Scaling Catalytic Contributions of Small Self‐Cleaving Ribozymes

Abstract

Nucleolytic ribozymes utilize general acid-base catalysis to perform phosphodiester cleavage. In most ribozyme classes, a conserved active site guanosine is positioned to act as general base, thereby activating the 2'-OH group to attack the scissile phosphate (γ-catalysis). Here, we present an atomic mutagenesis study for the pistol ribozyme class. Strikingly, "general base knockout" by replacement of the guanine N1 atom by carbon results in only 2.7-fold decreased rate. Therefore, the common view that γ-catalysis critically depends on the N1 moiety becomes challenged. For pistol ribozymes we found that γ-catalysis is subordinate in overall catalysis, made up by two other catalytic factors (α and δ). Our approach allows scaling of the different catalytic contributions (α, β, γ, δ) with unprecedented precision and paves the way for a thorough mechanistic understanding of nucleolytic ribozymes with active site guanines.

Robust synthesis of 2′-azido modified RNA from 2′-amino precursors by diazotransfer reaction

“…Hence, the 10 nt hairpin ( 16 ) was equipped with all four standard 2′-NH 2 nucleotides (A, C, G, U) at once, and a second hairpin ( 17 ) was prepared forming a base pair between 2′-NH 2 -A and 2′- O -(3-aminopropyl)uridine. 12,13,25 The modified RNAs were treated with FSO 2 N 3 for 24 h and clean conversions for the sequences were observed (Fig. 3A and B).…”

Rapid Kinetics of Pistol Ribozyme: Insights into Limits to RNA Catalysis

“…To investigate Psr kinetics, we used a fluorescence-based assay developed by Micura and colleagues that consists of a 16 nucleotide substrate, A57Ap, containing a 2-aminopurine (2Ap) nucleobase that anneals to the 32 nucleotide env25 Psr (Figure C). ,,, The reaction is initiated by mixing the pre-annealed complex with divalent metal ions, and product formation is quantified by monitoring the increase in 2Ap fluorescence upon cleavage and product dissociation (Scheme ). ,, This method has provided a highly valuable tool to establish Psr structure–function relationships and importantly enables analysis of rapid kinetics.…”

“…Additionally, the correlation between the apparent cleavage rate and divalent metal aquo ion p K a must be accepted as the basis for the effect on catalysis, rather than related properties such as radius and polarizability, which could affect transition state stabilization. Recently, it was demonstrated that a 1-deazaguanine modification at G42 which should knock out the nucleophile activating general base showed no large effect . This result was interpreted as showing minimal proton transfer prior to the rate-limiting transition state, or alternatively a nonchemical rate-limiting step.…”

A new route for the synthesis of 1-deazaguanine and 1-deazahypoxanthine

“…6-Iodo-9-(tetrahydro-2H-pyran-2-yl)-1deazapurine (17) 6-Iodo-1-deazapurine (2.89 g, 11.79 mmol), p-toluenesulfonic acid monohydrate (TsOH•H 2 O, 0.34 g, 1.77 mmol) and 3,4dihydro-2H-pyran (2.98 g, 3.21 mL, 35.38 mmol) were dissolved in 5 mL dry N,N-dimethylformamide and stirred overnight for 16 hours at 60 °C. The reaction was quenched by adding ammonia 25% (4 mL) and was stirred for further 5 minutes.…”

“…Previously published routes toward these compounds focused on ring closure of the imidazole part of the purine system after the pyrimidine core had been functionalized, however, the drawback of this approach is the passage of rather hazardous/explosive intermediates. Here, we present a new tactic for the syntheses of 1-deazaguanine and 1-deazahypoxanthine stimulated by a recently published route of our research group for the corresponding nucleosides [16,17], employing the same key reaction, namely the copper-catalyzed coupling of an aryl iodide with benzyl alcohol. We build on a commercially available imidazopyridine derivative and conceived a protecting group strategy to enhance solubility and selectivity to orchestrate the installation of the exocyclic amino and hydroxy groups.…”