Ribozyme Switch

A ribozyme switch is a part of an mRNA that can directly bind to a small target molecule and whose binding affects the gene's ability. There are two aspects of a riboswitch, the aptamer and the expression platform. The aptamer portion binds to a target molecules and changes shape, affecting the expression platform, which is how gene expression is regulated. Some types of riboswitch mechanisms include:

• Formation of transcription termination hairpins
• Blockage of translation by folding to isolate ribosome-binding sites
• Effect of folding on splicing of mRNA
• Self-cleavage ribozymes that cleave themselves in the presence of a target molecule
  • This paper focuses on self-cleaving ribozymes with an aptamer sequence and a hammerhead ribozyme sequence
    • Strand-Displacement based switch
    • Helix-Slipping based switch

See also Riboswitches for additional information

The lab of Maung Nyan Win and Christina D. Smolke uses ribozymes to control Post-transcriptional gene regulation regulation within a cell.

Experimental Design and Results

The setup of the ribozyme utilizes portability, utility and composability, all importants factors in the goal for this paper. The first component of this design is the placement of the ribozyme within the 3' UTR of a gene, in this case connected to GFP. The purpose of this design is to insure that any gene regulation occurs from cleavage of the ribozyme rather than from inhibition of translation iniation, which can occur with antisense RNA (Wikipedia). The second component ensures that there are no interactions between the ribozyme and the rest of the transcript by placing spacer sequences around the 3' and 5' end of the ribozyme. Lastly, the third component involves keeping loops I and II intact so that their tertiary interactions will be stable against Mg2+ concentrations (Figure 1A). For this reason, this paper uses a specific method to couple the ribozyme with an aptamer so that neither loop is destroyed (Figure 1B). http://www.pnas.org/content/vol104/issue36/images/large/zpq0340773700001.jpeg

Figure 1. "General design strategy for engineering ribozyme switches. The color scheme is as follows: catalytic core, purple; aptamer sequences, brown; loop sequences, blue; spacer sequences, yellow; brown arrow, cleavage site. (A) General compositional framework and design strategy for engineering cis-acting hammerhead ribozyme-based regulatory systems. Restriction enzyme sites are underlined. (B) Modular coupling strategies of the sensor and regulatory domains to maintain in vivo activity of the individual domains" (Win and Smolke 2007). Image Permission Pending.

Additional Applications

Modularity and Specificity of Strand-Displacement-based Ribozyme Switches

http://www.pnas.org/content/vol104/issue36/images/medium/zpq0340773700005.gif

Figure 5. "Modularity and specificity of the strand-displacement-based ribozyme switches. (A) Modular design strategies for the construction of new ribozyme switches. The theophylline (left dashed box) and tetracycline (right dashed box) aptamers are shown. (B) Regulatory activities of the modular ribozyme switch pair, L2bulge1 and L2bulge1tc, in response to their respective ligands, theophylline (theo) and tetracycline (tc), and closely related analogues, caffeine (caff) and doxycycline (doxy). Regulatory effects are reported in fold induction relative to the expression levels in the absence of effector as described in Fig. 2" (Win and Smolke, 2007). Image Permission Pending.

Examples of Modularity of Various Ribozyme switches in Cellular Engineering Applications

http://www.pnas.org/content/vol104/issue36/images/medium/zpq0340773700006.gif

Figure 7. "Fig. 6. System modularity of ribozyme switches enables implementation in diverse cellular engineering applications. (A) System design for ribozyme switch-based regulation of cell growth. Small molecule-mediated regulation of a gene required for cell growth is illustrated for a strand-displacement-based OFF switch. (B) Theophylline-mediated ribozyme switch-based regulation of cell growth. Changes in growth are reported as OD600 values for cells grown in 5 mM 3-aminotriazole (3AT) in media lacking histidine. (C) System design for ribozyme switch-based in vivo sensing of metabolite production. Xanthine was synthesized from cultures fed xanthosine, and product accumulation over time was detected through a strand-displacement-based xanthine-responsive ON switch coupled to the regulation of a reporter protein. (D) Ribozyme switch-based xanthine synthesis detection through L2bulge9. Metabolite sensing through L2bulge9 is reported in fold induction of GFP levels relative to the expression levels in the absence of xanthosine feeding as described in Fig. 2" (Win and Smolke, 2007). Image Permission Pending.

---