Numbering system for the hammerhead

Hertel, Klemens J.; Pardi, Arthur; Uhlenbeck, Oike C.; Koizumi, Makoto; Ohtsuka, Eiko; Uesugi, Seiichi; Cedergren, Robert; Eckstein, Fritz; Gerlach, W. L.; Hodgson, R. A. J.; Symons, Robert H.

doi:10.1093/nar/20.12.3252

Cited by 283 publications

(148 citation statements)

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“…The cleavage site in the target mRNA is indicated by an arrow; the GUC recognition triplet is underlined. The numbering of the hammerhead ribozyme is according to the system suggested by Hertel et al 36 For construction of a noncatalytic active control ribozyme, two point mutations were introduced in the sequence, G 5 !U and A 14 !C. The BCRP -encoding sequence and nt positions were derived from the BCRP cDNA sequence ( GenBank accession no.…”

Section: Confirmation Of Ribozyme Expressionmentioning

confidence: 99%

Modulation of the atypical multidrug-resistant phenotype by a hammerhead ribozyme directed against the ABC transporter BCRP/MXR/ABCG2

et al. 2002

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The phenomenon of multidrug resistance ( MDR ) in human cancers is one of the major causes of failure of chemotherapy. A recently identified new member of the superfamily of ATP -binding cassette transporters, breast cancer resistance protein ( BCRP ), was demonstrated to confer an atypical multidrug -resistant phenotype to tumor cells. To overcome the BCRP -mediated drug resistance, a specific anti -BCRP hammerhead ribozyme was introduced into the human gastric carcinoma cell line, EPG85 -257RNOV, exhibiting an atypical MDR phenotype. By this approach, the expression levels of the targeted BCRP -encoding mRNA and the BCRP transport protein were decreased to the low constitutive expression level that was observed in highly drug -sensitive parental gastric carcinoma cells. In addition, in the anti -BCRP ribozyme -treated cells, the cellular drug accumulation was dramatically increased to the level measured in drug -sensitive cells. These effects were accompanied by an extensive reversal of the drug -resistant phenotype of more than 80%. Because additional mechanisms contribute to the multimodal -mediated MDR phenotype exhibited by this gastric carcinoma cell line, the data suggest that the BCRP -mediated contingent to the drug resistance was overcome nearly completely. Moreover, the data indicate that ribozyme -based gene therapy may be clinically applicable in preventing and reversing BCRPmediated atypical MDR.

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Section: Confirmation Of Ribozyme Expressionmentioning

confidence: 99%

Modulation of the atypical multidrug-resistant phenotype by a hammerhead ribozyme directed against the ABC transporter BCRP/MXR/ABCG2

et al. 2002

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“…Thus, the self-cleaving reaction has been turned into a multiple turnover reaction, with separate oligonucleotides acting as the "ribozyme" and the "substrate" (Figure 1) (Sampson et al, 1987;Uhlenbeck, 1987;Haseloff & Gerlach, 1988;Koizumi et al, 1988 Koizumi et al, , 1989 Jeffries & Symons, 1989 p3WAACGUC>p; PI-G, GGGAACGUCG; P1-3'p, GGGAACGUC3'-p; P2, GUCGUCGC; P2-C, GUCGUCGCC; P2-p'zCp, GUCGUCGCp"Cp; S-C, GG-G AACGUCGUCGUCGCC; p3*S-C, p3WAACGUCGUCGUCGCC; S-p'zCp, GGGAACGUCGUCGUCGCpWp; S, GGGAACGUC-GUCGUCGC; p32S, p32GGGAACGUCGUCGUCGC; E, ribozyme; HH16, hammerhead cleavage motif comprised of separate ribozyme and substrate oligonucleotides ( (HH 16). Using standardized hammerhead nomenclature (Hertel et al, 1992), the ribozyme (E), comprised of 38 nucleotides, catalyzes thecleavageof a specific phosphodiester bond within its 1 8-nucleotidelong substrate (S-C), generating two 9-nucleotide-long products. P2-C binds to E to form helix I; P1 binds to E to form helix 111.…”

mentioning

confidence: 99%

“…Using standardized hammerhead nomenclature (Hertel et al, 1992), the ribozyme (E), comprised of 38 nucleotides, catalyzes thecleavageof a specific phosphodiester bond within its 1 8-nucleotidelong substrate (S-C), generating two 9-nucleotide-long products. P2-C binds to E to form helix I; P1 binds to E to form helix 111.…”

mentioning

confidence: 99%

A Kinetic and Thermodynamic Framework for the Hammerhead Ribozyme Reaction

1994

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A hammerhead ribozyme (HH16) with eight potential base pairs in each of the substrate recognition helices stabilized product binding sufficiently to enable investigation of the ligation of oligonucleotides bound to the ribozyme. All individual rate constants for product association and dissociation were determined. The following conclusions were obtained for HH16 from the analysis performed at 50 mM Tris, pH 7.5, 10 mM MgC12, and 25 "C.(1) HH16 cleaves bound substrate with a rate constant of k2 = 1 min-l, similar to rate constants obtained with other hammerhead ribozymes. (2) k-2, the rate of ligation of the 5' product and 3' product to form substrate, equaled 0.008 min-I, indicating an approximately 100-fold preference for the formation of products on the ribozyme. This internal equilibrium, compared with that for the overall solution reaction, gives an effective concentration (EC) of M for the two products bound to the ribozyme. This low EC suggests that upon cleavage of S the hammerhead complex acquires a "floppiness" which provides an entropic advantage for the formation of products on the ribozyme. (3) Product and substrate association rate constants were in the range of 107-108 M-1 min-l, comparable to values determined for short helices. (4) The stabilities of ribozyme/product complexes were similar to affinities predicted from helix-coil transitions of simple R N A duplexes, providing no indication of additional tertiary interactions. The products, P1 and P2, stabilize one another 4-fold on the ribozyme. ( 5 ) The dissociation constant for the binding of the substrate to the ribozyme was estimated to be about M.These results allowed the construction of a free energy profile for the reaction of HH16, and provide a basis for future mechanistic studies.Several plant viroids and virusoids autolytically cleave at a specific phosphodiester bond (Buzayan et al., 1986a,b;Hutchins et al., 1986; Prody et al., 1986). A consensus secondary structure of approximately 55 nucleotides termed the "hammerhead" domain has been identified (Buzayan et al., 1986b; Forster & Symons, 1987a,b) which can be assembled from 2 or 3 separate RNA molecules. Thus, the self-cleaving reaction has been turned into a multiple turnover reaction, with separate oligonucleotides acting as the "ribozyme" and the "substrate" (Figure 1) (Sampson et al., 1987;Uhlenbeck, 1987;Haseloff & Gerlach, 1988;Koizumi et al., 1988 Koizumi et al., , 1989 Jeffries & Symons, 1989 p3WAACGUC>p; PI-G, GGGAACGUCG; P1-3'p, GGGAACGUC3'-p; P2, GUCGUCGC; P2-C, GUCGUCGCC; P2-p'zCp, GUCGUCGCp"Cp; S-C, GG-G AACGUCGUCGUCGCC; p3*S-C, p3WAACGUCGUCGUCGCC; S-p'zCp, GGGAACGUCGUCGUCGCpWp; S, GGGAACGUC-GUCGUCGC; p32S, p32GGGAACGUCGUCGUCGC; E, ribozyme; HH16, hammerhead cleavage motif comprised of separate ribozyme and substrate oligonucleotides ( (HH 16). Using standardized hammerhead nomenclature (Hertel et al., 1992), the ribozyme (E), comprised of 38 nucleotides, catalyzes thecleavageof a specific phosphodiester bond within its 1 8-nucleotidelong substrate (S-C), ...

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“…Rz-MT2Ei has the same modifications as Rz-MT2E, but the substrate binding arms were inverted to prevent such binding. Asterisks indicate the phosphorothioates; numbering is according to Hertel et al 18 transfected with neo gene 13 were cultured in 1:1 Dulbecco's modified Eagle's medium:␣-minimal essential medium supplemented with 1.5 mg/mL G418 and 10% heat-inactivated (30 minutes at 56°C) fetal calf sera at 37°C in a humidified atmosphere containing 6% CO 2 . Under these conditions, CHO47 cells express 97,000 molecules of MGMT per cell, whereas CHO5 cells were devoid of any activity.…”

Section: Cell Line and Treatment Proceduresmentioning

confidence: 99%

Ribozyme and free alkylated base: a dual approach for sensitizing Mex+ cells to the alkylating antineoplastic drug

et al. 2000

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N-alkyl-nitrosoureas and alkyl-triazenes are alkylating antineoplastic drugs, the efficacy of which is strongly affected by the level of expression of the DNA-repair enzyme O 6 -methylguanine-DNA methyltransferase (MGMT). In tumors, MGMT activity reduces the chemotherapeutic potential of alkylating drugs; therefore, efforts have been made to down-regulate the protein. A partial sensitization of Mex ϩ cells to alkylating drugs has been obtained using either free alkylated bases or oligonucleotides targeted against MGMT mRNA. In the present work, O 6 -methylguanine and a chemically modified ribozyme, without a cationic liposome as a carrier, were coadministered to CHO47 cells, which express a high level of human MGMT protein. The reduction of MGMT mRNA and protein enhanced the genotoxicity of the alkylating drug mitozolomide. Furthermore, the sensitivity of CHO47 cells is the same as that of CHO5 cells, which lack MGMT protein. These data indicate that a strategy in which both mRNA and protein are degradation targets can be successfully applied to down-regulate the MGMT gene. Cancer Gene Therapy (2000) T he Mex ϩ phenotype defines a cell that expresses the O 6 -methylguanine (MeG)-DNA methyltransferase (MGMT) repair enzyme. The protein is able to repair DNA alkylation damage by transferring the alkyl group from the O 6 position of guanine to a catalytic cysteine residue in a stoichiometric reaction. 1 Because a high level of expression of the MGMT gene was observed in human tumors resistant to alkylating antineoplastic drugs such as N-alkyl-nitrosoureas and alkyl-triazenes, 2,3 down-regulation of the MGMT gene represents a possible strategy to bypass the resistance. A peculiarity of the repair protein is that each molecule that has reacted with either a free O 6 alkylated guanine or an alkylated base in DNA is inactivated and degraded. 4 -6 Therefore, pretreatment with an alkylated base such as benzylguanine or methylguanine provides a means to deplete MGMT protein and thus renders the tumor cells sensitive to lower doses of chemotherapeutic alkylating drugs. 7,8 We found, however, that the alkylating drug mitozolomide (MTZ) was less cytotoxic to Mex ϩ cells depleted of MGMT than to Mex Ϫ cells, which do not have MGMT activity. 9 This indicates that despite MGMT depletion, the cells retained the Mex ϩ phenotype. An alternative approach uses oligonucleotides such as antisense oligodeoxynucleotides 10 and hammerhead ribozymes 11 to cleave MGMT mRNA and potentiates the genotoxicity of MTZ while cells are still Mex ϩ . It seems that neither alkylated base nor oligonucleotides are independently adequate to sensitize Mex ϩ cells fully to alkylating drugs. Most likely, the very long half-life of MGMT mRNA 12 as well as the incomplete inactivation of the MGMT protein pool 9 influence the maintenance of the Mex ϩ phenotype. To test this hypothesis, we coadministered ribozyme and MeG to CHO47-transfected cell lines in which the expression of human MGMT and correlations with sister chromatid exchanges (SCEs) and other genet...

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Numbering system for the hammerhead

Cited by 283 publications

References 2 publications

Modulation of the atypical multidrug-resistant phenotype by a hammerhead ribozyme directed against the ABC transporter BCRP/MXR/ABCG2

Modulation of the atypical multidrug-resistant phenotype by a hammerhead ribozyme directed against the ABC transporter BCRP/MXR/ABCG2

A Kinetic and Thermodynamic Framework for the Hammerhead Ribozyme Reaction

Ribozyme and free alkylated base: a dual approach for sensitizing Mex+ cells to the alkylating antineoplastic drug

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