A strategy for developing a hammerhead ribozyme for selective RNA cleavage depending on substitutional RNA editing

Abstract

Substitutional RNA editing plays a crucial role in the regulation of biological processes. Cleavage of target RNA that depends on the specific site of substitutional RNA editing is a useful tool for analyzing and regulating intracellular processes related to RNA editing. Hammerhead ribozymes have been utilized as small catalytic RNAs for cleaving target RNA at a specific site and may be used for RNA-editing-specific RNA cleavage. Here we reveal a design strategy for a hammerhead ribozyme that specifically recognizes adenosine to inosine (A-to-I) and cytosine to uracil (C-to-U) substitutional RNA-editing sites and cleaves target RNA. Because the hammerhead ribozyme cleaves one base upstream of the target-editing site, the base that pairs with the targetediting site was utilized for recognition. RNA-editing-specific ribozymes were designed such that the recognition base paired only with the edited base. These ribozymes showed A-to-I and C-to-U editing-specific cleavage activity against synthetic serotonin receptor 2C and apolipoprotein B mRNA fragments in vitro, respectively. Additionally, the ribozyme designed for recognizing A-to-I RNA editing at the Q/R site on filamin A (FLNA) showed editing-specific cleavage activity against physiologically edited FLNA mRNA extracted from cells. We demonstrated that our strategy is effective for cleaving target RNA in an editing-dependent manner. The data in this study provided an experimental basis for the RNA-editing-dependent degradation of specific target RNA in vivo.

Improved design of hammerhead ribozyme for selective digestion of target RNA through recognition of site-specific adenosine-to-inosine RNA editing

“…Our results indicated that the saturation value and cleavage rate were similar to those obtained in the reaction against the HTR2C RNA fragment. In the case of minHHR, the in vitro trans-cleavage constant against the long substrate was >100-fold lower than that against the short substrate (Campbell et al 1997;Hormes and Sczakiel 2002;Fukuda et al 2012). The reason for this decrease in cleavage activity may be an inability to form the proper active conformation through base-paired, nonspecific interactions with ribozyme nucleotides.…”

“…The results of B and C are presented as averages and standard deviations from three independent experiments. developed a strategy for HHR cleavage of target RNA when a specific site is subjected to A-to-I RNA editing (Fukuda et al 2012). In this report, we next developed an HHR with an inverse sequence specificity, i.e., one that cleaves substrate RNA only at unedited target sites.…”

“…We have previously reported a method for constructing a trans-acting HHR for target RNA cleavage based on A-to-I RNA editing (Fukuda et al 2012). In this strategy, editing recognition ability was introduced into the canonical minHHR by substituting the recognition nucleotide, in which basepairing is highly sensitive to cleavage activity (Werner and FIGURE 1.…”

Neoepitope formation through the generation of RNA-derived “editopes”

“…Multiple studies have recently shown promising results in employing RNA editing to correct single point mutations, both in cells (19–21, 23, 24, 27–29, 31, 33–35, 42, 51–53) and in animal models (25, 26, 30, 32). These studies have revealed the following major weaknesses in the system: (a) editing can never reach 100% efficiency; (b) off-target (or bystander) editing is more of a problem than initially expected (though it can be ameliorated) (42, 46, 47).…”

Zn2+-dependent DNAzymes that cleave all combinations of ribonucleotides

“…In vitro selection. In vitro selection was basically performed using the methods reported by Lockett et al 52 and Fukuda et al 53 the 3′-biotinylated substrate strand (200 pmol) was incubated with Dynabeads™ MyOne™ Streptavidin C1 beads (Thermo Fisher Scientific Inc., Waltham, MA, USA) in a wash buffer (50 mM HEPES, pH 7.5, 1 M NaCl and 0.01% Tween 20) for 1 h at 25°C. After three times wash with the wash buffer, the N16 library (200 pmol) was annealed to the substrate by heating at 95°C for 5 min and gradually cooling to 4°C.…”