A Faster Triphosphorylation Ribozyme

Abstract

In support of the RNA world hypothesis, previous studies identified trimetaphosphate (Tmp) as a plausible energy source for RNA world organisms. In one of these studies, catalytic RNAs (ribozymes) that catalyze the triphosphorylation of RNA 5'-hydroxyl groups using Tmp were obtained by in vitro selection. One ribozyme (TPR1) was analyzed in more detail. TPR1 catalyzes the triphosphorylation reaction to a rate of 0.013 min-1 under selection conditions (50 mM Tmp, 100 mM MgCl2, 22°C). To identify a triphosphorylation ribozyme that catalyzes faster triphosphorylation, and possibly learn about its secondary structure TPR1 was subjected to a doped selection. The resulting ribozyme, TPR1e, contains seven mutations relative to TPR1, displays a previously unidentified duplex that constrains the ribozyme's structure, and reacts at a 24-fold faster rate than the parent ribozyme. Under optimal conditions (150 mM Tmp, 650 mM MgCl2, 40°C), the triphosphorylation rate of TRP1e reaches 6.8 min-1.

Fig 1. Proposed secondary structures for (A) the parent ribozyme, TPR1, and (B) the most active ribozyme identified in this study, TPR1e.

TPR1e contains seven beneficial mutations relative to TPR1 (mutations are in the figure).

Fig 2. Triphosphorylation kinetics of central ribozyme variants in this study.

The starting point of the doped selection was the ribozyme TPR1 (empty circles). It has a k_obs of 0.013 min^-1 under selection conditions (100 mM MgCl₂, 50 mM trimetaphosphate, 50 mM Tris/HCl pH 8.3). The most efficient isolate from the doped selection was a 16-mutation variant called clone 11 (filled triangles, k_obs of 0.21 min^-1). After removal of unnecessary mutations a 5-mutation variant called TPR1-II resulted (open squares, k_obs of 0.25 min^-1). Two mutations that arose independently were introduced to yield TPR1e (filled squares), a 7-mutation variant with a k_obs of 0.31 min^-1. Lines are single-exponential curve fits to the data. Error bars denote the standard deviations from triplicate experiments.

Fig 3. Experiments testing the formation of duplex P4.

(A) Proposed secondary structure for ribozyme TPR1e showing proposed paired regions labeled P1-P5. All duplexes with exception of P4 were identified previously [16]. Nucleotides found accessible by Shape probing are colored in red, while protected nucleotides are shown in blue. C-A pairs are shown with a dot. The numbering scheme corresponds to the cis-acting ribozyme (Fig 1). Note that the duplexes P1 and P2 are formed in trans because the experiments were performed using the trans reaction. Base pairs for duplex P1 are shown in grey because for TPR1e, nucleotides 78–80 appeared accessible by Shape probing. (B) Apparent triphosphorylation rates of the mutated ribozymes in the base covariation experiment of twelve bases in the proposed P4 duplex. The six column graphs are in the same order as the proposed based pairs in P4 of Fig 3A. Each graph shows the triphosphorylation rate of TPR1e on the left, the two single mutants in the middle, and the double mutant on the right. Error bars are standard deviations of triplicate experiments. (C) Shape probing experiments for nucleotides 17 to 46 of the ribozymes TPR1 (empty circles) and TPR1e (colored circles). The normalized Shape signal is shown as a function of nucleotide position. A threshold value (grey, dashed line) is used to distinguish between low Shape reactivity (blue) and high Shape reactivity (red). The colors match the color-coding in Fig 3A. The position of duplexes P1-P5 in TPR1e is indicated by thick, solid, grey, horizontal lines. (D) As in (C) but for nucleotides 57 to 94 in the ribozymes.

Fig 4. Determination of optimal TPR1e triphosphorylation conditions.

(A) Observed triphosphorylation rate as function of the temperature, at 50 mM Tmp, 100 mM MgCl₂, and 50 mM Tris/HCl pH 8.3 (B) Influence of the trimetaphosphate concentration on the observed reaction kinetics at 40°C, and with an excess of 400 mM MgCl₂ over Tmp. (C) Influence of the free Mg²⁺ concentration on the triphosphorylation rate at 40°C and 150 mM Tmp. The free Mg²⁺ concentration is the total Mg²⁺ concentration minus the concentration of Tmp because each Tmp appears to be coordinated by one Mg²⁺ at these concentrations and pH 8.3 [16]. The grey arrows indicate the optimum condition for each series of experiments. Note that the scale in (A) is different from the scale in (B) and (C). Error bars are standard deviations of triplicate experiments.

Fig 5. Triphosphorylation kinetics of TPR1e.

(A) At a Tmp concentration of 1 mM, the triphosphorylation kinetics are shown for synthetic seawater at 22°C (black triangles, k_obs = 0.0014 min^-1, max = 93%), and at 40°C (empty squares, k_obs = 0.0039 min^-1, max = 97%). For comparison, the reaction kinetics are shown for 54 mM MgCl₂ in Tris/HCl pH 8.3 at 22°C (black circles, k_obs = 0.020 min^-1, max = 93%). This latter condition lacks all seawater components with exception of Mg²⁺. Error bars are standard deviations from triplicate experiments, and are smaller than the symbols if not visible. Curves are single-exponential fits to the data. (B) Titration of the Tmp concentration in the reaction at 22°C, in synthetic seawater (black triangles) and in 54 mM MgCl₂ with 50 mM Tris/HCl pH 8.3 (black circles). The offset between the linear fits (grey lines) is 15-fold, on average. (C) Titration of sodium chloride into a triphosphorylation ribozyme reaction containing 50 mM Tmp and 140 mM MgCl₂. The grey line is a single-exponential fit to the data (with offset) and identifies a 1.9-fold reduction in k_obs at 470 mM [NaCl], the same NaCl concentration as in synthetic seawater (dashed line). Error bars are standard deviations from triplicate experiments, and are smaller than the symbols if not visible.