Design of hairpin ribozyme variants with improved activity for poorly processed substrates.

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.

Twin ribozyme mediated removal of nucleotides from an internal RNA site.

Over the past two decades, the structure and mechanism of catalytic RNA have been extensively studied; now ribozymes are understood well enough to turn them into useful tools. After we have demonstrated the twin ribozyme mediated insertion of additional nucleotides into a predefined position of a suitable substrate RNA, we here show that a similar type of twin ribozyme is also capable of mediating the opposite reaction: the site-specific removal of nucleotides. In particular, we have designed a twin ribozyme that supports the deletion of four uridine residues from a given RNA substrate.