Instantaneous Iodine-Assisted DNAzyme Cleavage of Phosphorothioate RNA

Metal ions play a critical role in the RNA-cleavage reaction by interacting with the scissile phosphate and stabilizing the highly negatively charged transition state. Many metal-dependent DNAzymes have been selected for RNA cleavage. Herein, we report that the Ce13d DNAzyme can use nonmetallic iodine (I₂) to cleave a phosphorothioate (PS)-modified substrate. The cleavage yield exceeded 60% for both the R_p and S_p stereoisomers in 10 s, while the yield without the enzyme strand was only ∼10%. The Ce13d cleavage with I₂ also required Na⁺, consistent with the property of Ce13d and confirming the similar role of I₂ as a metal ion. Ce13d had the highest yield among eight tested DNAzymes, with the second highest DNAzyme showing only 20% cleavage. The incomplete cleavage was due to competition from desulfurization and isomerization reactions. This DNAzyme was engineered for fluorescence-based I₂ detection. With EDTA for masking metal ions, I₂ was selectively detected down to 4.7 nM. Oxidation of I^- with Fe³⁺ produced I₂ in situ, allowing detection of Fe³⁺ down to 78 nM. By harnessing nonelectrostatic interactions, such as the I₂/sulfur interaction observed here, more nonmetal species might be discovered to assist DNAzyme-based RNA cleavage.