Toward a Home Test for COVID-19 Diagnosis: DNA Machine for Amplification-Free SARS-CoV-2 Detection in Clinical Samples

Abstract

Nucleic acid-based detection of RNA viruses requires an annealing procedure to obtain RNA/probe or RNA/primer complexes for unwinding stable structures of folded viral RNA. In this study, we designed a protein-enzyme-free nano-construction, named four-armed DNA machine (4DNM), that requires neither an amplification stage nor a high-temperature annealing step for SARS-CoV-2 detection. It uses a binary deoxyribozyme (BiDz) sensor incorporated in a DNA nanostructure equipped with a total of four RNA-binding arms. Additional arms were found to improve the limit of detection at least 10-fold. The sensor distinguished SARS-CoV-2 from other respiratory viruses and correctly identified five positive and six negative clinical samples verified by quantitative polymerase chain reaction (RT-qPCR). The strategy reported here can be used for the detection of long natural RNA and can become a basis for a point-of-care or home diagnostic test.

Keywords: 10-23 DNAzyme; DNA machine; binary hybridization probe; deoxyribozyme sensor; detection of folded RNA.

Figure 1

Design of the DNA constructs used in this study. A) BiDz probe: DNA strands Dz_a and Dz_b, bind RNA analyte and form the catalytic core that cleaves fluorophore and quencher labelled F sub. B) The four‐armed DNA nanomachine (4DNM) has Dz_a and Dz_b attached to a double stranded DNA scaffold (shaded grey). Arms 3 and 4 are designed to tightly bind natural RNA, thereby unwinding its secondary structure. F sub is not shown.

Figure 2

4DNM selectively detects CoV2 RNA. A) Comparison of fluorescent response of 4DNM with BiDz in the absence or presence (dark grey and light grey bars, respectively) of 125 pM CoV2 RNA. F‐sub (200 nM) was incubated with either BiDz (20 nM Dz_a and 5 nM Dz_b) or DNM (20 nM Dz_a and 5 nM of T1/T2 complex) in the presence 3.8×10⁹ of either CoV2 or OC43 RNA molecules in a reaction buffer 1 (50 mM HEPES, pH 7.5, 150 mM KCl, 15 mM NaCl, 200 mM MgCl₂). Fluorescence at 517 nm (λ_ex=485 nm) was registered after 1 or 3 h of incubation at 55 °C. The error bars represent one standard deviation from average values calculated after three independent experiments. B) Selectivity of 4DNM machine. Fluorescent response of 4DNM to 3.8×10⁹ molecules in 50 μL (125 pM) of CoV2 or OC43 RNA (See experimental section). The conditions were as described for panel A.

Figure 3

Analysis of clinical samples using 4DNM. All samples contained F‐sub (200 nM) and 4DNM (20 nM Dza and 5 nM of T1/T2 complex) in reaction buffer 1 in the absence (−) or presence of clinical samples. Positive control (last bar) contained 20 pM of the synthetic CoV2‐DNA1 analyte. Fluorescence at 517 nm (λ_ex=485 nm) was registered after 3 h of incubation at 55 °C. Nine CoV‐2 positive and five negative samples were collected from 14 different individuals and confirmed by RT‐qPCR with indicated threshold cycle (Ct) values. The Ct values for positive samples were as follows: 6–20.4; 7–21.0; 8–21.8, 9–22.0; 10–22.9; 11–24.2; 12–25.5; 13–25.9; 14–27.7. Clinical samples 1–14 lack standard deviation since all available RNA from each sample was fully used only in one measurement to ensure the highest available concentration of viral RNA. Standard deviation for the negative (−) and the positive (+) controls were determined after three independent measurements. The threshold, line corresponds to 3 standard deviations above average (−) control.