthia mandrillaris and Acanthamoeba), six bacterial pathogens (Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, Mycoplasma pneumoniae, Mycobacterium tuberculosis, and Bartonella), and 13 viruses (parechovirus, dengue virus, Nipah virus, varicella-zoster virus, mumps virus, measles virus, lyssavirus, herpes simplex viruses 1 and 2, Epstein-Barr virus, enterovirus, cytomegalovirus, and chikungunya virus). The card also includes human RNase P as a nucleic acid extraction control and an internal manufacturer control, GAPDH (glyceraldehyde-3-phosphate dehydrogenase). This CNS-TAC assay can test up to eight samples for all 21 agents within 2.5 h following nucleic acid extraction. The assay was validated for linearity, limit of detection, sensitivity, and specificity by using either live viruses (dengue, mumps, and measles viruses) or nucleic acid material (Nipah and chikungunya viruses). Of 120 samples tested by individual real-time PCR, 35 were positive for eight different targets, whereas the CNS-TAC assay detected 37 positive samples across nine different targets. The CNS-TAC assays showed 85.6% sensitivity and 96.7% specificity. Therefore, the CNS-TAC assay may be useful for outbreak investigation and surveillance of suspected neurological disease.
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PMID:Evaluation of a TaqMan Array Card for Detection of Central Nervous System Infections. 2840 79


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