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rdac:C10001 bibo:Article

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n7:nucleicacidenzymes

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Mechanisms of covalent self-assembly of the Azoarcus ribozyme from four fragment oligonucleotides

rdaw:P10072

n9:web n9:print

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n2:draperwille n2:haydenericj n2:lehmanniles

dcterms:abstract

RNA oligomers of length 40-60 nt can self-assemble into covalent versions of the Azoarcus group I intron ribozyme. This process requires a series of recombination reactions in which the internal guide sequence of a nascent catalytic complex makes specific interactions with a complement triplet, CAU, in the oligomers. However, if the CAU were mutated, promiscuous self-assembly may be possible, lessening the dependence on a particular set of oligomer sequences. Here, we assayed whether oligomers containing mutations in the CAU triplet could still self-construct Azoarcus ribozymes. The mutations CAC, CAG, CUU and GAU all inhibited self-assembly to some degree, but did not block it completely in 100 mM MgCl2. Oligomers containing the CAC mutation retained the most self-assembly activity, while those containing GAU retained the least, indicating that mutations more 5′ in this triplet are the most deleterious. Self-assembly systems containing additional mutant locations were progressively less functional. Analyses of properly self-assembled ribozymes revealed that, of two recombination mechanisms possible for self-assembly, termed ‘tF2’ and ‘R2F2’, the simpler one-step ‘tF2’ mechanism is utilized when mutations exist. These data suggest that self-assembling systems are more facile than previously believed, and have relevance to the origin of complex ribozymes during the RNA World.

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gkm1055

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