Chemical evolution of ASO-like DNAzymes for effective and extended gene silencing in cells.

Antisense oligonucleotides (ASOs) and small interfering RNA (siRNA) therapeutics highlight the power of oligonucleotides in silencing disease-causing messenger RNAs (mRNAs). Another promising class of gene-silencing oligonucleotides is RNA-cleaving nucleic acid enzymes, which offer the potential for allele-specific RNA inhibition with greater precision than ASOs and siRNAs. Herein, we chemically evolved the nucleolytic DNA enzyme (DNAzyme) 10-23, by incorporating the modifications that are essential to the success of ASO drugs, including 2'-fluoro, 2'-O-methyl, and 2'-O-methoxyethyl RNA analogues, and backbone phosphorothioate, to enhance catalytic efficiency by promoting RNA substrate binding and preventing dimerization of 10-23. These ASO-like DNAzymes cleaved structured RNA targets in long transcripts, showed prolonged intracellular stability, and downregulated mRNA and protein levels of both exogenously transfected eGFP and endogenously elevated oncogenic c-MYC. In colon cancer HCT116 cells, the downregulation of oncogenic c-MYC RNA resulted in cell cycle arrest, reduced proliferation, and increased apoptosis. RACE (rapid amplification of cDNA ends) polymerase chain reaction and Sanger sequencing confirmed precise, site-specific mRNA transcript cleavage with minimal RNase H activation in cells. By merging ASO structural and pharmacokinetic advantages with DNAzyme catalytic versatility, these ASO-like 10-23 variants offer a promising new class of potent gene-silencing agents, representing a significant step toward therapeutic DNAzyme development.