Highly specific enrichment of rare nucleic acid fractions using Thermus thermophilus argonaute with applications in cancer diagnostics

• Published in: Nucleic acids research 2020-02, Vol.48 (4), p.e19-e19
• Main Authors: Song, Jinzhao, Hegge, Jorrit W, Mauk, Michael G, Chen, Junman, Till, Jacob E, Bhagwat, Neha, Azink, Lotte T, Peng, Jing, Sen, Moen, Mays, Jazmine, Carpenter, Erica L, van der Oost, John, Bau, Haim H
• Format: Article
• Language: English
• Subjects: Alleles; Argonaute Proteins - genetics; Cell-Free Nucleic Acids - genetics; Humans; Methods Online; Mutation - genetics; Neoplasms - diagnosis; Neoplasms - genetics; Neoplasms - pathology; Peptide Nucleic Acids - genetics; Peptide Nucleic Acids - isolation & purification; Thermus thermophilus - genetics
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkz1165

Detection of disease-associated, cell-free nucleic acids in body fluids enables early diagnostics, genotyping and personalized therapy, but is challenged by the low concentrations of clinically significant nucleic acids and their sequence homology with abundant wild-type nucleic acids. We describe a novel approach, dubbed NAVIGATER, for increasing the fractions of Nucleic Acids of clinical interest Via DNA-Guided Argonaute from Thermus thermophilus (TtAgo). TtAgo cleaves specifically guide-complementary DNA and RNA with single nucleotide precision, greatly increasing the fractions of rare alleles and, enhancing the sensitivity of downstream detection methods such as ddPCR, sequencing, and clamped enzymatic amplification. We demonstrated 60-fold enrichment of the cancer biomarker KRAS G12D and ∼100-fold increased sensitivity of Peptide Nucleic Acid (PNA) and Xenonucleic Acid (XNA) clamp PCR, enabling detection of low-frequency (