Dumbbell-type triplex molecular switch-based logic molecular assays of SARS-CoV-2

Abstract

Accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is of great importance to control the COVID-19 pandemic. The gold standard assays for COVID-19 diagnostics are mainly based on separately detecting open reading frame 1ab (ORF1ab) and nucleoprotein (N) genes by RT-PCR. However, the current approaches often obtain false positive-misdiagnose caused by cross-contamination or undesired amplification. To address this issue, herein, we proposed a dumbbell-type triplex molecular switch (DTMS)-based, logic-gated strategy for high-fidelity SARS-CoV-2 RNA detection. The DTMS consists of a triple-helical stem region and two-loop regions for recognizing the ORF1ab and N genes of SARS-CoV-2. Only when the ORF1ab and N gene are concurrent, DTMS experiences a structural rearrangement, thus, bringing the two pyrenes into spacer proximity and leading to a new signal readout. This strategy allows detecting SARS-CoV-2 RNA with a detection limit of 1.3 nM, independent of nucleic acid amplification, holding great potential as an indicator probe for screening of COVID-19 and other population-wide epidemics.

Keywords: Dumbbell-type triplex molecular switch; High-fidelity; Logic-gate; SARS-CoV-2 RNA.

Scheme 1

Illustration of DTMS-based AND-logic-gated identification of the ORF1ab and N genes of SARS-CoV-2 RNA genome.

Fig. 1

(A) Fluorescence intensity of FIS (400 nM) without (red) and with (black) the addition of DTMS (100 nM) in the phosphate buffer saline. (B) CD spectrograph of DTMS (2 μM) (black) and the precursor of DTMS (2 μM) (red) in the phosphate buffer saline.

Fig. 2

PAGE analysis. (A) Characterization of the nuclease resistance to DNase I cleavage of MB and DTMS. (B) Verification of the hybridization of DTMS with the ORF1ab and N gene. Lane 1: ORF1ab gene; lane 2: N gene; lane 3: the precursor of DTMS; lane 4: DTMS; lane 5: DTMS + ORF1ab gene + N gene; lane 6: DNA marker.

Fig. 3

(A) Fluorescence emission spectra of Py-labeled DTMS (black); Py-DTMS + N gene (blue); Py-DTMS + ORF1ab gene (red); Py-DTMS + N gene + ORF1ab gene (green) in the phosphate buffer saline. (B) The fluorescence intensity at 490 nm which from a to d: Py-DTMS; Py-DTMS + N gene; Py-DTMS + ORF1ab gene; Py-DTMS + ORF1ab + N gene. The concentrations of Py-DTMS, N gene and ORF1ab gene were 100 nM. Error bars stand for SD (n = 3). * , P < 0.05, * *, P < 0.01, * ** , P < 0.001, * ** *, P < 0.0001; ns, not signifificant.

Fig. 4

Optimization of experiment conditions. Concentrations of Spermidine (A), Mg²⁺ (B), γ-CD (C); (D) pH value; a: Py-DTMS + ORF1ab + N gene, b: Py-DTMS + N gene, c: Py-DTMS + ORF1ab. (E) hybridization time of DTMS with ORF1ab gene and N gene. Error bars stand for SD (n = 3).

Fig. 5

(A) Fluorescence emission spectra of Py-DTMS in the presence of increasing concentrations of ORF1ab and N genes. The arrow indicates the signal changes with increases in target concentrations (0, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, and 200 nM). (B) S/B of Py-DTMS in the presence of increasing concentrations of ORF1ab and N genes. (C). The linear relationship between the S/B value and the concentration of targets ranges from 0 to 60 nM. (D) The S/B value of Py-DTMS response to different combinations of targets. The concentrations of Py-DTMS and targets were 100 nM. From 1–6: ORF1ab + N genes；M + N gene；S + ORF1ab；S + N gene；M + ORF1ab；M + S. The above experiments were all performed in the phosphate buffer saline (500 nM Spermine, 5 mM MgCl₂, 5 mM γ-CD, pH 6.5), and the reaction time was 20 min. Error bars stand for SD (n = 3). * , P < 0.05, * *, P < 0.01, * ** , P < 0.001, * ** *, P < 0.0001; ns, not signifificant.