Pseudo-Luciferase Activity of the SARS-CoV-2 Spike Protein for Cypridina Luciferin

Enzymatic reactions that involve a luminescent substrate (luciferin) and enzyme (luciferase) from luminous organisms enable a luminescence detection of target proteins and cells with high specificity, albeit that conventional assay design requires a prelabeling of target molecules with luciferase. Here, we report a luciferase-independent luminescence assay in which the target protein directly catalyzes the oxidative luminescence reaction of luciferin. The SARS-CoV-2 antigen (spike) protein catalyzes the light emission of Cypridina luciferin, whereas no such catalytic function was observed for salivary proteins. This selective luminescence reaction is due to the enzymatic recognition of the 3-(1-guanidino)propyl group in luciferin at the interfaces between the units of the spike protein, allowing a specific detection of the spike protein in human saliva without sample pretreatment. This method offers a novel platform to detect virus antigens simply and rapidly without genetic manipulation or antibodies.


Synthesis and characterization of the Cypridina luciferin analogue (CLA) series
General: All reagents and solvents for organic synthesis were purchased from common commercial suppliers (Tokyo Kasei, Sigma Aldrich, or FUJIFILM Wako Pure Chemical) and were used without purification.All moisture-sensitive reactions were carried out under an argon atmosphere.The composition of mixed solvents is given as the volume ratio (v/v). 1 H NMR spectra were recorded on an Avance III-500 (Bruker Ltd.) spectrometer at room temperature.The 1 H NMR measurements were performed at 500 MHz.All chemical shifts are expressed relative to tetramethylsilane ( = 0.0 ppm) as the internal standard or residual undeuterated solvent peaks (CHD2OD in CD3OD:  = 3.31 ppm for 1 H); all coupling constants are given in Hz.
General synthetic procedure for the CLA series: Pyruvaldehyde (2 eq.) in ethanol (2.0 mL) and Milli-Q water (0.2 mL) was added to the amino-pyrazine analogue (0.13-0.19 mmol), before the mixture was stirred at room temperature (RT).After vacuum deaeration, the solution was cooled to 0 °C and hydrochloric acid (0.1 mL) was added.Once the solution reached RT, it was heated and stirred for 3 h at 80 °C.Then, the solvent was removed under reduced pressure, and the crude material was purified by column chromatography on silica gel (eluent composition: dichloromethane/methanol = 20/1), affording the luciferin as a yellow or brown solid.This synthetic scheme is a slight modification of a reported one

Luminescence intensities of 36 IPT luciferins with the monomeric S protein (full length) of SARS-CoV-2
To 5 μL of an aliquot of the monomeric S protein (722 nM) in a 384-well microplate (PerkinElmer, Massachusetts, USA), 45 μL of 20 μM luciferin in PBS buffer (pH = 7.4) was added, and the luminescence signals were read immediately for 60 s using a plate reader (Glomax ® Explorer; Promega, Wisconsin, USA).

Luminescence intensities of luciferins with the trimeric S protein (full length) of SARS-CoV-2
To 5 μL of an aliquot of the trimeric S protein (722 nM) in a 96-well microplate (PerkinElmer), 45 μL of 20 μM luciferin in PBS buffer (pH = 7.4) was added using an injector, and the luminescence signals were read immediately for 60 s using a plate reader (Glomax ® Explorer).

Kinetic parameters (Km and Vmax) of luciferin with S protein
To 5 μL of an aliquot of the monomeric or trimeric S protein (722 nM) in a 96-well microplate (PerkinElmer), 45 μL of 0-100 μM or 0-50 μM luciferin in PBS buffer (pH = 7.4) was added using an eight-channel pipette, and the luminescence signals were read immediately for 60 s using a plate reader (Spark Cyto; TECAN, Männedorf, Switzerland) with the following settings: mode, kinetic; kinetic cycles, 60 or 30; integration time, 1 s.The kinetic parameters (Km and Vmax) were calculated by fitting the total luminescence intensity for 30 s, excluding the background signals obtained with only luciferin, using the Michaelis-Menten equation in GraphPad Prism 9.

Luminescence spectra
To 5 μL of an aliquot of the protein or organic solvent in a 96-well microplate (PerkinElmer), 45 μL of 20 μM luciferin in PBS buffer (pH = 7.4) was added using an injector, and the luminescence spectra were read immediately using a plate reader (Spark Cyto) with the following settings: integration time, 1 s; central wavelength start, 398 nm; and central wavelength end, 653 nm.

Luminescence intensities of luciferin with the salivary proteins
To 5 μL of an aliquot of the salivary proteins (0.1 mg/mL) in a 96-well microplate (PerkinElmer), 45 μL of 20 μM Cypridina luciferin in PBS buffer (pH = 7.4) was added using an injector, and the luminescence signals were read immediately for 60 s using a plate reader (Glomax ® Explorer).

SARS-CoV-2 trimeric S protein assay in human saliva
A human saliva sample was centrifuged at 1000g for 10 min at 4 °C to remove floating material according to a reported procedure 7 .In the luminescence assay, to 5 μL of an aliquot of 10% human saliva spiked with the trimeric S protein (25-250 μg/mL) in a 96-well microplate (PerkinElmer), 45 μL of 20 μM Cypridina luciferin in HEPES buffer (pH = 7.4) was added using an injector, and the luminescence signals were read immediately for 60 s using a plate reader (Glomax ® Explorer).
To calculate the concentration of S protein in the saliva from the luminescence signals, we used a calibration curve obtained by measurement in a buffer system spiked with the same S6 concentration of protein.In the ELISA, trimeric S protein from Bio-Serv, Co. was used as standard sample and the concentration of S protein in saliva was measured according to the manufacturer's protocol.S11

Docking-simulation studies
3 3 .The cluster centers of S protein's Down-to-Up transitions from the previous MD simulations 4 were used as receptors.Multiple conformations of Spike including, Down, Down-like and Down-with-slightlyincreased-hinge-angle (I1) of the receptor binding domain (RBD) were used.All seven conformations of the trimeric S protein are glycosylated.The two ligands (Luciferin and CLA1)were constructed, and energy-minimized using the Merck molecular force field (MMFF94s) as implemented in ChemDoodle 3D software5 .Two docking steps were performed.Initially, the whole protein was considered as a receptor (blind docking), using a large box size of 140 x 126x 126 Å 3 with a grid spacing of 1.00 Å.The monomeric S protein was located in a box the size of 140 x 94 x126 Å 3 .The top-20 poses were generated while based on docking scores, and only the top ten were used for further analysis.Based on the blind-docking results, refined docking with a default grid spacing of 0.375 Å were performed, using six potential receptor regions.This includes the N-terminal domain (NTD), NTD/RBD interface, small subdomain 1 (SD1)/ subunit 2 (S2) interface, and the three regions of S2/S2/S2 including top and bottom interfaces as well as the S2 outer region.The top-three poses from all six docking simulations (72 poses) of the S protein Down conformations (symmetric and asymmetric) were further analyzed and energy averaged.The PyMOL software was used to investigate all binding poses interactions and visualization6 .

Figure S7 .Figure S8 .
Figure S7.Predicted binding affinity and pocket.(a) Comparison of the average binding affinity using the top-three-ranked poses from six specified regions, including four interfaces (NTD/RBD, SD1/S2, S2/S2/S2 at the top region closer to RBD, S2/S2/S2 at the bottom region), the NTD domain, and the S2 unit.(B) Binding pose of Cypridina luciferin at the SD1/S2 interface in the symmetric D1 conformation. 2.

Table S1 .
Vmax values of Cypridina luciferin with the S1+S2 full-length protein or fragment proteins

Table S2 .
Vmax values of Cypridina luciferin with the fragment proteins

Table S3 .
Vmax values of Cypridina luciferin with the trimeric S protein

Table S5 .
Vmax values of CLA1 with the monomeric S protein or the trimeric S protein

Table S6 .
Comparison of the binding affinities of the top-ten-ranked poses of luciferin with the trimeric S protein