BRET-based biosensors for SARS-CoV-2 oligonucleotide detection

Abstract

The need for the early detection of emerging pathogenic viruses and their newer variants has driven the urgent demand for developing point-of-care diagnostic tools. Although nucleic acid-based methods such as reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and loop-mediated isothermal amplification (LAMP) have been developed, a more facile and robust platform is still required. To address this need, as a proof-of-principle study, we engineered a prototype-the versatile, sensitive, rapid, and cost-effective bioluminescence resonance energy transfer (BRET)-based biosensor for oligonucleotide detection (BioOD). Specifically, we designed BioODs against the SARS-CoV-2 parental (Wuhan strain) and B.1.617.2 Delta variant through the conjugation of specific, fluorescently modified molecular beacons (sensor module) through a complementary oligonucleotide handle DNA functionalized with the NanoLuc (NLuc) luciferase protein such that the dissolution of the molecular beacon loop upon the binding of the viral oligonucleotide will result in a decrease in BRET efficiency and, thus, a change in the bioluminescence spectra. Following the assembly of the BioODs, we determined their kinetics response, affinity for variant-specific oligonucleotides, and specificity, and found them to be rapid and highly specific. Furthermore, the decrease in BRET efficiency of the BioODs in the presence of viral oligonucleotides can be detected as a change in color in cell phone camera images. We envisage that the BioODs developed here will find application in detecting viral infections with variant specificity in a point-of-care-testing format, thus aiding in large-scale viral infection surveillance.

Keywords: COVID-19; SARS-CoV-2; bioluminescence; bioluminescence resonance energy transfer; biosensor; molecular beacon.

FIGURE 1

Bioluminescence resonance energy transfer (BRET)-based biosensor for viral oligonucleotide detection (BioOD). Schematic representation of the BRET-based biosensor (BioOD) design for SARS-CoV-2 parental and Delta variant nucleic acid detection. The biosensor consists of a reporter module (constant DNA handle conjugated to a luciferase protein) and a DNA stem loop-based sensor module (viral strain and variant nucleic acid, RNA in this case, binding oligonucleotide sequence). Close positioning of the luciferase (NLuc; BRET donor) and fluorophore (BRET acceptor) proteins results in significant BRET in the absence of a viral oligonucleotide. Binding of the viral oligonucleotide will result in the dissolution of the loop, leading to a decrease in BRET.

FIGURE 2

Comparison of GLuc and NLuc as the BRET donor luciferase in the BioOD. (A and B) Surface and schematic representation of SNAP-GLuc-His₁₀ (left panel) and NLuc(C166S/G182C)-His₁₀ (right panel) constructs. (B) Graph showing bioluminescence spectra (left panel) of the SNAP-GLuc and NLuc(C166S/G182C) (right panel) proteins. Insets show the total bioluminescence of the proteins.

FIGURE 3

SDS-PAGE shows NLuc-constant handle DNA oligonucleotide conjugation. (A) SDS-PAGE image showing a shift in the electrophoretic mobility of the NLuc(C166S/G182C)-His₁₀ protein after handle DNA conjugation. (B) Graph showing line-scan intensity profiles of the lanes in image (A). Numbers 01 and 02 in panels A and B indicate reaction numbers (performed twice).

FIGURE 4

Time- and concentration-dependent change in the kinetics and affinity of the BioOD. (A and B) Schematic showing BRET-based parental and Delta variant BioODs. Data were fit to two Gaussian model reflecting Alexa 488 fluorescence and NLuc bioluminescence peaks (A) and Alexa 532 and NLuc bioluminescence peaks (B). (C and D) Graphs showing time-dependent decrease in BRET. (E and F) Graphs showing the oligonucleotide concentration-dependent change in bioluminescence spectra. (G and H) Graphs showing the BRET of parental (G) and Delta variants (H). Data are shown as the mean ± SD obtained from three independent experiments, with each experiment performed in triplicate.

FIGURE 5

Specificity of the BRET response of parental and Delta variant-specific BioODs. (A–O) Schematic diagram showing the binding and the region of complementarity of complete (A), stem + loop (D), stem + half-loop (G), loop (J), and half-loop (M) Graphs showing the % change in BRET of the parental (B,E,H,K,N) and Delta variant (C,F,I,L,O) BioODs in the presence of the indicated concentrations of the complete (B and C), stem + loop (E and F), stem + half-loop (H and I), loop (K,L), and half-loop (N,O) specific to the parental (B,E,H,K,N; left panels) and Delta variant (B,E,H,K,N; right panels) BioODs and Delta variant (C,F,I,L,O; left panel) and parental (C,F,I,L,O; right panel) BioODs. Data are shown as the mean ± SD from three independent experiments, with each experiment performed in triplicate.

FIGURE 6

BioOD enables cell phone camera-based detection of the viral DNA oligonucleotide. (A) Image showing the presence and absence (control) of the complementary oligonucleotides of parental and Delta variant in the RGB composite. (B) Graphs showing the parental BioOD (right panel) and Delta BioOD expressing higher BRET, with green in the channel with the control, change in the BRET with blue, and less green in the presence of the complementary oligonucleotides. Graph showing the intensity ratio of red to blue in the presence of control and no change in color in the presence of complementary oligonucleotides. Data shown are the mean ± SD obtained from three independent experiments.