In vitro selection of glmS ribozymes

Abstract

Riboswitches modulate gene expression in eubacteria and eukaryotes in response to changing concentrations of small molecule metabolites. In most examples studied to date, riboswitches achieve both metabolite sensing and gene control functions without the obligate involvement of protein factors. These findings validate the hypothesis that RNA molecules could be engineered to function as designer gene control elements that sense and respond to different ligands. We believe that reverse engineering natural riboswitches could provide an intellectual foundation for those who wish to build synthetic riboswitches. Also, natural riboswitches might serve as starting points for efforts to change ligand specificity or gene control function through mutation and selection in vitro. In this chapter, we describe how in vitro selection can be used to create variant glmS ribozymes. Additionally, we discuss how these techniques can be extended to other riboswitch classes.

Fig. 1

Consensus and Bacillus cereus glms ribozymes. (A) Consensus sequence and secondary structure model for glmS ribozymes. Nucleotide numbers conform to those for the B. cereus RNA (Fig. 1B). Boxed nucleotide is absent from the B. cereus sequence, and circles represent the two extra nucleotides present in B. cereus and Bacillus anthracis ribozymes (Fig. 1B). Nucleotides in black circles are conserved in at least 97% of natural glmS ribozyme representatives. N represents any nucleotide identity, where some stretches are variable in length. Nucleotides denoted Y are either C or U, and nucleotides denoted R are either G or A. Arrowhead identifies the site of ribozyme cleavage. (B) The sequence and secondary structure model for the glmS ribozyme from B. cereus based on comparative sequence analysis (6,7) with revisions based on additional bioinformatics (unpublished data) and x-ray structural data (17,18). Nucleotides 61 and 62 (circled) are found in strains of B. cereus and B. anthracis, but are typically not present in glmS ribozymes from other bacteria (6,9). Nucleotides depicted in gray were mutagenized to a degeneracy level of 0.09 (11). Mutations were not introduced at nucleotide 52 due to restrictions of the method used to generate the DNA templates for transcription of the G0 RNA population. Arrowhead identifies the site of ribozyme cleavage and the boxed nucleotides identify the minimal functional core of the ribozyme (6). Images reproduced from reference with permission.

Fig. 2

Outline of the basic glmS selection scheme. (A) Two synthetic oligonucleotides are extended using SSII RT to generate double stranded DNAs encoding the mutagenized glmS ribozymes. (B) The double stranded DNAs encoding the mutagenized glmS ribozymes are transcribed using T7 RNA polymerase. (C) The full-length RNAs are purified and the RNAs are subjected to positive selection in the presence of the effectors. (D) The 3′ cleavage products carrying the randomized ribozyme domains are gel purified and reverse transcribed. (E) The cDNA is then amplified in a PCR reaction that generates double stranded DNAs encoding the selected glmS ribozyme variants.

Fig. 3

Construct design for the in vitro selection of allosteric ribozymes. A mutagenized riboswitch aptamer can be grafted onto stem II of the hammerhead ribozyme via a communication module to create a population of RNAs. Allosteric ribozymes that are controlled by ligand binding to the aptamer undergo self-cleavage at the site indicated by an arrowhead.