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  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
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  • C12N9/485—Exopeptidases (3.4.11-3.4.19)
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  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
  • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
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  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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  • C12Y102/04—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with a disulfide as acceptor (1.2.4)
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  • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
  • C12Y304/17—Metallocarboxypeptidases (3.4.17)
  • C12Y304/17023—Angiotensin-converting enzyme 2 (3.4.17.23)

Definitions

• a folding-unfolding property of proteins wherein a protein folds and unfolds gradually (one-state downhill folding), can be utilized to develop analog singlemolecule devices.
• a selected protein may be purposely engineered to fold downhill and configured to output corresponding fluorescent signals.
• engineered proteins may be configured as ultra-high sensitive and specific sensors, and may display wide dynamic range, ultrafast response and analog readouts at the single-molecule level.
• FIG. 10A shows an illustration of SARS-CoV-2 spike trimer inserted into nanodisks or solubilized in micelles and an outline of procedure to produce spike-trimer decorated liposomes.
• FIG. 9 shows a schematic illustration of an example multivalent adhesion approach for detecting an example biological agent, such as SARS- Cov-2 in an one-step fluorescence read-out.
• Micrometer-sized polystyrene beads (larger spheres) are coated at a designed density with an engineered peptide binding moiety (protrusions from larger sphere) that recreates the binding site on ACE2 that is recognized by the spike protein of SARS-CoV-2.
• the peptide binding moiety is tagged with a fluorescent moiety (tips of protrusions) for fluorescence-based detection of binding events from viral particles to form a sensor peptide.
• the fluorescent moiety contains a two-color readout for accurate quantitative ratiometric detection of viral particles.
• B-Hel-B is a fluorescence biosensor for measuring pH that can operate in analog fashion at the singlemolecule level.
• a series of stochastic simulations were performed based on the sm-FRET experimental data that we obtained for B-Hel-B (see previous section).
• the sm-FRET data contains bursts of 0.5 ms because they come from free diffusion experiments.
• the liposome-encapsulated B-Hel-B biosensor molecules at various pH values was produced and their fluorescence properties was characterized using a TIRF microscope.
• a total of 120 images were collected in series (13.407 Hz) with 50 ms exposure time to get a continuous stream of data corresponding to 6 s of continuous pH monitoring from individual molecules.
• the 512x512 pixel image was separated onto the donor and acceptor halves, and the two images were overlaid.
• a count threshold of 5,000 was applied to remove the background. Locations of single-molecules were identified from the pixels with maximum intensity, and areas of 5 * 5 pixels surrounding the maximum intensity pixel were used to encompass the complete image from each molecule (optical resolution with our 1.39 N.A.
• Downhill (Un)folding coupled to binding is a powerful mechanism for engineering conformational rheostat transducers that convert the ligand-binding event into an analog signal with an extended detecting range.
• a pH transducer may be engineered by coupling the histidine (un)protonation with a downhill folding protein (gpW) based on the histidine grafting approach, which showed a sensitivity range from 3 to 9.
• the coupling of binding with downhill (un)folding is described by Nagpal, S., Luong, T. D. N., Sadqi, M., and Munoz, V, in ACS Synth.
• a fluorescence-based biosensor may be used for optical sensing due to their high sensitivity and great potential in biomolecule imaging to study the organization and functions of living systems. Among all the advantages, these fluorescence-based biosensors will be of great interest to detect the analytes at the single-molecule level and generate realtime responses.
• the L7H-Cter exhibited a gradual reduction in fluorescence intensity as pH decreases (pH3 to pH9), indicating that the end-to-end distance was shortened as pH increases. Especially, the intensity change was significantly high within 2-folds, which is considering a good signal change.
• a signal readout system may be produced that can process the conformational pH transducing to an optical output using a highly sensitive technique.
• pH fluorescence-based biosensor shows a broadband analog sensing that extends over 8 orders of magnitude of [H+],
• the (un)folding is coupled to binding principle and further, a protein scaffold that exhibits (or is engineered to exhibit) downhill folding characteristics is needed.
• the Calnuc double EF- hand domain from Calbindin a human protein that is unstructured in absence of Ca +2 , but it folds up onto a characteristic double EF-hand domain in presence of Ca +2 in pM concentrations may be selected.
• the EF hands are specific Ca2+ binding motifs that are widespread in Biology (FIG. 4A). Calnuc was cloned as a fusion protein with SUMO, which provided very high expression.
• the Calnuc FRET and PET variants were expressed, purified (using the His-tag of the SUMO construct and a Ni 2+ column, then removal of SUMO by ULP-1 protease, and second Ni 2+ purification step), and properly labelled with the required fluorophores.
• the Ca 2+ sensing properties of both sensors may be characterized via fluorescence in bulk.
• the bulk fluorescence experiments demonstrated sensitivity to Ca 2+ concentrations in the micromolar range, mimicking the changes in Calnuc folding (measured by circular dichroism in the unlabeled protein) upon increases in [Ca 2+ ], Interestingly, in this system the FRET and PET readouts produce sensors with distinct properties due to the different distance sensitivity of the two approaches.
• fluorescence systems that participate in Forster resonance energy transfer (FRET) are used.
• the first emission property is a ratio of fluorescence emission intensity at two different wavelengths
• the second emission property is an increase in emission intensity at one of the two wavelengths, and a decrease in emission intensity at the other wavelength; /. ⁇ ., a change in the ratio of emission intensities.
• Many fluorophore pairs that participate in FRET are known; one example being the combination of Alexa488 and Alexa594.
• the target is a cell.
• the cell can be a prokaryotic cell (e.g., a pathogenic bacterium) or a eukaryotic cell e.g., a pathogenic fungus).
• Additional cellular targets can be animal cells (e.g., human cells) such as, for example, tumor cells.
• AVCHPTA (SEQ ID NO: 8);
• AVCHPTA SEQ ID NOTO
• a functional method for defining common properties of individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz, G. E. and R. H. Schirmer, Principles of Protein Structure, Springer-Verlag, 1979). According to such analyses, groups of amino acids can be defined in which amino acids within a group are preferentially substituted for one another in homologous proteins, and therefore have similar impact on overall protein structure (Schulz & Schirmer, supra). According to this type of analysis, the following groups of amino acids can be conservatively substituted for one another:
• non-naturally occurring amino acid also includes, but is not limited to, amino acids that occur by modification (e.g., post-translational modifications) of a naturally occurring amino acid (including but not limited to, the 20 common amino acids, pyrolysine or selenocysteine).
• a naturally occurring amino acid including but not limited to, the 20 common amino acids, pyrolysine or selenocysteine.
• non-naturally-occurring amino acid include, but are not limited to, N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and O- phosphotyrosine.
• amino acid sequences that have greater than 55%, greater than 60%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, greater than 99.5%, and greater than 99.9% identity with the sequences disclosed herein are also provided.
• a fusion protein comprising both the a-helix or a derivative or portion thereof) and the P-hairpin or a derivative or portion thereof is used as a binding moiety for detection of SARS-CoV-2.
• the a-helix and the P- hairpin sequences can be joined (or are joined) with a flexible linker to generate a fusion peptide that recapitulates the entire viral S-protein binding site (shown in FIG. 8A).
• the helix and hairpin portions of the biosensor are separated, because they do not have sufficient molecular interactions with each other to form a stable complex.
• ESIPT excited state intramolecular proton transfer
• ESIPT fluorophores change their properties depending on the polarity of the environment (i.e.. they exhibit solvatochromism). For example, an ESIPT fluorophore will emit fluorescence at a first wavelength when exposed to water, and at a second, different wavelength when buried inside a protein; or when internalized in a protein-protein, protein- peptide or peptide-peptide complex.
• a binding moiety/fluorescent moiety composition when a binding moiety/fluorescent moiety composition, as described herein, binds to a target (e.g., a viral particle); the fluorescence emission shifts to a higher wavelength.
• a binding moiety/fluorescent moiety composition as described herein, binds to a target (e.g., a viral particle); the fluorescence emission shifts to a lower wavelength.
• the fluorescence emission has a higher wavelength (and, optionally, a greater intensity) when the biosensor is bound to target, than when the biosensor is unbound.
• compositions comprising a binding moiety and a fluorescent moiety are attached to a solid support.
• the solid support is a sphere or is spheroidal, approximating the shape of a cell.
• a spherical or spheroidal solid support has a diameter commensurate with the size of a cell (e.g., a eukaryotic cell; e.g.,. a mammalian cell; e.g., a human cell).
• a solid support will have the approximate size and shape of a human lung epithelial cell.
• the diameter (or nominal diameter for a spheroidal support) is greater than 0.5 pm; or is between 0.5 pm and 1.0 pm; or is between 0.2 and 0.8 pm, or is between 0.3 and 0.7 pm, or is between 0.4 and 0.6 pm, or is between 0.5 pm and 1.5 pm; or is between 1.0 pm and 1.5 pm or is between 0.8 pm and 1.2 pm or is between 0.9 pm and 1.1 pm.
• Example 6 provides sample calculations for determining coverage density of biosensors containing the ACE2 a-helix on a 1 pm polystyrene bead that will provide maximal sensitivity. The same formulae can be used for determining coverage density for other binding moieties, providing that the monovalent dissociation constant of the complex between the binding moiety and the target molecule is known.
• a solid support comprising biosensors can be designed to approximate the size, shape and receptor density of the host cell (hence the term “cell decoy”). Approximating the size and shape of the host cell is accomplished by choosing the size and shape of the solid support. For example, spherical ( and spheroidal) polystyrene beads (as well as beads made of other materials) are available in a variety of sizes, such that it is possible to choose the size of the solid surface to be roughly the size of the host cell. Thus, binding of the target to the cell decoy occurs under steric conditions that are similar to binding of the target to its host cell.
• Fluorescence microscopes with plate-reading capability i.e., plate reader fluorescence imagers
• plate reader fluorescence imagers capable of single-bead resolution
• ImageXpress Pico Molecular Devices, San Jose, CA
• Cytation 5 BioTek Instruments, Santa Clara, CA
• Spark Cyto TECAN US, Morrisville, NC
• Nyone Advanced Robotics, Tarzana, CA, Synentec GmbH, Elmshorn, Germany.
• code will be added to that software (using, e.g., Matlab) to interpret the results; e.g., to convert fluorescence signal to viral load.
• the density of the solid support per unit area of the surface is between one support per 320 pm 2 and one support per 32 mm 2 of the surface (e.g., for a 96-well microtiter plate. In other embodiments, the density of the solid support per unit area of the surface is between one support per 112 pm 2 and one support per 11.2 mm 2 of the surface (e.g., for a 384-well microtiter plate.
• Controls can include sensor beads that have not been contacted with sample, from which a first emission property can be measured. Changes in fluorescence (i.e., differences between a first emission property and a second emission property) are determined and converted to a result (e.g., viral load) by methods known in the art (e.g., a standard curve).
• a result e.g., viral load
• any type of biological sample that can be obtained from a subject is suitable for testing using the methods and compositions disclosed herein.
• respiratory tract samples such as nasopharyngeal swabs, throat swabs, buccal swabs, mucus, saliva and sputum can be used.
• Additional sample types include, without limitation, blood, plasma, serum, cerebrospinal fluid, lymph, urine, stool and semen.
• sensor beads containing different densities of biosensor are arrayed along an axis of the matrix. Because the viral load in subjects with COVID-19 varies so widely (10 3 -10 8 viral particles per ml of saliva), this type of matrix will ensure that one or more sensor beads in the array has a sufficient biosensor density to detect a low virus concentration, while other sensor beads in the array have a biosensor density that will not be saturated by a high virus concentration.
• the F-moc derivatives are incorporated into the a-helix peptide (as shown in Example 6, above) during the solid-phase synthesis at position 21 (indicated by X). This position is completely solvent exposed in absence of virus, thus providing very low fluorescence emission.
• the binding moiety becomes covered by the viral S-protein, resulting in brighter emission and a spectral shift (solvatochromism).
• Computer modeling indicates that the fluorophore will not interfere with the binding between the peptide and the spike protein, but rather will enhance binding through additional hydrophobic interactions.
• the a-helix portion of this hybrid binding domain is similar to the a-helix peptide used for the ESIPT probe described in Examples 6 and 7, with Phel4 of the helix sequence shown in Example 5 substituted by Ala, no change at His 16 of the helix sequence shown in Example 1, Phe22 of the helix sequence shown in Example 5 substituted by Cys to provide a site for attachment of the fluorophore (see below), and Trp30 of the helix sequence shown in Example 5 substituted with tyrosine, to remove a potential (binding unspecific) PET donor.
• the N-terminal linker sequence (SSGSG) is slightly different from that present in the al- helix binding domain described in Examples 6 and 7.
• a sortase recognition sequence is present at the C-terminus of this peptide. Accordingly, the amino acid sequence of the helical portion of the PET binding moiety is:
• the peptide is esterified at its C-terminus, during solid-phase synthesis, to contain a N-hydroxysuccinimide ester (NHS ester).
• NHS-esterified peptide is combined with aminated beads in an aqueous solution at neutral pH (e.g., 50 mM PBS, 150 mM NaCl, at a pH of 7.0-7.5 or 50 mM Tris, 150 mM NaCl, at a pH of 7.0-7.5) and incubated for two hours at ambient temperature, during which time the NHS-activated carboxyl groups of the peptide react spontaneously with amino groups on the bead. Coated beads are then separated from the reaction mixture by centrifugation.
• neutral pH e.g., 50 mM PBS, 150 mM NaCl, at a pH of 7.0-7.5
• Tris 150 mM NaCl
• Binding of the ACE2 receptor a-helix binding moiety (Example 6) to the spike protein with a Kd of 50 nM yields a AG of -41 kJ/mol.

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