Ribonuclease H1 Hijacking: Oncotherapeutics through MicroRNA-Triggered Intracellular Construction of Long DNA/RNA Heteroduplexes

Ribonuclease H1 (RNase H1) cleaves the RNA strand of DNA/RNA heteroduplexes and plays essential roles in R-loop resolution, mitochondrial DNA replication, and antisense oligonucleotide (ASO) activity. Because RNase H1 has also been implicated in ASO-induced cytotoxicity, considerable effort has been focused on minimizing its activation. Here we present a reverse strategy that harnesses RNase H1-mediated cytotoxicity for cancer therapy using an oncogenic microRNA (miR-21) as an intracellular trigger. A designed hairpin DNA/hairpin RNA pair undergoes a hybridization chain reaction to assemble long-nicked DNA/RNA heteroduplexes selectively in miR-21-overexpressing cells. The resulting heteroduplexes induce RNase H1-dependent cytotoxicity in cancer cells while exhibiting minimal effects on normal cells. Treatment also caused redistribution of the paraspeckle protein P54nrb from the nucleoplasm to the nucleoli, consistent with RNase-H1-associated nucleolar stress. Furthermore, lipid-nanoparticle-mediated delivery of the DNA/RNA heterohairpin pair significantly suppressed tumor growth in mice. These findings establish a therapeutic strategy that converts RNase H1, traditionally regarded as a source of oligonucleotide toxicity, into a mechanism for selective anticancer activity through the intracellular assembly of long DNA/RNA heteroduplexes.