The number of self-cleaving small ribozymes has increased sharply in recent years. Advances have been made in describing these ribozymes in terms of four catalytic strategies: α describes in-line attack, β describes neutralization of the nonbridging oxygens, γ describes activation of the nucleophile, and δ describes stabilization of the leaving group. Current literature presents the rapid self-cleavage of the twister ribozyme in terms of all four of these classic catalytic strategies. Herein, we describe the nonspecific contribution of small molecules to ribozyme catalysis. At biological pH, the rate of the wild-type twister ribozyme is enhanced up to 5-fold in the presence of moderate buffer concentrations, similar to the 3-5-fold effects reported previously for buffer catalysis for protein enzymes. We observe this catalytic enhancement not only with standard laboratory buffers, but also with diverse biological small molecules, including imidazole, amino acids, and amino sugars. Brønsted plots suggest that small molecules assist in proton transfer, most likely with δ catalysis. Cellular small molecules provide a simple way to overcome the limited functional diversity of RNA and have the potential to participate in the catalytic mechanisms of many ribozymes in vivo.

Reaction kinetics on the millisecond timescale pervade the protein and RNA fields. To study such reactions, investigators often perturb the system with abiological solution conditions or substrates in order to slow the rate to timescales accessible by hand-mixing; however, such perturbations can change the rate-limiting step and obscure key folding and chemical steps that are found under biological conditions. Mechanical methods for collecting data on the millisecond timescale, which allow these perturbations to be avoided, have been developed over the last few decades. These methods are relatively simple and can be conducted on affordable and commercially available instruments. Here, we focus on using the rapid quench-flow technique to study the fast reaction kinetics of RNA enzymes, or ribozymes, which often react on the millisecond timescale under biological conditions. Rapid quench of ribozymes is completely parallel to the familiar hand-mixing approach, including the use of radiolabeled RNAs and fractionation of reactions on polyacrylamide gels. We provide tips on addressing and preventing common problems that can arise with the rapid-quench technique. Guidance is also offered on ensuring the ribozyme is properly folded and fast-reacting. We hope that this article will facilitate the broader use of rapid-quench instrumentation to study fast-reacting ribozymes under biological reaction conditions.

The number of self-cleaving ribozymes has increased sharply in recent years, giving rise to elaborations of the four known ribozyme catalytic strategies, α, β, γ, and δ. One such extension is utilized by the twister ribozyme, which is hypothesized to conduct δ, or general acid catalysis, via N3 of the syn adenine +1 nucleobase indirectly via buffer catalysis at biological pH and directly at lower pH. Herein, we test the δ catalysis role of A1 via chemical rescue and the catalytic relevance of the syn orientation of the nucleobase by conformational analysis. Using inhibited twister ribozyme variants with A1(N3) deaza or A1 abasic modifications, we observe >100-fold chemical rescue effects in the presence of protonatable biological small molecules such as imidazole and histidine, similar to observed rescue values previously reported for C75U/C76Δ in the HDV ribozyme. Brønsted plots for the twister variants support a model in which small molecules rescue catalytic activity via a proton transfer mechanism, suggesting that A1 in the wild type is involved in proton transfer, most likely general acid catalysis. Additionally, through glycosidic conformational analysis in an appropriate background that accommodates the bromine atom, we observe that an 8BrA1-modified twister ribozyme is up to 10-fold faster than a nonmodified A1 ribozyme, supporting crystallographic data that show that A1 is syn when conducting proton transfer. Overall, this study provides functional evidence that the nucleotide immediately downstream of the cleavage site participates directly or indirectly in general acid−base catalysis in the twister ribozyme while occupying the syn conformation.

Gel electrophoresis and subsequent imaging using phosphorimagers is one of the most important and widely used techniques in RNA and DNA analysis. Radiolabeling nucleic acids with P and detecting bands using a phoshorimager are useful both in a qualitative sense for nucleic acid detection and in a quantitative sense for structural, kinetic, or binding-based assays. Because of this, good resolution of gel bands based on molecular weight and size of RNA or DNA is essential for analysis. The appearance of blurry gel bands ofP-labeled RNA and DNA thus represents a serious problem in the laboratory. A quick search on the Internet uncovers numerous reports begrudging the appearance of blurry bands, as well as attempts to fix them without success. Indeed, our laboratories were beset by the intermittent problem of blurry gels for over one year before we found a solution. Herein we describe a simple and cost-effective solution to a problem that we show originates from the phosphorimager cassettes rather than the integrity of the gel itself. We hope that the information provided here will lead to immediate help for other laboratories experiencing similar issues with labeled nucleic acid gel-based assays. The improvement in the clarity of the gels is nothing short of astonishing in many instances and will lead to higher resolution images for analysis and publications.

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