Journal
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
Volume 54, Issue 50, Pages 15128-15133Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/anie.201506601
Keywords
metal ion rescue; nucleoside modifications; oligoribonucleotides; perturbed pK(a); solid-phase synthesis
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Funding
- Austrian Science Fund FWF [I1040, P27947, P26550, P27347]
- US National Institutes of Health [1 U19 CA179564]
- EU FP7 Marie Curie ITN RNPnet program [289007]
- Austrian Science Fund (FWF) [P27347, P26550, I1040] Funding Source: Austrian Science Fund (FWF)
- Austrian Science Fund (FWF) [Y 372, I 1040, P 27347] Funding Source: researchfish
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Nucleolytic ribozymes catalyze site-specific cleavage of their phosphodiester backbones. A minimal version of the twister ribozyme is reported that lacks the phylogenetically conserved stem P1 while retaining wild-type activity. Atomic mutagenesis revealed that nitrogen atoms N1 and N3 of the adenine-6 at the cleavage site are indispensable for cleavage. By NMR spectroscopy, a pK(a) value of 5.1 was determined for a (13)C2-labeled adenine at this position in the twister ribozyme, which is significantly shifted compared to the pKa of the same adenine in the substrate alone. This finding pinpoints at a potential role for adenine-6 in the catalytic mechanism besides the previously identified invariant guanine-48 and a Mg2+ ion, both of which are directly coordinated to the non-bridging oxygen atoms of the scissile phosphate; for the latter, additional evidence stems from the observation that Mn2+ or Cd2+ accelerated cleavage of phosphorothioate substrates. The relevance of this metal ion binding site is further emphasized by a new 2.6 angstrom X-ray structure of a 2'-OCH3- U5 modified twister ribozyme.
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