In vitro selection of active hairpin ribozymes by sequential RNA-catalyzed cleavage and ligation reactions.

Abstract

In vitro selection methods provide rapid and extremely powerful tools for elucidating interactions within and between macromolecules. Here, we describe the development of an in vitro selection procedure that permits the rapid isolation and evaluation of functional hairpin ribozymes from a complex pool of sequence variants containing an extremely low frequency of catalytically proficient molecules. We have used this method to analyze the sequence requirements of two regions of the ribozyme-substrate complex: a 7-nucleotide internal loop within the ribozyme that is essential for catalytic function and substrate sequences surrounding the cleavage-ligation site. Results indicate that only 3 of the 16,384 internal loop variants examined have high cleavage and ligation activity and that the ribozyme has a strong requirement for guanosine immediately 3' to the cleavage-ligation site.

Substrate selection rules for the hairpin ribozyme determined by in vitro selection, mutation, and analysis of mismatched substrates.

“…Results of studies reported here and elsewhere {Chow-rira et al 1991;Berzal-Herranz et al 1992) indicate that hairpin ribozymes can be engineered to efficiently cleave RNA sequences containing the sequence 5'-nnrynSghybnnnnn-3' (Fig. 7).…”

“…Both active and inactive clones were selected from each experiment for further analysis by cloning, sequencing, and in vitro self-cleavage assays {Fig. 1B) (Berzal-Herranz et al 1992). …”

“…Three complementary experimental strategies have been employed: (1) We have analyzed the ability of the ribozyme to cleave a series of variant substrates, each containing single-or multiplebase substitutions; (2) an in vitro selection method (Fig. 1B) (Berzal-Herranz et al 1992) has been applied to identify sequences necessary for optimal cleavage and ligation activities in cis; and (3) sequence preferences resulting from the selection experiments have been critically tested by assaying trans-cleavage activity of panels of variant ribozymes against a series of variant substrates. Our results permit the formulation of a set of comprehensive substrate selection rules for the hairpin ribozyme.…”

Systematic substitution of individual bases in two important single‐stranded regions of the HDV ribozyme for evaluation of the role of specific bases

“…By analyzing self-cleavage activities of various mutants of SSrA and SSrB regions we could determine the preferred sequences for these regions and the sequences are 5'-G-G-(G/A/U)-N-(A/U/G)-Pu-Y and 5'-(G/U)-C-N-(A/G/U)-A-3' for SSrA and SSrB regions, respectively. Recently, we employed in vitro selection proceedure [21,22] to find the important bases at a remained single-stranded region, SSrC (U708-U715) and could find a few bases that are important for the cleavage activity (unpublished data). Thus, several bases in the SSrC region, together with C763 and other bases in the SSrA and the SSrB regions, appear to represent the core structure of the genomic HDV ribozyme.…”

Inhibition of gene expression in human cells using RNase P-derived ribozymes and external guide sequences

“…Since its introduction, the technology found itself in many different laboratories, used for different purposes from enhancing catalytic activity to enzyme engineering to isolating nucleic acids with novel functions. [58,[62][63][64] We have used the in vitro selection technique to successfully isolate highly active M1GS RNA variants targeting the mRNA sequence encoding the thymidine kinase (TK) of herpes simplex virus type 1 (HSV-1) [65]. Our initial library of ribozymes contained random mutations in regions that are known to be conserved across all species of RNase P catalytic RNAs and are important for catalysis and substrate binding [66][67][68].…”