Chemical and enzymatic probing of effector-mediated changes in the conformation of a maxizyme

Zhou, Jing; Nakamatsu, Yuka; Kuwabara, Tomoko; Warashina, Masaki; Tanaka, Yoshiyuki; Yoshinari, Koichi; Taira, Kazunari

doi:10.1016/s0162-0134(00)00005-2

Cited by 9 publications

(8 citation statements)

References 29 publications

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“…The rAAV vector backbone contained the intronic sequence derived from the chicken β-actin (CBA) gene placed between the CBA promoter and the ribozyme cloning site. The inclusion of the intron at this position stimulates transport of the ribozyme RNA to the cytoplasm (Zhou et al, 2000). An internal self-cleaving hairpin ribozyme was located downstream of the ribozyme cloning site.…”

Section: Generation Of Recombinant Raav Vectors Expressing Hd6 and Hdmentioning

confidence: 99%

Unexpected off-targeting effects of anti-huntingtin ribozymes and siRNA in vivo

Denovan‐Wright

Rodríguez-Lebrón

Lewin

et al. 2008

Neurobiology of Disease

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Gene transfer strategies to reduce levels of mutant huntingtin (mHtt) mRNA and protein by targeting human Htt have shown therapeutic promise in vivo. Previously, we have reported that a specific, adeno-associated viral vector (rAAV)-delivered short-hairpin RNA (siHUNT-2) targeting human Htt mRNA unexpectedly decreased levels of striatal-specific transcripts in both wild-type and R6/1 transgenic HD mice. The goal of this study was to determine whether the siHUNT-2-mediated effect was due to adverse effects of RNA interference (RNAi) expression in the brain. To this end, we designed two catalytically active hammerhead ribozymes directed against the same region of human Htt mRNA targeted by siHUNT-2 and delivered them to wild-type and R6/1 transgenic HD mice. After 10 weeks of continuous expression, these ribozymes, like siHUNT-2, negatively impacted the expression of a subset of genes in the striatum. This effect was independent of rAAV transduction and specific to the targeting of a unique sequence in human Htt mRNA. After consideration of the known potential RNAi-specific toxic mechanisms, only cleavage of an unintended RNA target can account for the data reported herein. Thus, long-term rAAV-mediated RNAi in the brain does not, in and of itself, negatively affect striatal gene expression. These findings have important implications in the development of therapeutic RNAi for the treatment of neurological disease.

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Section: Generation Of Recombinant Raav Vectors Expressing Hd6 and Hdmentioning

confidence: 99%

Unexpected off-targeting effects of anti-huntingtin ribozymes and siRNA in vivo

Denovan‐Wright

Rodríguez-Lebrón

Lewin

et al. 2008

Neurobiology of Disease

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“…It is well known that, at high pH, all phosphodiester bonds in RNAs are cleaved rapidly because of intramolecular anchimeric attack by 2‘-OH groups. We previously demonstrated that, in weakly alkaline solution, rates of hydrolysis of phosphodiester bonds in the duplex region were significantly lower, because of the rigid conformation, than corresponding rates in single-stranded regions, and thus this property was used to probe the secondary structure of RNAs . In view of the probable advantages of the chemical probing of functional RNAs by simple weak-alkaline hydrolysis, we performed such analysis using our maxizyme complex that consisted of four strands of RNA 8d…”

Section: Resultsmentioning

confidence: 99%

“…Probing of the Secondary Structures of Natural and Mutant Ribozymes. To evaluate the structures of Rz, we used a weakly alkaline solution, in which phosphodiester bonds in single-stranded regions were more susceptible to alkaline attack than those within double-stranded regions. , The reaction mixture for hydrolysis contained 50 mM Na 2 CO 3 /NaHCO 3 (pH 9.2), 25 mM MgCl 2 , and 5‘- 32 P-labeled Rz (10 kcpm). The 5‘- 32 P-labeled Rz was first heated to 95 °C for 2.5 min, slowly cooled to room temperature, and allowed equilibrate for 30 min to avoid formation of local minimum-energy structures.…”

Section: Methodsmentioning

confidence: 99%

Analysis of the Conserved P9-G10.1 Metal-Binding Motif in Hammerhead Ribozymes with an Extra Nucleotide Inserted between A9 and G10.1 Residues

et al. 2004

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Hammerhead ribozymes (Rz) have catalytically important tandem G:A pairs in the core region, and we recently demonstrated that the P9-G10.1 motif (a sheared-type G:A pair with a guanine residue on the 3' side of the adenine residue) with several flanking base pairs is sufficient for capture of divalent cations, such as Mg(2+) and Cd(2+) ions that are important to maintain full activities (Tanaka et al. J. Am. Chem. Soc. 2002, 124, 4595-4601; Tanaka et al. J. Am. Chem. Soc. 2004, 126, 744-752). We also found that mutant hammerhead ribozymes that have an additional G residue inserted between A9 and G10.1 residues (the metal-binding P9-G10.1 motif) have significant catalytic activities. In this study, we demonstrate that the hammerhead ribozymes are capable of maintaining the catalytically competent structure even when the tandem, sheared-type G:A pairs were perturbed by an insertion of an additional nucleotide, whereas the chirality of the phosphorothioate at the P9 position significantly influenced the enzymatic activity for both the natural and G-inserted ribozymes.

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“…Kinetic and NMR analyses indicated that forms generated in vitro, the cleavage activities of hetthe shortened ribozyme was essentially inactive as a monoerodimeric maxizymes examined previously were lower mer but exhibited strong catalytic activity as a dimer (Fig. than those of parental hammerhead ribozymes and of the IB) (5,34). We also designed a heterodimer composed of homodimeric maxizyme (35)(36)(37)(38)(39)(40)(41)(42)(43). Nonetheless, the intraceltwo different monomers, designated maxizyme left (MzL) lular activities of heterodimeric maxizymes were as high as and maxizyme right (MzR) (Fig.…”

mentioning

confidence: 97%

“…zyme, we developed the maxizyme, which functions as a 1C) (37)(38)(39). Because of the mixed populations of dimeric dimer (5,(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43). Kinetic and NMR analyses indicated that forms generated in vitro, the cleavage activities of hetthe shortened ribozyme was essentially inactive as a monoerodimeric maxizymes examined previously were lower mer but exhibited strong catalytic activity as a dimer (Fig. than those of parental hammerhead ribozymes and of the IB) (5,34).…”

mentioning

confidence: 99%

Cleavage of an Inaccessible Site by the Maxizyme with Two Independent Binding Arms: An Alternative Approach to the Recruitment of RNA Helicases

Kuwabara

Warashina

Taira

2002

Journal of Biochemistry

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To overcome obstacles to target site selection, we recently created a novel hybrid ribozyme that could access any chosen site by the recruitment of intracellular RNA helicases [Warashina et al. (2001) Proc. Natl. Acad. Sci. USA 98, 5572-5577; Kawasaki et al. (2002) Nat. Biotech. 20, 376-380]. We also demonstrated previously that pol III-driven maxizymes with two substrate-binding arms that were directed against two different sites within a target mRNA formed very active heterodimers in vivo [Kuwabara, et al. (2000) Trends Biotechnol. 18, 462-468; Tanabe et al. (2001) Nature 406, 473-474]. Despite the complicated dimerization process, all the maxizymes that we tested in cultured cells had greater catalytic activity than the parental ribozymes. To investigate the action of maxizymes in cells, we designed a specific maxizyme with two substrate-binding arms that was directed against endogenously expressed LTR-luciferase chimeric mRNA, where LTR refers to the long terminal repeat of HIV-1. One substrate-binding arm of the maxizyme was designed to bind to a site within HIV-1 TAR RNA that is known to form a stable stem structure that normally prevents binding of a ribozyme. The other substrate-binding arm was directed against a relatively accessible site within the luciferase gene. As expected, the conventional ribozyme failed to cleave the TAR region in vivo because of the latter's stable secondary structure. However, to our surprise, the maxizyme cleaved the TAR region within the stem with high efficiency in vivo. The enhanced cleavage in vivo by the maxizyme might have resulted from an entropically favorable, intramolecular, second binding process that occurred during the breathing of the stem structure of the target mRNA. Importantly, our data suggest that this maxizyme technology might be used as an alternative approach to the recruitment of RNA helicases in cleaving sites previously found to be inaccessible.

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Chemical and enzymatic probing of effector-mediated changes in the conformation of a maxizyme

Cited by 9 publications

References 29 publications

Unexpected off-targeting effects of anti-huntingtin ribozymes and siRNA in vivo

Unexpected off-targeting effects of anti-huntingtin ribozymes and siRNA in vivo

Analysis of the Conserved P9-G10.1 Metal-Binding Motif in Hammerhead Ribozymes with an Extra Nucleotide Inserted between A9 and G10.1 Residues

Cleavage of an Inaccessible Site by the Maxizyme with Two Independent Binding Arms: An Alternative Approach to the Recruitment of RNA Helicases

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