Comparative analysis of cleavage rates after systematic permutation of the NUX↓ consensus target motif for hammerhead ribozymes

Zoumadakis, Michalis; Tabler, Martin

doi:10.1093/nar/23.7.1192

Cited by 89 publications

(55 citation statements)

References 42 publications

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“…[35][36][37] However, as stem III is only three base pairs in length, any modification in this domain could cause dramatic structural change which could impact the catalytic activity of the ribozyme. Our work reveals that the loss of catalytic activity of the ST-HHRz to a RNA substrate with a GUG cleaving site was caused by a shift of stem III.…”

Section: Discussionmentioning

confidence: 99%

Re-characterization of hammerhead ribozymes as molecular tools for intermolecular RNA cleavage

Liu

Huang

et al. 2017

Org. Biomol. Chem.

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Section: Discussionmentioning

confidence: 99%

Re-characterization of hammerhead ribozymes as molecular tools for intermolecular RNA cleavage

Liu

Huang

et al. 2017

Org. Biomol. Chem.

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“…These molecules were subsequently subjected to controlled kinetic analyses. As an internal size marker, we used a G17 variant of the HHRz motif that is strongly impaired in its cleavage reaction, as numerous previous analyses of minimal HHRzs have shown (Ruffner et al 1990;Shimayama et al 1995;Zoumadakis and Tabler 1995;Kore et al 1998Kore et al , 2000Przybilski and Hammann 2007b;Nelson and Uhlenbeck 2008b;Shepotinovskaya and Uhlenbeck 2010). To our surprise, the G17 variant of the X. tropicalis motif Xetr5 (Seehafer et al 2011) exhibited significant self-cleavage activity under the conditions of in vitro transcription (Fig.…”

Section: Substantial Cleavage Activity Of Hammerhead Ribozymes With Amentioning

confidence: 95%

“…One of the most conserved positions in the HHRz core is H17, as documented in a high number of studies (Ruffner et al 1990;Shimayama et al 1995;Zoumadakis and Tabler 1995;Kore et al 1998Kore et al , 2000Przybilski and Hammann 2007b;Nelson and Uhlenbeck 2008b;Shepotinovskaya and Uhlenbeck 2010), leading to the establishment of the NUH rule for the HHRz cleavage site. As reason for the dramatically reduced functionality of HHRzs with a guanosine at position 17, the formation of a Watson-Crick base pair with C3 was presumed ( Fig.…”

Section: Introductionmentioning

confidence: 99%

G17-modified hammerhead ribozymes are active in vitro and in vivo

Kalweit

Hammann

2013

RNA

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Natural hammerhead ribozymes (HHRz) feature tertiary interactions between hairpin loops or bulges in two of three helices that surround the catalytic core of conserved nucleotides. Their conservation was originally established on minimal versions lacking the tertiary interactions. While those sequence requirements in general also apply to natural versions, we show here differences for the HHRz cleavage site N17. A guanosine at this position strongly impairs cleavage activity in minimal versions, whereas we observe for the G17 variants of four tertiary stabilized HHRz significant cleavage and ligation activity in vitro. Kinetic analyses of these variants revealed a reduced rate and extent of cleavage, compared with wild-type sequences, while variants with distorted tertiary interactions cleaved at a reduced rate, but to the same extent. Contrary to this, G17 variants exhibit similar in vitro ligation activity as compared with the respective wild-type motif. To also address the catalytic performance of these motifs in vivo, we have inserted HHRz cassettes in the lacZ gene and tested this β-galactosidase reporter in Dictyostelium discoideum. In colorimetric assays, we observe differences in the enzymatic activity of β-galactosidase, which correlate well with the activity of the different HHRz variants in vitro and which can be unambiguously attributed to ribozyme cleavage by primer extension analysis.

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“…Among the different sequences fulfilling the required NUX cleavage motif for hammerhead ribozymes GUC appears to be the optimal triplet (20). In vitro the efficacy of cleavage of the GUC motif is generally higher compared with the cleavage efficiency of the GUU motif (5,12,21). Differences in cleavage activity of ribozymes can also be related to different flanking sequences and/or different secondary structures of the target RNAs (17,22).…”

Section: Single Cells Colonies Colonies/ (Single Cells + Colonies) (%)mentioning

confidence: 99%

Enzymatic and antisense effects of a specific anti-Ki-ras ribozyme in vitro and in cell culture

Giannini¹,

Roth

Piiper³

et al. 1999

Nucleic Acids Research

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Due to their mode of action, ribozymes show antisense effects in addition to their specific cleavage activity. In the present study we investigated whether a hammerhead ribozyme is capable of cleaving mutated Ki-ras mRNA in a pancreatic carcinoma cell line and whether antisense effects contribute to the activity of the ribozyme. A 2'-O-allyl modified hammerhead ribozyme was designed to cleave specifically the mutated form of the Ki-ras mRNA (GUU motif in codon 12). The activity was monitored by RT-PCR on Ki-ras RNA expression by determination of the relative amount of wild type to mutant Ki-ras mRNA, by 5-bromo-2'-deoxy-uridine incorporation on cell proliferation and by colony formation in soft agar on malignancy in the human pancreatic adenocarcinoma cell line CFPAC-1, which is heterozygous for the Ki-ras mutation. A catalytically inactive ribozyme was used as control to differentiate between antisense and cleavage activity and a ribozyme with random guide sequences as negative control. The catalytically active anti-Ki-ras ribozyme was at least 2-fold more potent in decreasing cellular Ki-ras mRNA levels, inhibiting cell proliferation and colony formation in soft agar than the catalytically inactive ribozyme. The catalytically active anti-Ki-ras ribozyme, but not the catalytically inactive or random ribozyme, increased the ratio of wild type to mutated Ki-ras mRNA in CFPAC-1 cells. In conclusion, both cleavage activity and antisense effects contribute to the activity of the catalytically active anti-Ki-ras hammerhead ribozyme. Specific ribozymes might be useful in the treatment of pancreatic carcinomas containing an oncogenic GTT mutation in codon 12 of the Ki-ras gene.

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Comparative analysis of cleavage rates after systematic permutation of the NUX↓ consensus target motif for hammerhead ribozymes

Cited by 89 publications

References 42 publications

Re-characterization of hammerhead ribozymes as molecular tools for intermolecular RNA cleavage

Re-characterization of hammerhead ribozymes as molecular tools for intermolecular RNA cleavage

G17-modified hammerhead ribozymes are active in vitro and in vivo

Enzymatic and antisense effects of a specific anti-Ki-ras ribozyme in vitro and in cell culture

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