Hatchet ribozyme

Ribozyme catalysis experiments were done by the addition of MgCl₂ and stopped for measurement at each time point by the addition of a stop solution containing urea and EDTA.

A plot of the kobs values measured at pH 7.5 with increasing concentrations of Mg²⁺. There is a sharp increase in ribozyme function that plateaus as the concentration approaches 10 mM. The steep slope observed at lower Mg²⁺ concentrations suggests that more than one metal ion is necessary for each RNA to achieve maximal ribozyme activity. Moreover, this suggests that the construct requires higher than normal physiological concentrations of Mg²⁺ to become completely saturated with Mg²⁺ as the cofactor. It is possible that native unimolecular constructs, also carrying P0, might achieve saturation at concentrations of Mg²⁺ that are closer to normal physiological levels.

The effect of pH on ribozyme rate constant in reactions containing 10 mM Mg²⁺ was also experimentally measured. pH-dependent ribozyme activity increases linearly with a slope of 1 until reaching a kobs, of a Michaelis-Menten plot, plateau of ~4/min near a pH value of 7.5. Any higher pH has the same catalytic effect and more acidic pH's begin denaturing the ribozyme and thus reducing catalytic function. Both the pH dependency and the maximum rate constant have interesting implications for the possible catalytic strategies used by this ribozyme class.^[3]

The Hatchet ribozyme construct remains completely inactive when incubated in the absence of Mg²⁺ in reactions containing only other monovalent cations at 1 M (Na⁺, K⁺, Rb⁺, Li⁺, Cs⁺), 2.5 M (Na⁺, K⁺), or 3 M (Li⁺). In contrast, other divalent metal ions such as Mn²⁺, Co²⁺, Zn²⁺, and Cd²⁺ support ribozyme function with varying levels of efficiency. Furthermore, two metal ions (Zn²⁺, Cd²⁺) function only at low concentrations, and three metal ions (Ba²⁺, Ni²⁺, and Cu²⁺) inhibit activity at 0.5 mM, even when Mg²⁺ is present. These results indicate that Hatchet ribozymes are relatively restrictive in their use of cations to promote catalysis, perhaps indicating that one or more specialized binding sites that accommodate a limited number of divalent cations are present in the RNA structure or perhaps even at the active site. Inhibition by certain divalent metal ions could be due to the displacement of critical Mg²⁺ ions or by general disruption of RNA folding.^[3]

One standard application is to use flanking self-cleaving ribozymes to generate precisely cut out sequences of functional RNA molecules (i.e. shRNA, saiRNA, sgRNA). This is especially useful for in vivo expression of gene editing systems (i.e. CRISPR/Cas sgRNA) and inhibitory systems.^[6]

Another method is for in vivo transcription of siRNA. This design uses multiple self-cleaving ribozymes, which are all transcribed from the same gene. After cleavage, both parts of the precursor siRNA (siRNA 1 and 2) can form a double strand and act as intended. To see the setup, see saiRNA graphic^[7]

Lastly, if you want to combine self-cleaving ribozymes with protein sequences, it is important to know that the self-cleaving mechanism of the ribozymes will modify the mRNA. A 5' ribozyme will modify the downstream 5' end of the pre-mRNA, disabling the cell from creating a 5' cap. This decreases the stability of the pre-mRNA and prevents it from being fully functional mature mRNA. On the other side, a 3' ribozyme would prevent polyadenylation of the upstream pre-mRNA, again decreasing stability and preventing maturation. Both interfere with translation as well.^[5]