Selection of M2+-Independent RNA-Cleaving DNAzymes with Side-Chains Mimicking Arginine and Lysine

Sequence-specific cleavage of RNA by nucleic acid catalysts in the absence of a divalent metal cation (M2+) has remained an important goal in biomimicry with potential therapeutic applications. Given the lack of functional group diversity in canonical nucleotides, modified nucleotides with amino acid-like side chains were used to enhance self-cleavage rates at a single embedded ribonucleoside site. Previous works relied on three functional groups: an amine, a guanidine and an imidazole ensconced on three different nucleosides. However, to date, few studies have systematically addressed the necessity of all three modifications, as the value of any single modified nucleoside is contextualized at the outset of selection. Herein, we report on the use of only two modified dNTPs, excluding an imidazole, i. e. 5-(3-guanidinoallyl)-2'-dUTP (dU(ga)TP) and 5-aminoallyl-2 '-dCTP (dC(aa)TP), to select in-vitro self-cleaving DNAzymes that cleave in the absence of M2+ in a pH-independent fashion. Cleavage shows biphasic kinetics with rate constants that are significantly higher than in unmodified DNAzymes and compare favorably to certain DNAzymes involving an imidazole.

Automated summary

This study reports on the specific cleavage of RNA by nucleic acid catalysts without the need for a divalent metal cation. Using modified nucleotides with amino acid-like side chains, the researchers were able to enhance the self-cleavage rates of DNAzymes. By excluding an imidazole and using only two modified dNTPs, they selected DNAzymes that cleave in a pH-independent fashion. The cleavage rates were significantly higher than unmodified DNAzymes and comparable to certain DNAzymes involving an imidazole.