Ribozyme motif structure mapped using random recombination and selection

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Abstract
Isolating the core functional elements of an RNA is normally performed during the characterization of a new RNA in order to simplify further biochemical analysis. The removal of extraneous sequence is challenging and can lead to biases that result from the incomplete sampling of deletion variants. An impartial solution to this problem is to construct a library containing a large number of deletion constructs and to select functional RNA isolates that are at least as efficient as their full-length progenitors. Here, we use nonhomologous recombination and selection to isolate the catalytic core of a pyrimidine nucleotide synthase ribozyme. A variable-length pool of ~108 recombinant molecules that included deletions, inversions, and translocations of a 271-nucleotide-long ribozyme isolate was constructed by digesting and randomly religating its DNA genome. In vitro selection for functional ribozymes was then performed in a size-dependent and a size-independent manner. The final pools had nearly equivalent catalytic rates even though their length distributions were completely different, indicating that a diverse range of deletion constructs were functionally active. Four short sequence islands, requiring as little as 81 nt of sequence, were found within all of the truncated ribozymes and could be folded into a secondary structure consisting of three helix–loops. Our findings suggest that nonhomologous recombination is a highly efficient way to isolate a ribozyme’s core motif and could prove to be a useful method for evolving new ribozyme functions from pre-existing sequences in a manner that may have played an important role early in evolution.