Mutagenesis of the hairpin ribozyme

Abstract

Extensive in vitro mutagenesis studies have been performed on the hairpin ribozyme and substrate in an effort to refine the overall secondary structure of the molecule and provide further insight into what elements are essential for activity. A secondary structure consisting of four helices and five loop regions remains the basic model as originally proposed. Two helices, helix 1 and 2, form between the substrate and ribozyme while helices 3 and 4 are within the ribozyme itself. Our results suggest that helices 3 and 4 are smaller than previously proposed, consisting of four base pairs and three base pairs respectively. Helix 4 can be extended without loss of activity and loop 3 at the closed end of the hairpin model can be varied in sequence with retention of activity. There is an unpaired nucleotide between helices 2 and 3 consisting of a single A base, suggesting the opportunity for flexibility within the tertiary structure at this point. Comparisons are made between the new data and previously published mutagenesis and phylogenetic data. Substrate targeting rules require base pairing between helices 1 and 2 with cleavage (*) occurring in a preferred 5'(g/c/u)n*guc3' sequence of the substrate.

Insights into functional modulation of catalytic RNA activity

“…Between the substrate and the ribozyme, two helices are formed, which allow the specificity of binding. Located between these two helices in the substrate are an N ; GUC sequence, where GUC is a required sequence, and the arrow marks the cleavage site (144). In the engineered minimum hairpin motif, the connecting junction is minimised, forming a hinge (two-way junction) (Fig.…”

The sCYMV1 hairpin ribozyme: targeting rules and cleavage of heterologous RNA

“…For catalysis, the nucleotide preceding the substrate loop sequence, the C in position 4 (s4), needs to be the B nucleotide in the BN*GUC substrate sequence of sTRSV. 16 The targeting rules for the sTRSV-based hairpin ribozyme are BN*GUC where * is the site of cleavage, N is any nucleotide, and B represents G, U or C, but not A. The A nucleotide had little or no activity in this position for the sTRSV ribozyme.…”

“…The A nucleotide had little or no activity in this position for the sTRSV ribozyme. 7 It was therefore logical to determine if the A nucleotide in position s4 also had low activity with the sCYMV1-based ribozyme. Mutations were made in the native sequence of the sCYMV1 conventional ribozyme of G 11 U and substrate was prepared with a mutation C s4 A.…”

“…The reason for inverting the first base pair, which when done restores the original C 27 :G 37 base pair in the ribozyme, was that for the sTRSV ribozyme, it was found that when this C:G base pair was inverted to G:C, 90% of the catalytic activity was lost. 16 Therefore the original C 27 :G 37 base pair was maintained for the sCYMV1 HALT ribozyme as shown in Figure 3.…”

“…6 The sTRSV hairpin ribozyme prefers to cleave substrates containing a BN*GUC sequence where B is any nucleotide except A and * is the site of cleavage. 7,8 By using these targeting rules, the sTRSV system has been successfully applied to down-regulate gene expression in HIV-1, 6,9,10 hepatitis B virus, 11 hepatitis C virus 12 and human papillomavirus. 13 In this report we determined the sCYMV1-based hairpin ribozyme can be engineered to cleave heterologous substrates as well.…”