Length Variation of Helix III in a Hammerhead Ribozyme and Its Influence on Cleavage Activity (original) (raw)
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Status of Ribozyme and Antisense-Based Developmental Approaches for Anti-HIV-1 Therapya
Annals of the New York Academy of Sciences, 1992
The observation that the complementary interaction of nucleotide sequences exists in nature for the regulation of cellular functions led to the concept of "antisense" as a biological entity.' Antisense is any DNA or RNA that inhibits a gene function by methods that require complementary base-pairing to the genetic target. In the last 15 years, it has been shown that the expression of antisense RNA as well as the exogenous introduction of antisense DNA can affect a variety of cell function^.^-^ The observation that RNA has autocatalytic function that is mediated in part by complementary base-pairing has led to a second gcneration of antisense which is called ribozyme. Ribozyme is an RNA molecule having catalytic enzyme activity that cleaves RNA in a sequence-dependent manner, thereby inactivating the RNA. There are several types of ribozymes. (For a review see ref. 6 . The first of these, from the Tetrahymena group one intervening sequence, was shown by Zaug and Cech7 to be essential for cleaving an intron from messenger RNA. A second ribozyme was noted by Altmann and co-workers to function as the RNA component of RNAseP in the maturation of tRNAsX RNAseP consists of both RNA and protein moieties that function together to complete the 5' cleavage of pre-tRNAs. In this situation, the RNA component can function independently, but the 14-kD protein moiety is thought to create a microenvironment that optimizes the catalytic reaction . A third type of ribozyme, the hammerhead ribozyme, is the one that exists in certain viroids and virusoids as well as in Neurospora. These ribozymes require specific flanking sequences that target the ribozyme and bring together an active center, causing cleavage at that specific site. Synthetic ribozyme can be made synthetically, and the use of ribozyme for inhibition of HIV infection will be discussed in detail here.
Status of Ribozyme and Antisense-Based Developmental Approaches for Anti-HIV-1 Therapy
Annals of The New York Academy of Sciences - ANN N Y ACAD SCI, 1992
The observation that the complementary interaction of nucleotide sequences exists in nature for the regulation of cellular functions led to the concept of "antisense" as a biological entity.' Antisense is any DNA or RNA that inhibits a gene function by methods that require complementary base-pairing to the genetic target. In the last 15 years, it has been shown that the expression of antisense RNA as well as the exogenous introduction of antisense DNA can affect a variety of cell function^.^-^ The observation that RNA has autocatalytic function that is mediated in part by complementary base-pairing has led to a second gcneration of antisense which is called ribozyme. Ribozyme is an RNA molecule having catalytic enzyme activity that cleaves RNA in a sequence-dependent manner, thereby inactivating the RNA. There are several types of ribozymes. (For a review see ref. 6 . The first of these, from the Tetrahymena group one intervening sequence, was shown by Zaug and Cech7 to be essential for cleaving an intron from messenger RNA. A second ribozyme was noted by Altmann and co-workers to function as the RNA component of RNAseP in the maturation of tRNAsX RNAseP consists of both RNA and protein moieties that function together to complete the 5' cleavage of pre-tRNAs. In this situation, the RNA component can function independently, but the 14-kD protein moiety is thought to create a microenvironment that optimizes the catalytic reaction . A third type of ribozyme, the hammerhead ribozyme, is the one that exists in certain viroids and virusoids as well as in Neurospora. These ribozymes require specific flanking sequences that target the ribozyme and bring together an active center, causing cleavage at that specific site. Synthetic ribozyme can be made synthetically, and the use of ribozyme for inhibition of HIV infection will be discussed in detail here.
Inhibition of HIV-1 Replication by an Improved Hairpin Ribozyme That Includes an RNA Decoy
RNA Biology, 2005
An anti-Tat hairpin ribozyme and a TAR RNA decoy were combined in one molecule. The chimeric molecule strongly inhibited HIV-1 replication (measured as changes in p24 levels in viral replication assays). The inhibitory action of the ribodecozyme (85%) was significantly greater than that shown by ribozyme and a non-catalytic variant carrying the functional decoy RNA domain (55% and 35%, respectively). This represents a significant improvement of the inhibitory efficiency of the ribozyme, suggesting there is an additive inhibitory effect on HIV-1 replication by the catalytic and decoy domains. This strategy could be used to create new inhibitor RNAs with enhanced in vivo performance.
Gene Therapy, 1997
Retroviral vectors were engineered to express monomeric be detected in these cells and in their culture supernatants and multimeric hammerhead ribozymes targeting one and for up to 60 days after infection. The genomic DNA from nine highly conserved sites within the HIV-1 envelope Rz Env1-9-expressing cells was shown to contain HIV-1 pro-(Env) coding region. In vitro, both the monomeric and multi-viral DNA sequences at days 3 and 60 after HIV infection. meric ribozymes were shown to be active and cleave the HIV-1 used in the challenge experiments was found to contarget RNA containing the cleavage sites. A human CD4 + tain fully reverse transcribed '−' strand DNA which should T lymphocyte-derived MT4 cell line was stably transduced have been able to infect, complete proviral DNA synthesis with retroviral vectors expressing these ribozymes. and integrate within the cellular genome without being Ribozyme expression in stably transduced cells was con-affected by pre-existing ribozymes. Therefore, the proviral firmed by Northern blot analysis and reverse-transcription DNA present at day 3 after infection may have originated polymerase chain reaction (RT-PCR). As compared with from infection by such DNA-containing virus particles. The the control cells lacking any ribozyme, HIV-1 replication results obtained with the retroviral vector expressing was delayed in monomeric Rz Env-expressing cells. Virus Rz Env1-9 are very encouraging and we envisage its future replication was almost completely inhibited in multimeric use in anti-HIV-1 gene therapy. Rz Env1-9-expressing cells as no viral RNA or protein could
FEBS Letters, 1995
Two hammerhead chimeric RNA/DNA ribozymes (HRz) were synthesized in pure form. Both were 30 nucleotides long, and the sequences were such that they could be targeted to cleave the HIV-I gag RNA. Named HRz-W and HRz-M, the former had its invariable core region conserved, the latter had a uridine in the invariable region replaced by a guanine. Their secodary structures were determined by 2D NOESY IH 500 MHz NMR spectroscopy in 90% water and 10% D20, following the imino protons. The data show that both HRz-M and HRz-W form identical secondary structures with stem regions consisting of continuous stacks of AT and GT pairs. An energy mimimized computer model of this stem region is provided. The results suggest that the loss of catalytic activity that is known to result when an invariant core residue is replaced is not related to the secondary structure of the ribozymes in the absence of substrate.
Effective Inhibition of Human Immunodeficiency Virus 1 Replication by Engineered RNase P Ribozyme
PLoS ONE, 2012
Using an in vitro selection procedure, we have previously isolated RNase P ribozyme variants that efficiently cleave an mRNA sequence in vitro. In this study, a variant was used to target the HIV RNA sequence in the tat region. The variant cleaved the tat RNA sequence in vitro about 20 times more efficiently than the wild type ribozyme. Our results provide the first direct evidence that combined mutations at nucleotide 83 and 340 of RNase P catalytic RNA from Escherichia coli (G 83-. U 83 and G 340-. A 340) increase the overall efficiency of the ribozyme in cleaving an HIV RNA sequence. Moreover, the variant is more effective in reducing HIV-1 p24 expression and intracellular viral RNA level in cells than the wild type ribozyme. A reduction of about 90% in viral RNA level and a reduction of 150 fold in viral growth were observed in cells that expressed the variant, while a reduction of less than 10% was observed in cells that either did not express the ribozyme or produced a catalytically inactive ribozyme mutant. Thus, engineered ribozyme variants are effective in inhibiting HIV infection. These results also demonstrate the potential of engineering RNase P ribozymes for anti-HIV application.
International Journal of Biological Macromolecules, 2001
The main transcriptional regulator of the human immunodeficiency virus is the Tat protein, which recognises and binds to a fragment RNA at the 5% end of viral mRNA, named transactivation response element (TAR) RNA. Extensive mutagenesis studies have shown that a region of TAR RNA important for Tat binding involves a set of nucleotides surrounding a characteristic UCU nucleotide bulge. The specific Tat-TAR complex formation enhances the rate of transcription elongation but inhibition of that interaction prevents the human immunodeficiency virus type 1 (HIV-1) replication. If so, a possibility of virus inactivation would be a site specific degradation of the TAR RNA element. To break down and inactivate TAR RNA, we designated the anti-hammerhead (HH) ribozyme to cleave nucleosides within the bulge. We showed for the first time the new type of the AUC hammerhead ribozyme, which hydrolyses specifically the TAR RNA element at C8 nucleotide in the bulge (C24 in the standard TAR RNA numbering). The cleavage reaction has broad magnesium requirements. Mn and particularly Ca are less efficient. Argininamide interferes with the cleavage of TAR RNA induced by the ribozyme. These results have two implications; (i) structural, where the HIV-1 TAR RNA element in solution occurs in equilibrium of only two forms, one of which, a double stranded RNA, meets structural requirements for ribozyme pairing and cleavage, and (ii) functional, the HH ribozyme can be explored for an inactivation of HIV-1 through the TAR RNA element deintegration.
Frontiers in Bioscience, 2006
Introduction 3. Materials and Methods 3.1. Construction of retroviral vectors 3.2. Transduction and selection of stable MT4 transductants 3.3. PCR analysis of genomic DNA from MT4 transductants 3.4. RT-PCR analysis of total RNA from MT4 transductants 3.5. In vitro cleavage activity of multimeric ribozymes amplified from the pools of MT4 transductants 3.6. HIV-1 susceptibility of MT4 transductants 4. Results 4.1. MGIN-based vectors expressing the multimeric ribozymes, the antisense RNA, or the multimeric ribozymes and the antisense RNA 4.2. Development of pools of stable MT4 transductants expressing the interfering RNAs 4.3. HIV-1 susceptibility of MT4 transductants expressing the interfering RNAs 5. Discussion 6. Acknowledgements 7. References
Journal of General Virology, 2008
Rz 1-7 is a multimeric hammerhead ribozyme targeting seven unique sites within the human CCR5 mRNA that is active in vitro. Mouse stem cell virus-based MGIN and human immunodeficiency virus (HIV)-1-based HEG1 vectors were used to express Rz 1-7 in a human CD4 + T lymphoid cell line. Stable transductants expressed Rz 1-7 , which was further shown to be active, since CCR5 mRNA and surface CCR5 protein expression levels decreased. High levels of progeny virus were produced when the transduced cells were challenged with an X4-tropic HIV-1 (NL4-3) strain, suggesting that Rz 1-7 expression does not affect X4-tropic virus replication. When the transduced cells expressing Rz 1-7 were challenged with the R5-tropic HIV-1 (BaL) strain, 99-100 % inhibition of progeny virus production was observed for the duration of the experiment (~2 months). When the cells were precultured for 2-3 months prior to HIV-1 infection, inhibition was more prominent in cells transduced with MGIN-Rz 1-7 than with HEG1-Rz 1-7 . Inhibition occurred at the level of viral entry, as no HIV-1 DNA could be detected. These results demonstrate that Rz 1-7 confers excellent inhibition of R5-tropic HIV-1 replication at the level of entry. Therefore, we anticipate that this multimeric ribozyme will be beneficial for HIV-1 gene therapy.
The effect of structure in a long target RNA on ribozyme cleavage efficiency
Nucleic Acids Research, 1997
Inhibition of gene expression by catalytic RNA (ribozymes) requires that ribozymes efficiently cleave specific sites within large target RNAs. However, the cleavage of long target RNAs by ribozymes is much less efficient than cleavage of short oligonucleotide substrates because of higher order structure in the long target RNA. To further study the effects of long target RNA structure on ribozyme cleavage efficiency, we determined the accessibility of seven hammerhead ribozyme cleavage sites in a target RNA that contained human immunodeficiency virus type 1 (HIV-1) vif-vpr. The base pairing-availability of individual nucleotides at each cleavage site was then assessed by chemical modification mapping. The ability of hammerhead ribozymes to cleave the long target RNA was most strongly correlated with the availability of nucleotides near the cleavage site for base pairing with the ribozyme. Moreover, the accessibility of the seven hammerhead ribozyme cleavage sites in the long target RNA varied by up to 400-fold but was directly determined by the availability of cleavage sites for base pairing with the ribozyme. It is therefore unlikely that steric interference affected hammerhead ribozyme cleavage. Chemical modification mapping of cleavage site structure may therefore provide a means to identify efficient hammerhead ribozyme cleavage sites in long target RNAs.