Sequence-dependent RNA cleavage by the 8-17 DNAzyme: Identification of preferred tetranucleotide motifs

In recent years, deoxyribozymes (DNAzymes) capable of catalyzing RNA cleavage have attracted growing attention for their potential therapeutic applications. Among these, the 8-17 DNAzyme is one of the most extensively studied motifs, owing to its compact catalytic core and robust catalytic activity. Although it is well established that the 8-17 DNAzyme preferentially cleaves GG and AG (5' → 3') dinucleotide junctions within substrate RNA strands, the influence of flanking nucleotides on catalytic efficiency has not been systematically examined. Here, we comprehensively evaluated the tetranucleotide sequence preferences of the 8-17 DNAzyme. By assessing cleavage efficiencies across diverse tetranucleotide contexts in which the scissile phosphodiester bond lies between the second and third ribonucleosides, we identified three motifs-UGGG, UGGU, and UGGA-that were cleaved with markedly enhanced efficiency. Molecular dynamics and quantum chemical calculations suggested that stable formation of the noncanonical rG12·dA24 base pair in the UGGG motif facilitates a cleavage-competent structure, whereas this base pair is relatively disrupted in the GGGG motif, a less cleavable sequence. This sequence preference was further validated using 8-17 DNAzymes targeting mouse coagulation factor XI (FXI) mRNA. Collectively, these findings reveal a practical sequence preference of the 8-17 DNAzyme and provide a rational framework for designing highly active DNAzyme-based therapeutics.