Fig. 1: XNAzymes specifically cleave individual members of the miR-17~92 / OncomiR-1 microRNA cluster.
From: Targeting non-coding RNA family members with artificial endonuclease XNAzymes
a Chemical structure of a 2’-deoxy-2’-fluoro-β-D-arabino nucleic acid (FANA) nucleotide and schematic showing the putative secondary structure of the FR6_1 RNA endonuclease XNAzyme composed of FANA (purple) bound to an RNA substrate (red). b Sequence similarity between mature microRNAs of the human miR-17 family; note that although alternative sequences have been reported, in this study we use those from ref. 42 as proof of concept. Variants of the FR6_1 XNAzyme engineered to cleave (c–f) miR-17 (“Fz_miR_17”) or (g–j) miR-20a (“Fz_miR_20a”): (c, g) putative secondary structures, (d–f, h–j) urea-PAGE gels and graphs showing (d, h) RNA substrate cleavage, (e, i) catalytic rate constant (kobs) and (f, j) XNAzyme specificity of pseudo first-order single-turnover reactions between cognate miRNA substrates and XNAzymes for (d, h) 15 h, (f, j) 5 h, or the times indicated, under quasi-physiological conditions (37 °C, 1 mM Mg2+, pH 7.4) (d, e, h, i: 1 μM substrate, 5 μM enzyme. f, j 0.25 μM substrate, 1.25 μM enzyme). Black arrows indicate site of substrate cleavage. (-OH) indicates RNA substrates subjected to partial alkaline hydrolysis. Black circles and error bars represent mean and standard error (SEM) for n = 3 independent experiments, red crosses represent individual data points.
