Mg2+-independent hairpin ribozyme catalysis in hydrated RNA films

The hammerhead cleavage reaction in monovalent cations

“…where F is the maximum fraction reacted (typically 0+9), k is the observed rate constant, and t is time+ All rates were measured at least twice, and independent determinations differed by less than 15% for catalyzed rates, and less than twofold for nonenzymatic cleavage rates+ To confirm that accurate rates could be determined using our modified protocol, some rates were also determined using a standard annealing protocol (Stage-Zimmermann & Uhlenbeck, 1998)+ The two protocols yielded indistinguishable values, and these values were similar to those previously reported (Clouet-d'Orval & Uhlenbeck, 1997;Murray et al+, 1998)+ One difference between our modified protocol and the standard protocol is that, when using the modified protocol, a small amount of cleavage (about 5%) is typically observed during the evaporation+ Such cleavage has been observed by others (Seyhan & Burke, 2000), and, as mentioned above, we confirmed that it had no effect on observed rates+ We also note that reactions in 4 M salt were not effectively terminated when 10 vol of stop solution were used to quench the reaction+ A primary role of the stop solution was to reduce the concentration of monovalent salt by dilution, because urea does not effectively denature the hammerhead in the presence of high concentrations of monovalent salt+ For example, in 4 M Li ϩ the reaction rate is reduced only about 40-fold in 8 M urea, whereas in 10 mM Mg 2ϩ the reaction rate is reduced about 200-fold in 2 M urea, and about 60,000-fold in 8 M urea (Fig+ 3)+ Consequently, we stopped reactions by diluting in 20 vol of stop solution and freezing in dry ice+ Background rates of RNA cleavage were measured in the same way as ribozyme-catalyzed rates, but in the absence of 1 The hypothesis that the N 1 nitrogen of G 5 acts as a base in the reaction is consistent with a recent crystal structure, in which the keto oxygen of G 5 is positioned 3 Å away from the 29-OH at the cleavage site, suggesting that it, or a nearby functional group, could abstract a proton from this -OH (Murray et al+, 2000)+ Furthermore, replacement of G 5 with 1-methylguanosine reduces the hammerhead cleavage rate to background levels (Limauro et al+, 1994), and binding of Tb 3ϩ to the Watson-Crick face of G 5 similarly inhibits hammerhead activity (Feig et al+, 1998)+ To test this idea, we examined the activity of a hammerhead in which the guanosine at G 5 (pK a ϭ 9+4) was replaced by 7-methylguanosine (pK a ϭ 6+7; Hendler et al+, 1970)+ If the N 1 nitrogen of G 5 acts as a base in the reaction, its lowered pK a with the 7-methyl substitution might lead to a faster rate+ However, at pH 6+0, substitution at G 5 was inhibitory, although at G 8 this substitution increased the hammerhead cleavage rate fivefold (data not shown)+ Inhibition at G 5 is likely due to either the positive charge introduced at N 7 or to the methyl group at N 7 , so this result is inconclusive+ But because N 7 appears to be one of the few positions of G 5 that can be modified without loss of function (Fu et al+, 1993;McKay 1996), perhaps other substitutions at this position could better address our hypothesis+ enzyme-strand RNA+ A ladder of cleavage products was observed when time points were run on denaturing polyacrylamide gels, and rate constants were calculated for the hammerhead cleavage site as well as eight neighboring phosphodiester linkages+ For each metal, cleavage rates at different linkages systematically var...…”

An efficient method for long-term room temperature storage of RNA

“…As expected, dehydration of RNA strongly inhibits its degradation. 13 However, partial rehydration by atmospheric water restores the initial instability while still in the solid state. 13 Another characteristic of the reaction is that it is highly dependent on the geometry of the molecule.…”

“…13 However, partial rehydration by atmospheric water restores the initial instability while still in the solid state. 13 Another characteristic of the reaction is that it is highly dependent on the geometry of the molecule. Indeed, in the transition state, the 2 0 oxygen, the phosphorus and a negatively charged oxygen of the phosphate group must be 'in line' .…”

Computed Energetics of Nucleotides in Spatial Ribozyme Structures: An Accurate Identification of Functional Regions from Structure

“…In other words, results produced by the implicit solvent model and the explicit solvent and counterions are similar in terms of the identified nucleic bases, as it is illustrated by the example of the hammerhead considered above. The role of water in molecular events and enzymatic active sites in a low-water environment is not well understood [49]. However, it is interesting to notice that hairpin ribozyme (and, to some extent, hammerhead ribozyme) catalyzes RNA cleavage in partially hydrated RNA films [49].…”

“…The role of water in molecular events and enzymatic active sites in a low-water environment is not well understood [49]. However, it is interesting to notice that hairpin ribozyme (and, to some extent, hammerhead ribozyme) catalyzes RNA cleavage in partially hydrated RNA films [49]. Catalysis is minimal but still occurs under conditions of extreme dehydration; mutations of the known catalytic bases of ribozyme abolish catalysis in the dehydrated RNA films.…”