Single Mutation in Hammerhead Ribozyme Favors Cleavage Activity with Manganese over Magnesium

Abstract

Hammerhead ribozymes are one of the most studied classes of ribozymes so far, from both the structural and biochemical point of views. The activity of most hammerhead ribozymes is cation-dependent. Mg2+ is one of the most abundant divalent cations in the cell and therefore plays a major role in cleavage activity for most hammerhead ribozymes. Besides Mg2+, cleavage can also occur in the presence of other cations such as Mn2+. The catalytic core of hammerhead ribozymes is highly conserved, which could contribute to a preference of hammerhead ribozymes toward certain cations. Here, we show a naturally occurring variation in the catalytic core of hammerhead ribozymes, A6C, that can favor one metallic ion, Mn2+, over several other cations.

Toehold-mediated strand displacement to measure released product from self-cleaving ribozymes

“…The previously published A6C mutation in the core of a HHR ( Fig. 5 A) was shown to have a k obs of 0.18 min −1 in the presence of Mn 2+ ( Naghdi et al 2020 ), much faster than the 0.005 min −1 of the example described above ( Fig. 1 A).…”

“…( D ) As in Figure 5 C, interpolated fluorescence values were mapped using the standard curve to concentrations of released output strand. It should be noted that the ribozyme adapted from Naghdi et al (2020) was described as having a pseudoknot (as was the original version of this HHR described previously [ Perreault et al 2011 ] which had an A at position 6 from the core, instead of a C in this “Mn 2+ -HHR”). However, secondary structure prediction also suggests an alternative structure ( Supplemental Fig.…”

“…A second ribozyme example, which cleaves in the presence of Mn 2+ (but not Mg 2+ ), was selected from the literature ( Naghdi et al 2020 ). The fact that this ribozyme is inactive in the presence of Mg 2+ allowed us to prepare self-cleaving ribozymes easily for assays in TMSDR.…”

Ribozymes as Therapeutic Agents against Infectious Diseases

“…Earlier, the 2′-hydroxyl group of G8 was thought to be the acid in this acid-base catalysis. However, a recent study reports that Mn 2+ -bound water is the general acid during cleavage [25].…”