This project was supported by a research grant from the National Aeronautics and Space Administration (NNG04-GM20G) to N.L. Funding to pay the Open Access publication charges for this article was provided by NASA
Nucleic Acids Research
Catalytic RNA, Oligonucleotides -- Synthesis, RNA-protein interactions
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 100mM 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 selfassembly, 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.
Draper, W.E.; Hayden, E.J.; Lehman, N. (2008). Mechanisms of covalent self-assembly of the Azoarcus ribozyme from four fragment oligonucleotides. Nucleic Acids Research 36:520-531.