Highly specific enrichment of rare nucleic acids using Thermus thermophilus Argonaute

Abstract

ABSTRACTCharacterization of disease-associated, cell-free nucleic acids (liquid biopsy) provides a powerful, minimally-invasive means for early detection, genotyping, and personalized therapy; but is challenged by alleles of interest differing by single nucleotide from and residing among large abundance of wild-type alleles. We describe a new multiplexed enrichment assay, dubbed NAVIGATER, that utilizes short nucleic acid-guided endonucleases Argonaute (Ago), derived from the bacterium Thermus thermophilus (TtAgo), to specifically cleave complementary DNA and RNA while sparing alleles having single nucleotide mismatches with the guides. NAVIGATER greatly increases the fractions of rare alleles of interest in samples and enhances sensitivity of downstream procedures such ddPCR, sequencing, and clamped enzymatic amplification. We demonstrate 60-fold enrichment of KRAS G12D in blood samples from pancreatic cancer patients and detection of KRAS, EGFR, and BRAF mutants with XNA-PCR at 0.01% fraction.