key: cord-0711150-ypheoxs3 authors: Tripathy, Soumya P.; Ponnapati, Manvitha; Jacobson, Joseph; Chatterjee, Pranam title: Sub-Picomolar Detection of SARS-CoV-2 RBD via Computationally-Optimized Peptide Beacons date: 2021-06-04 journal: bioRxiv DOI: 10.1101/2021.06.04.447114 sha: d2d5151179d7f1c8b6ca8fbedf1e52e460935bcf doc_id: 711150 cord_uid: ypheoxs3 The novel coronavirus SARS-CoV-2 continues to pose a significant global health threat. Along with vaccines and targeted therapeutics, there is a critical need for rapid diagnostic solutions. In this work, we employ deep learning-based protein design to engineer molecular beacons that function as conformational switches for high sensitivity detection of the SARS-CoV-2 spike protein receptor binding domain (S-RBD). The beacons contain two peptides, together forming a heterodimer, and a binding ligand between them to detect the presence of S-RBD. In the absence of S-RBD (OFF), the peptide beacons adopt a closed conformation that opens when bound to the S-RBD and produces a fluorescence signal (ON), utilizing a fluorophore-quencher pair at the two ends of the heterodimer stems. Two candidate beacons, C17LC21 and C21LC21, can detect the S-RBD with limits of detection (LoD) in the sub-picomolar range. We envision that these beacons can be easily integrated with on-chip optical sensors to construct a point-of-care diagnostic platform for SARS-CoV-2. in the test volume (6). RT-PCR tests, however, require laborious and expensive nucleic 29 acid isolation, purification, and processing steps, which increases both the turnaround 30 time of detection and the cost of testing (6, 7). Alternatively, there are FDA-authorized 31 low-sensitivity, inexpensive, and rapid diagnostics. These tests, which often rely on anti-32 gen detection, have LoDs of 10 5 -10 7 RNA copies/ml, or around 1-100 femtomolar (fM) (8). Recently, there has been significant effort to detect SARS-CoV-2 via fluorescence-based 35 readouts to allow for specific signal amplification (9-12). Such methods largely rely on 36 binding to SARS-CoV-2 RNA or DNA, which requires isolation of nucleic acids, as de-37 scribed above. In this study, we develop a molecular assay to detect the spike protein 38 receptor binding domain (S-RBD) of SARS-CoV-2 using computationally-validated pep-39 tide beacons, which enable single-step detection of S-RBD presence through the produc-40 tion of a fluorescence signal. Our eventual goal is to integrate these optimized beacons 41 within miniaturized total internal reflection fluorescence (TIRF) microscopes, which pro-Our molecular beacon design includes two heterodimer-forming peptides, a binding ligand 48 to the S-RBD, as well as a fluorophore-quencher pair at the terminal ends of the beacon 49 ( Figure 1A ). This fluorophore-quencher pair induces fluorescence quenching through the 50 mechanism of Förster resonance energy transfer (FRET), where the efficiency of energy 51 transfer between the fluorophore and quencher is proportional to their spatial distance. Hence, a small change in spatial distance between the two beacon arms can drastically The three peptide beacon sequences were folded in the absence of S-RBD using trRosetta. 134 trRosetta is a deep learning tool to predict structures from sequence information (17). The three peptide beacon sequences were also folded using Rosetta Abinitio folding. Abi-136 nitio folding solves the protein structure from sequence through physics-based constraints 137 rather than relying on previously solved structures like trRosetta (18) . The three peptide beacon sequences were docked against the S-RBD using HDOCK. HDOCK is a protein-protein docking platform that combines ab-initio docking and template- All samples were performed in independent duplicates (n=2), triplicates (n=3), or quin-References 220 Detection of SARS-CoV-2 in different types of clinical specimens Clinical validation of a cas13-based assay for the detection of 223 SARS-CoV-2 RNA SARS-CoV-2 detection with CRISPR diagnostics CRISPR-cas12-based detection of SARS-CoV-2 Rapid point-of-care detection of SARS-CoV-2 using reverse tran-228 scription loop-mediated isothermal amplification (RT-LAMP) Diagnostics for SARS-CoV-2 infections RT-qPCR testing of SARS-CoV-2: A primer Comparison of seven commercial SARS-CoV-2 rapid point-of-235 care antigen tests: a single-centre laboratory evaluation study Point-of-care bulk testing for SARS-CoV-2 by combining hy-238 bridization capture with improved colorimetric LAMP Rosetta in CASP4: Progress in ab initio protein structure predic-263 tion The HDOCK server for integrated pro-265 tein-protein docking Versatile and multivalent nanobodies efficiently neutralize SARS-267 Influenza hemagglutinin protein stability, 269 activation, and pandemic risk De 271 novo protein design by deep network hallucination A) Low-fluorescent state is the closed heterodimer state of the peptide beacon in the absence of S-RBD. B) High-fluorescent state is the opencoil state after binding of S-RBD with the loop of the peptide beacon. C) CxxL (25 uM All samples were performed in independent transfection duplicates (n=2) and gated on GFP+ fluorescence. Mean percentage of GFP+ cell depletion was calculated in comparison to the S-RBD-sfGFP only control