Research Article: A Faster Triphosphorylation Ribozyme

Date Published: November 6, 2015

Publisher: Public Library of Science

Author(s): Gregory F. Dolan, Arvin Akoopie, Ulrich F. Müller, Alfred S Lewin.


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.

Partial Text

The RNA world hypothesis states that an early stage in the evolution of life used RNA as the genome and as the only genome-encoded catalyst [1–3]. This hypothesis has gained support from several directions, including the findings that RNA molecules can indeed catalyze reactions [4], that the ribosome is a catalytic RNA [5], and that deoxyribonucleotides are synthesized in cells from ribonucleotide precursors [6]. To find out how an RNA world organism could have functioned, several laboratories are trying to generate an RNA world organism in the laboratory. An important part of producing an RNA world organism is the generation of catalytic RNAs (ribozymes) that could support a self-replicating and evolving ribozyme system. Since the inception of in vitro selection [7, 8] and the demonstration that novel catalytic RNAs can be isolated from randomized RNA pools [9], several types of ribozymes with possible involvement in an RNA world organism have been isolated [10], most importantly a ribozyme that catalyzes RNA-dependent RNA polymerization [11]. The substrates for this ribozyme are nucleoside 5′-triphosphates; these nucleotides contain the perhaps most prebiotically plausible chemical activation group [12]. Nucleoside 5′-triphosphates can be generated from nucleosides and trimetaphosphate (Tmp) [13]. Tmp likely existed prebiotically, as suggested from the finding of large amounts of its chemical precursor in 3.5 billion year old marine sediments [14, 15]. However, the uncatalyzed triphosphorylation of nucleosides with Tmp occurs efficiently only at pH values above 12 [13], which would quickly hydrolyze the RNA polymers of an RNA world organism. We previously showed that the triphosphorylation of RNA 5′-hydroxyl groups is possible at neutral pH, with ribozymes that were obtained by in vitro selection [16].

To identify a more active sequence variant of the previously isolated triphosphorylation ribozyme, TPR1 (Fig 1A), a ‘doped selection’ was performed. A partially randomized library was generated that contained the sequence of TPR1 at each position with a frequency of 76% while containing each of the other three nucleotides at a frequency of 8%. With a total of 82 nucleotides being partially randomized, around 103 wild-type sequences were contained in a library of 1013 sequences (0.7682× 1013 ~ 1,700). The selection was performed as described previously [16] and started with an effective complexity of 7.0 × 1013 sequences. After two rounds of selection the number of PCR cycles required to amplify the reverse transcription product dropped from 15 to 6. Such a drop was previously found to be a good indicator that active clones dominated the library [16]. To isolate the most efficient catalysts, higher selection pressure was used in additional rounds of selection by changing the Tmp incubation conditions (shorter incubation time, lower Tmp concentration, or higher incubation temperature; S1 Fig). After three or four rounds of selection, eighty-three clones were isolated and analyzed for triphosphorylation activity (see materials and methods for details on how many clones were tested from each condition/round).

In the present study, a ribozyme catalyzing the triphosphorylation of RNA 5′-hydroxyl groups with Tmp was subjected to a doped selection, which resulted in the improved ribozyme, TPR1e. A previously unidentified duplex was identified in this ribozyme, indicating a very compact structure. TPR1e shows triphosphorylation kinetics of 6.8 min-1 under optimal conditions. In synthetic seawater with 1 mM Tmp at 40°C, a triphosphorylation rate of 0.004 min-1 was measured.