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

Abstract

Detection of viruses is critical for controlling disease spread. Recent emerging viral threats, including Zika virus, Ebola virus, and SARS-CoV-2 responsible for coronavirus disease 2019 (COVID-19) highlight the cost and difficulty in responding rapidly. To address these challenges, we develop a platform for low-cost and rapid detection of viral RNA with DNA nanoswitches that mechanically reconfigure in response to specific viruses. Using Zika virus as a model system, we show nonenzymatic detection of viral RNA with selective and multiplexed detection between related viruses and viral strains. For clinical-level sensitivity in biological fluids, we paired the assay with sample preparation using either RNA extraction or isothermal preamplification. Our assay requires minimal laboratory infrastructure and is adaptable to other viruses, as demonstrated by quickly developing DNA nanoswitches to detect SARS-CoV-2 RNA in saliva. Further development and field implementation will improve our ability to detect emergent viral threats and ultimately limit their impact.

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.