In vitro selection of the Naegleria GIR1 ribozyme identifies three base changes that dramatically improve activity

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Abstract
NanGIR1 is a member of a new class of group I ribozymes whose putative biological function is site-specific hydrolysis at an internal processing site (IPS). We have previously shown that NanGIR1 requires 1 M KCl for maximal activity, which is nevertheless slow (0.03 min–1). We used in vitro selection and an RNA pool with approximately nine mutations per molecule to select for faster hydrolysis at the IPS in 100 mM KCl. After eight rounds of selection, GIR1 variants were isolated that catalyzed hydrolysis at 300-fold greater rates than NanGIR1 RNA. Although not required by the selection, many of the resultant RNAs had increased thermal stability relative to the parent RNA, and had a more compact structure as evidenced by their faster migration in native gels. Although a wide spectrum of mutations was found in generation 8 clones, only two mutations, U149C and U153C, were common to greater than 95% of the molecules. These and one other mutation, G32A, are sufficient to increase activity 50-fold. All three mutations lie within or proximal to the P15 pseudoknot, a structural signature of GIR1 RNAs that was previously shown to be important for catalytic activity. Overall, our findings show that variants of the Naegleria GIR1 ribozyme with dramatically improved activity lie very close to the natural GIR1 in sequence space. Furthermore, the selection for higher activity appeared to select for increased structural stability.