Novel In Vitro Selection of trans-Acting BCL-2 mRNA Cleaving Deoxyribozymes for Cancer Therapy

The B Cell Lymphoma-2 (BCL-2) family proteins are central regulators of apoptosis, and their dysregulation is frequently associated with cancer progression and resistance to therapy. While small molecules like venetoclax have shown promise, nucleic acid-based therapeutics targeting BCL-2 remain underexplored. Here, we report a novel in vitro evolution strategy to generate trans-acting RNA-cleaving DNAzymes targeting natural BCL-2 mRNA without requiring covalent substrate-linking. Using a 50-base region of BCL-2 mRNA as a selection target, we evolved several DNAzymes, that demonstrate significant RNA cleavage activity. These DNAzymes downregulated BCL-2 expression, induced apoptosis, and reduced cell viability in HepG2 and MCF-7 cancer cells. In vivo, our novel DNAzymes significantly suppressed tumor growth in a syngeneic mouse breast cancer model, with efficacy comparable to 5-Fluorouracil. This study presents a proof-of-concept for a novel strategy to evolve functional DNAzymes against native mRNA sequences and highlights their potential as gene-silencing tools in cancer therapy.