Recent developments in the hammerhead ribozyme field

Vaish, Narendra K.; Kore, Anilkumar R.; Eckstein, Fritz

doi:10.1093/nar/26.23.5237

Cited by 78 publications

(38 citation statements)

References 70 publications

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“…The modified nucleotide is not among the conserved core of nucleotides. 8,9 It is known that any base pair can be accommodated at the modification positions. 9 Since 2AP forms Watson-Crick-type hydrogen bonds with uridine resulting in a base pair that is isostructural to A=U, a negligible structural perturbation is anticipated (Fig.…”

Section: Designmentioning

confidence: 99%

2-Aminopurine as a real-time probe of enzymatic cleavage and inhibition of hammerhead ribozymes

Kirk

2001

Bioorganic &Amp; Medicinal Chemistry

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Abstract-The design, synthesis and study of internally fluorescent hammerhead (HH) ribozymes, where changes in fluorescence parameters directly reflect the progress of the ribozyme's cleavage chemistry, are described. The approach relies on a HH substrate modified at position 1.1, proximal to the cleavage site, with 2-aminopurine (2AP), an intensely fluorescent adenosine isoster. The incorporation of 2AP, an unnatural nucleoside, does not interfere with the ribozyme folding and catalysis. Since 2AP is highly sensitive to environmental changes, its fluorescence is dramatically altered upon ribozyme-mediated cleavage of the substrate. This generates a measurable signal that directly reflects the progress of the ribozyme's reaction in real time. Identical pseudo first order rate constants are obtained for HH constructs using both continuous fluorescence monitoring and radioactive labeling. This rapid and real-time monitoring facilitates the study of ribozyme activity under different conditions (e.g., ionic strength, pH, etc.), and provides a useful assay to rapidly screen potential inhibitors. Three hitherto unknown HH inhibitors are presented and compared to neomycin B and chlortetracycline, two previously studied HH inhibitors. All three new small molecules, neo-acridine, guanidinoneomycin B, and [Á-(Eilatin)Ru(bpy) 2 ] 2+ , prove to be better inhibitors than neomycin B or chlortetracycline. Investigating HH inhibition under different ionic strengths reveals that the binding of neo-acridine, [Á-(Eilatin)Ru(bpy) 2 ] 2+ , and chlortetracycline to the HH involves hydrophobic interactions as their RNA affinities are largely unaffected by increasing salt concentrations. In contrast, neomycin B loses more than 50-fold of its inhibitory ability as the NaCl concentration is increased from 50 to 500 mM. #

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Section: Designmentioning

confidence: 99%

2-Aminopurine as a real-time probe of enzymatic cleavage and inhibition of hammerhead ribozymes

Kirk

2001

Bioorganic &Amp; Medicinal Chemistry

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“…9 One method that has been used to protect the ribozymes from intracellular degradation is to connect them to a vector RNA such as tRNA. 20 Nevertheless, new approaches in using RNA as a vector to escort and chaperone ribozymes in vivo should be further investigated in order to improve this technique, which has shown great potential for improving human health and agricultural productivity.…”

Section: Introductionmentioning

confidence: 99%

Bacterial virus phi29 pRNA as a hammerhead ribozyme escort to destroy hepatitis B virus

et al. 2003

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The DNA-packaging pRNA of bacterial virus phi29, which forms dimers and then hexamers, contains two independent tightly self-folded domains. Circularly permuted pRNAs were constructed without impacting pRNA folding. Connecting the pRNA 5 0 /3 0 ends with variable sequences did not disturb its folding and function. These unique features, which help prevent two common problems -exonuclease degradation and misfolding in the cell, make pRNA an ideal vector to carry therapeutic RNAs. A pRNA-based vector was designed to carry hammerhead ribozymes that cleave the hepatitis B virus (HBV) polyA signal. The chimeric HBV-targeting ribozyme was connected to the pRNA 5 0 /3 0 ends as circularly permuted pRNA. Two cis-cleaving ribozymes were used to flank and process the chimeric ribozyme. The hammerhead ribozyme including its two arms for HBV targeting was able to fold correctly while escorted by the pRNA. The chimeric ribozyme cleaved the polyA signal of HBV mRNA in vitro almost completely. Cell culture studies showed that the chimeric ribozyme was able to enhance the inhibition of HBV replication when compared with the ribozyme not escorted by pRNA, as demonstrated by Northern blot and e-antigen assays. pRNA could also carry another hammerhead ribozyme to cleave other RNA substrate. These findings suggest that pRNA can be used as a vector for imparting stability to ribozymes, antisense, and other therapeutic RNA molecules in vivo.

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“…In the present study, we designed and synthesized hammerhead ribozymes against caspase-12 mRNA under the control of a U6 snRNA promoter. The U6 SnRNA Pol III transcripts were normally retained in the nucleus and expressed at high levels, transcripts were protected at their 3'-ends and Rev-binding RNAs and had certain advantages (49). It is a strong promoter for the expression of small RNAs in vivo.…”

Section: Discussionmentioning

confidence: 99%

Ribozyme-mediated inhibition of caspase-12 activity reduces apoptosis induced by endoplasmic reticulum stress in primary mouse hepatocytes

Jiang

Xie²,

Zhou³

et al. 1998

Int J Mol Med

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Abstract. Apoptosis via endoplasmic reticulum (ER) stress has been reported in many cell lines. ER stress plays an important role in many liver diseases and caspase-12 is the central player in ER stress-induced apoptosis. We conducted an investigation to determine whether catalytic cleavage of caspase-12 mRNA by hammerhead ribozymes can protect liver cells from apoptosis induced by ER stress. Thapsigargin (TG) was used to induce primary mouse hepatocytes apoptosis to establish the experimental system of ER stressmediated apoptosis. The effective ribozyme-Rz138 selected in vitro was embedded in eukaryotic expression vector and transfected into cultured cells. The activity of Rz138 in primary mouse hepatocytes was determined by testing the expression of caspase-12 mRNA and procaspase-12 protein in ribozyme treated cells compared with the control. The anti-apoptotic effect was assayed by the nuclear morphological features of primary mouse hepatocytes stained with Hoechst 33258 under the fluorescence microscope. Primary mouse hepatocytes were incubated with 4 μmol/l TG, the percentage of apoptotic cells increased along with the treatment time, obvious apoptosis was observed after 4 μmol/l TG treatment for 30 h. Expression of caspase-12 mRNA and procaspase-12 protein were decreased significantly in hepatocytes transfected with pRz138 compared with those untransfected. The percentage of apoptotic cells was also decreased in pRz138 treated cells measured by staining with Hoechst 33258. Rz138, as a specific inhibitor of caspase-12 can down-regulate the expression of caspase-12 in primary mouse hepatocytes and protect the cells from apoptosis induced by TG. These results further elucidated the new treatment for diseases associated with ER stress-mediated apoptosis.

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Recent developments in the hammerhead ribozyme field

Cited by 78 publications

References 70 publications

2-Aminopurine as a real-time probe of enzymatic cleavage and inhibition of hammerhead ribozymes

2-Aminopurine as a real-time probe of enzymatic cleavage and inhibition of hammerhead ribozymes

Bacterial virus phi29 pRNA as a hammerhead ribozyme escort to destroy hepatitis B virus

Ribozyme-mediated inhibition of caspase-12 activity reduces apoptosis induced by endoplasmic reticulum stress in primary mouse hepatocytes

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