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Design of hairpin ribozyme variants with improved activity for poorly processed substrates
Author(s) -
Drude Irene,
Strahl Anne,
Galla Daniel,
Müller Oliver,
Müller Sabine
Publication year - 2011
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2010.07983.x
Subject(s) - ribozyme , hairpin ribozyme , vs ribozyme , mammalian cpeb3 ribozyme , ligase ribozyme , rna , cleavage (geology) , base pair , biology , nucleobase , context (archaeology) , stereochemistry , microbiology and biotechnology , chemistry , biochemistry , genetics , dna , gene , paleontology , fracture (geology)
Application of ribozymes for knockdown of RNA targets requires the identification of suitable target sites according to the consensus sequence. For the hairpin ribozyme, this was originally defined as Y −2 N −1 *G +1 U +2 Y +3 B +4 , with Y = U or C, and B = U, C or G, and C being the preferred nucleobase at positions −2 and +4. In the context of development of ribozymes for destruction of an oncogenic mRNA, we have designed ribozyme variants that efficiently process RNA substrates at U −2 G −1 *G +1 U +2 A +3 A +4 sites. Substrates with G −1 *G +1 U +2 A +3 sites were previously shown to be processed by the wild‐type hairpin ribozyme. However, our study demonstrates that, in the specific sequence context of the substrate studied herein, compensatory base changes in the ribozyme improve activity for cleavage (eight‐fold) and ligation (100‐fold). In particular, we show that A +3 and A +4 are well tolerated if compensatory mutations are made at positions 6 and 7 of the ribozyme strand. Adenine at position +4 is neutralized by G 6 →U, owing to restoration of a Watson–Crick base pair in helix 1. In this ribozyme–substrate complex, adenine at position +3 is also tolerated, with a slightly decreased cleavage rate. Additional substitution of A 7 with uracil doubled the cleavage rate and restored ligation, which was lost in variants with A 7 , C 7 and G 7 . The ability to cleave, in conjunction with the inability to ligate RNA, makes these ribozyme variants particularly suitable candidates for RNA destruction.