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Volumn 279, Issue 5347, 1998, Pages 81-84

Inhibition of the hammerhead ribozyme cleavage reaction by site-specific binding of Tb(III)

Author keywords

[No Author keywords available]

Indexed keywords

RIBOZYME; RNA;

EID: 0032472205     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.279.5347.81     Document Type: Article
Times cited : (122)

References (45)
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    • note
    • 2 to the appropriate final concentration. When Tb(III) was present, it was added concomitantly with the Mg(II). Reactions were quenched by diluting a 10-μl portion of the reaction mixture into 20 μl of stop buffer (100 mM EDTA, 7 Murea, 1 × tris-borate-EDTA, 0.1% bromophenolblue, 0.1% xylene cyanol, 0.1% orange G). Reaction products were analyzed by electrophoresis on denaturing 20% polyacrylamide gels and quantitated with a Molecular Dynamics Phosphorimager and ImageQuant software. Pseudo-first order rate constants were calculated with Kaleidagraph (Synergy Software) from nonlinear least-squares fitting of plots of the fraction product [product/(product + substrate)] versus time. For extremely slow reactions, initial rates derived from linear fits of the same plots were used to estimate the rate constants. Protracted reactions (>4 to 6 hours) in the presence of Tb(III) were problematic because of nonspecific degradation of the RNA under the conditions used for kinetic analysis (22). Standard 1-hour time courses showed no substantial degradation. Reactions were usually performed in triplicate and the data averaged to give the observed rate constant. Errors are reported as standard deviations of the mean from replicate experiments.
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    • note
    • 2 was 0.9 ± 0.2 μM.
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    • 2′-Acetamido modification of RNA has been previously described [C. Hendrix et al., Biochem. Biophys. Res. Commun. 210, 67 (1995); C. Hendrix et al., Nucleic Acids Res. 23, 51 (1995)]; however, we adopted a postsynthetic approach in which a 2′-amino substrate RNA was treated with 25 mM acetic acid and 25 mM EDC in 200 mM MES buffer (pH 6.5) for 30 min at 37°C. An additional 10 μl of 250 mM EDC was then added to the 100-μl reaction mixture and allowed to react for 30 min. The reaction product was isolated by precipitation with ethanol and examined by matrix-assisted laser desorption/ionization-time-of-flight mass spectroscopy, with 3-hydroxypicolinic acid as a matrix. On the basis of this analysis, the reaction was >90% complete, with no residual starting material evident in the spectrum.
    • (1995) Biochem. Biophys. Res. Commun. , vol.210 , pp. 67
    • Hendrix, C.1
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    • 2′-Acetamido modification of RNA has been previously described [C. Hendrix et al., Biochem. Biophys. Res. Commun. 210, 67 (1995); C. Hendrix et al., Nucleic Acids Res. 23, 51 (1995)]; however, we adopted a postsynthetic approach in which a 2′-amino substrate RNA was treated with 25 mM acetic acid and 25 mM EDC in 200 mM MES buffer (pH 6.5) for 30 min at 37°C. An additional 10 μl of 250 mM EDC was then added to the 100-μl reaction mixture and allowed to react for 30 min. The reaction product was isolated by precipitation with ethanol and examined by matrix-assisted laser desorption/ionization-time-of-flight mass spectroscopy, with 3-hydroxypicolinic acid as a matrix. On the basis of this analysis, the reaction was >90% complete, with no residual starting material evident in the spectrum.
    • (1995) Nucleic Acids Res. , vol.23 , pp. 51
    • Hendrix, C.1
  • 38
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    • note
    • Supported by NSF (CHE-9504698 to A.L.F.) and NIH (GM-36944 to O.C.U.). We thank L. Behlen and J. Murray for preparing several of the synthetic oligonucleotides; E. Jabri, S. Cohen, and A. Klug for discussions; S. Price for help with data collection at Daresbury Laboratory; and W. Pieken for the 2′-amino cytosine phosphoramidite. Coordinates from the Tb(III) cocrystallization experiment have been deposited in the Nucleic Acids Database (NDB ID URX067).


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