Non-enzymatic cleavage and ligation of RNAs complementary to a plant virus satellite RNA

Buzayan, Jamal M.; Gerlach, W. L.; Bruening, George

doi:10.1038/323349a0

Cited by 354 publications

(243 citation statements)

References 15 publications

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“…The internal equilibrium has been measured to be approximately 1 (Buzayan et al, 1986a;Chowrira et al, 1993;Feldstein & Bruening, 1993). Therefore, the effective concentration of the hairpin ribozyme is about 1 M, or about 100-fold higher than that for the hammerhead ribozyme.…”

Section: Discussionmentioning

confidence: 99%

“…However, the relatively low affinities of the products to the ribozymes used in that study prevented an analysis of the reverse reaction and the determination of individual product association and dissoci- ation rate constants. In contrast, other ribozymes such as the hairpin ribozyme or the group I intron from Tetrahymena thermophilia pre-rRNA have been observed to catalyze the reverse reaction (Buzayan et al, 1986a;Chowrira et al, 1993;Feldstein & Bruening, 1993;Woodson & Cech, 1989), and it was possible to determine product affinities for the Tetrahymena ribozyme (Herschlag & Cech, 1990;Pyle et al, 1990;Bevilacqua & Turner, 1991).…”

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confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

A Kinetic and Thermodynamic Framework for the Hammerhead Ribozyme Reaction

1994

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“…A new class of self-cleavage RNA has been found in satellite RNA of the tobacco ring spot virus (STobRV RNA) [1,2], avocado sunblotch viroid (ASBV) [3] and lucerne transient streak virusoid (VLTSV) [4]. Transcripts of satellite 2 DNA of the newt were also found to be cleaved at a consensus sequence similar to the one found in infectious RNA in plants [5].…”

Section: Introductionmentioning

confidence: 99%

Construction of a series of several self‐cleaving RNA duplexes using synthetic 21‐mers

1988

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Two fragments (21-mers) containing consensus sequences for the self-cleavage domain in transcripts of satellite DNA of the newt were chemically synthesized and found to be cleaved in the presence of M$+. The cleaved product contained the 3'-terminal 2',3'-cyclic phosphate. Twenty-five combinations of partially double-stranded 21-mer RNA which contained different bases within the consensus sequences and at the cleavage sites were tested for self-cleavage. It seemed that guanosine 3'-phosphate was not susceptible to transesterification at the cleavage site.

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“…The hammerhead-type ribozyme was originally discovered in satellite RNA of tobacco ringspot virus (Buzayan et al, 1986). Recent investigations reported that a hammerhead ribozyme targeting hTER reduced telomerase activity against tumor cells (Kanazawa et al, 1996;Yokoyama et al, 1998), although no change in proliferation rate was observed.…”

Section: Hammerhead Ribozymesmentioning

confidence: 99%

Telomerase as a therapeutic target for malignant gliomas

et al. 2002

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Telomerase, a ribonucleoprotein enzyme, is considered as a potential target of cancer therapy because of its preferential expression in tumors. In particular, malignant gliomas are one of the best candidates for telomerasetargeted therapy. It is because malignant gliomas are predominantly telomerase-positive, while normal brain tissues do not express telomerase. In theory, there are two telomerase-associated therapeutic approaches for telomerase-positive tumors. One approach is the antitelomerase cancer therapy to directly inhibit telomerase activity, resulting in apoptotic cell death or growth arrest. Two major components of the telomerase holoenzyme complex, the RNA template (hTER) and catalytic subunit (reverse transcriptase, hTERT) are well considered as therapeutic targets. The other approach is the telomerasespeci®c cancer therapy by targeting telomerase-expressing tumor cells as a means to directly kill the cells. Strategies using the transfer of therapeutic gene under the hTERT promoter system as well as immunotherapy directed against telomerase-positive cells are generally included. These telomerase-associated therapies are very promising for the treatment of malignant gliomas.

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Non-enzymatic cleavage and ligation of RNAs complementary to a plant virus satellite RNA

Cited by 354 publications

References 15 publications

A Kinetic and Thermodynamic Framework for the Hammerhead Ribozyme Reaction

A Kinetic and Thermodynamic Framework for the Hammerhead Ribozyme Reaction

Construction of a series of several self‐cleaving RNA duplexes using synthetic 21‐mers

Telomerase as a therapeutic target for malignant gliomas

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