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

Abstract

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 (<0.01%) mutant alleles (∼1 copy) in blood samples of pancreatic cancer patients. NAVIGATER surpasses Cas9-based assays (e.g. DASH, Depletion of Abundant Sequences by Hybridization), identifying more mutation-positive samples when combined with XNA-PCR. Moreover, TtAgo does not require targets to contain any specific protospacer-adjacent motifs (PAM); is a multi-turnover enzyme; cleaves ssDNA, dsDNA and RNA targets in a single assay; and operates at elevated temperatures, providing high selectivity and compatibility with polymerases.

A programmable omnipotent Argonaute nuclease from mesophilic bacteria Kurthia massiliensis

“…However, MpAgo ( Marinitoga piezophila ) was reported to use small RNA guides to cleave DNA and RNA substrates ( 12 ). In analogy with the Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated (CRISPR–Cas) enzymes, pAgos may potentially be used in nucleic acid detection ( 22 , 23 ), molecular cloning ( 24 ) and genome editing applications ( 10 ). However, all the pAgos mentioned above are from thermophilic prokaryotes and they only have low levels of DNA-guided ssDNA and RNA cleavage activity at moderate temperatures (20–50°C) ( 16 , 18 , 21 ), which limits the application of pAgos.…”

A programmable pAgo nuclease with universal guide and target specificity from the mesophilic bacterium Kurthia massiliensis

“…In particular, degradation of one of the target variants can be used for enrichment of rare nucleic acid variants, both DNA and RNA, for their further detection. Thermophilic TtAgo and PfAgo were previously applied for precise recognition and cleavage of complementary targets and their closely related variants depending on the presence of mismatches ( 34 , 35 ). Similarly, thermophilic MpAgo, programmed with RNA guides, was used for selective binding of rare RNA variants with single-nucleotide changes without their cleavage ( 31 ).…”

A programmable pAgo nuclease with universal guide and target specificity from the mesophilic bacteriumKurthia massiliensis

“…In particular, specific degradation of one of the target variants can be used for enrichment of rare nucleic acid variants, both DNA and RNA, for their further detection. Thermophilic TtAgo and PfAgo were previously applied for precise recognition and cleavage of complementary targets and their closely related variants depending on the presence of mismatches (28,29). Similarly, thermophilic MpAgo, programmed with RNA guides, was used for selective binding of rare RNA variants with single-nucleotide changes without their cleavage (30).…”