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Programmable low-cost DNA-based platform for viral RNA detection

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Science Advances  25 Sep 2020:
Vol. 6, no. 39, eabc6246
DOI: 10.1126/sciadv.abc6246
  • Fig. 1 DNA nanoswitch strategy for viral RNA sensing.

    (A) Schematic of the DNA nanoswitch and detection of a viral RNA sequence. nt, nucleotide. (B) Fast development cycle of nanoswitches for RNA viruses. (C) Nanoswitch-based assay allows direct detection using a nonenzymatic approach (top) and can optionally be combined with an isothermal amplification step like NASBA (nucleic acid sequence–based amplification) (bottom).

  • Fig. 2 Detection of viral RNA using DNA nanoswitches.

    (A) Schematic of the fragmentation of viral RNA and subsequent detection by the DNA nanoswitch. (B) Fragmentation analysis of ZIKV RNA that was fragmented at 94°C for 1, 3, 6, and 9 min. (C) Proof of concept showing detection of a target region chosen from the literature (22) (0.8% agarose gel in 0.5× tris borate EDTA buffer). (D) Schematic of the design of multiple nanoswitches for detection with the signal multiplication strategy. T1 through Tn are specific targets in an “n” size target pool, that are responsive to nanoswitches NS1 through NSn. (E) Validation of the signal multiplication strategy: The detection signal was increased for a fixed pool of DNA targets when using multiple targeting nanoswitches. (F) Detection sensitivity of the pooled nanoswitches for ZIKV RNA in 10-μl reaction. Error bars represent SD from triplicate experiments.

  • Fig. 3 DNA nanoswitches specifically and differentially detect RNA from two different flaviviruses and between two highly similar ZIKV isolates.

    (A) ZIKV nanoswitches specifically detect ZIKV RNA but not DENV RNA, and vice versa. (B) Multiplexed detection of ZIKV and DENV RNA. (C) Illustration showing culture and RNA extraction of ZIKV Cambodia and Uganda strains. The mismatches in a representative target sequence between the two strains are shown. (D) Specificity test of Cambodia and Uganda strains of ZIKV RNA. * denotes a band of contaminating cellular DNA following RNA isolation.

  • Fig. 4 DNA nanoswitches directly detect ZIKV RNA extracted from infected human liver cells.

    (A) RNA isolated from mock infected Huh7 cells at 1, 2, and 3 days after infection shows no ZIKV detection. (B) RNA isolated from Zika-infected Huh7 cells at 1, 2, and 3 days after infection shows increasing detection of ZIKV RNA over time, with red arrows denoting detection bands. * denotes a band of contaminating cellular DNA following RNA extraction. (C) Quantification of nanoswitch detection signal, with error bars representing SD from triplicate experiments.

  • Fig. 5 Prior extraction or preamplification of target RNA facilitates detection of ZIKV and SARS-CoV-2 RNA at clinically relevant levels in biofluids.

    (A) Positive identification of ZIKV RNA in spiked urine by first isolating in vitro transcribed target RNA using a commercially available viral RNA extraction kit, followed by direct, nonenzymatic detection using DNA nanoswitches. (B) Positive identification of ZIKV RNA from virus particles spiked into urine based on NASBA. (C) Positive detection of in vitro transcribed SARS-CoV-2 RNA in human saliva based on NASBA. Error bars represent SD from triplicate experiments.

Supplementary Materials

  • Supplementary Materials

    Programmable low-cost DNA-based platform for viral RNA detection

    Lifeng Zhou, Arun Richard Chandrasekaran, Jibin Abraham Punnoose, Gaston Bonenfant, Stephon Charles, Oksana Levchenko, Pheonah Badu, Cassandra Cavaliere, Cara T. Pager and Ken Halvorsen

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