Mays, J. | Search bioRxiv

bioRxiv2018

Highly specific enrichment of rare nucleic acids using Thermus thermophilus Argonaute

Characterization 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.

molecular biology↗

bioRxiv2018

Influenza A Virus Negative Strand RNA is Translated for CD8+ T Cell Immunosurveillance

To probe the limits of CD8+ T cell immunosurveillance, we inserted the model peptide SIINFEKL into influenza A virus (IAV) negative strand gene segments. Although IAV genomic RNA is widely considered as non-coding, there is a conserved, relatively long open reading frame present in the genomic strand of segment eight, encoding a potential protein termed NEG8. The biosynthesis of NEG8 from IAV has yet to be demonstrated. While we failed to detect NEG8 protein expression in IAV infected cells, cell surface Kb-SIINFEKL complexes are generated when SIINFEKL is genetically appended to the predicted COOH-terminus of NEG8, as shown by activation of OT-I T cells in vitro and in vivo. Moreover, recombinant IAV encoding SIINFEKL embedded in the negative strand of the NA-stalk coding sequence also activates OT-I T cells in vivo. Together, our findings demonstrate both the translation of sequences on the negative strand of a single stranded RNA virus and its relevance anti-viral immunosurveillance.\n\nSignificanceEvery gene encodes complementary information on the opposite strand that can potentially be used for immunosurveillance. In this study, we show that the influenza A virus \"non-coding\" strand translated into polypeptides during a viral infection of either cultured cells or mice that can be recognized by CD8+ T cells. Our findings raise the possibility that influenza virus uses its negative strand to generate proteins useful to the virus. More generally, it adds to a growing literature showing that immunosurveillance extends to gene sequences generally thought not to be converted into proteins. The relevance of translating this \"dark\" information extends from viral immunity to cancer immunotherapy and autoimmunity.

immunology↗