REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The official copy of the Sequence Listing is submitted concurrently with the specification as an ASCII formatted text file via EFS-Web, with a file name of “ARX007_ST25.txt”, a creation date of Feb. 23, 2017, and a size of 45 kilobytes. The Sequence Listing filed via EFS-Web is part of the specification and is incorporated in its entirety by reference herein.

BACKGROUND OF THE INVENTION

The olfactory receptor genes have been characterized through homology as seven transmembrane domain G protein-coupled receptors (GPCR). It is estimated that there are probably 500-750 olfactory receptor gene sequences in humans, while there are 500-1000 olfactory genes in rat and mouse. Olfactory receptors are concentrated on the surface of the mucus coated cilia and odorant molecules bind to the olfactory receptors in the olfactory epithelium. Since mammals can detect at least 10,000 odors and there are approximately 1,000 or fewer olfactory receptors, many odorants must interact with multiple olfactory receptors.

The discriminatory power of olfactory receptors is such that it can perceive thousands of volatile chemicals as having different odors. It is known that the olfactory system uses a combinatorial receptor coding scheme to decipher the odor molecules. One olfactory receptor can recognize multiple odorants and one odorant is recognized by multiple olfactory receptors. A slight structural change in the odorant or a change in the concentration of the odorant in the environment results in a change in the odor-code of these receptors.

Each mammalian olfactory receptor neuron encodes only one olfactory receptor. The axons of the neurons expressing the same olfactory receptor converge to one olfactory bulb, which then processes the information to the brain. Olfactory receptors are structurally similar to G-Protein Coupled Receptors (GPCRs) and contain seven transmembrane (TM) domains connected by loops. The functionally important residues are present on the transmembrane helices 2-7.

Odor molecules belong to a variety of chemical classes: from alcohols, aldehydes, ketones and carboxylic acids to sulphur-containing compounds and essential oils. The physicochemical descriptors of odor molecules play an important role in the prediction of odor response by the olfactory receptor. Very identical olfactory receptor sequences can have a structural bias for ligand specificity on the basis of the number of carbon atoms present in the ligands. About 8000 odorants have been identified in food. About 400 food odorants have been characterized and this number approximately equals the number of olfactory receptors found in humans. The response of mixtures of odorants is neither the additive nor an average of its components. Some mixtures lead to the emergence of novel perceptual qualities that were not present in the individual components.

It is an object of this invention to provide a biosensor that produces an aromagraph for a mixture, composition, or molecule. It is an object of the invention to provide a biosensor that is used to deconstruct the components of a mixture, composition or molecule that are responsible for the scent, smell, odor, aroma, and/or taste of the mixture, composition or molecule. It is also an object of the invention to provide a biosensor that is used to diagnose and remediate malodors.

SUMMARY OF THE INVENTION

The invention relates to biosensors for the detection of interactions at an Olfactory Receptor. In some embodiments, the invention relates to the use of the biosensors of the invention to detect the interaction of odorants at Olfactory Receptors. In some embodiments, a plurality of biosensors are used to detect the interaction of an odorant at a plurality of Olfactory Receptors. In some embodiments, the plurality of biosensors represent the repertoire or a portion of the repertoire of an animals Olfactory Receptors. In some embodiments, the plurality of biosensors represent the repertoire or a portion of the repertoire of human Olfactory Receptors. In some embodiments, the plurality of biosensors represent the portion of the repertoire of human Olfactory Receptors that detect odorants in solution. In some embodiments, the plurality of biosensors represent the portion of the repertoire of human Olfactory Receptors that detect odorants in the gaseous phase. In some embodiments, the plurality of biosensors represent the portion of the repertoire of human Olfactory Receptors that detect a particular odorant. In some embodiments, the plurality of biosensors represent the portion of the repertoire of human Olfactory Receptors that detect a particular class or type of odorant.

In some embodiments, individual biosensors are comprised of an Olfactory Receptor that is fused in its N-terminal region to a polypeptide sequence that targets the nascent polypeptide to the host cell membrane, and fused in its C-terminal region to a polypeptide that stabilizes the receptor. In some embodiments, the polypeptide fused to the C-terminal region of the Olfactory Receptor targets the receptor to the outermembrane of the host cell. In some embodiments, the N-terminal portion of the biosensor is from positions 1-55 of the rat olfactory receptor RI7 (RI7). In some embodiments, full length olfactory receptors are used without fusing to another protein. In some embodiments, the nucleic acids encoding the RI7 portion of the fusion receptor or the full length olfactory receptor are preceded by a nucleic acid encoding an eight amino acid FLAG tag (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (SEQ ID NO:1)), e.g., used for identification and purification of the olfactory receptor fusion protein. In some embodiments, the Olfactory Receptor sequences are followed by a cassette encoding Green Fluorescent Protein or Red Fluorescent protein. The GFP or RFP is fused to the olfactory receptor to allow detection of the location of the olfactory receptor when it is expressed in host cells. In some embodiments, the Olfactory Receptor is a mammalian olfactory receptor. In some embodiments, the Olfactory Receptor is a human Olfactory Receptor. In some embodiments, the human olfactory receptor is OR1A1, OR2W1, OR2J2, OR5P3, or OR6A2. In some embodiments, the ORF sequence fused in the construct comprises nucleic acids encoding the amino acids sequence of the Olfactory Receptor from amino acid position 56 to the end of the amino acid sequence.

In some embodiments, a construct is used for accepting ORF cassettes to make new biosensors of the invention. In some embodiments the ORF cassette is fused into the construct so full length human receptor protein is encoded in the construct. In some embodiments, the nucleic acid encoding the full length human olfactory receptor is fused in frame with a nucleic acid encoding an eight amino acid FLAG tag, (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (SEQ ID NO:1)). In some embodiments, the Olfactory Receptor sequences are followed by a cassette encoding Green Fluorescent Protein or Red Fluorescent protein.

In some embodiments, the biosensors of the invention are also comprised of a G-protein and an adenylate cyclase. In some embodiments, the G-protein is comprised of three subunits the Gα subunit, Gβ subunit, and Gγ subunit. In some embodiments, the adenylate cyclase and the G protein are from the same species. In some embodiments, the adenylate cyclase and the G protein are from different species. In some embodiments, the G protein subunits are from the same or from different species. In some embodiments, the Olfactory Receptor, G protein and adenylate cyclase are from the same species, and in some embodiments, one or more of the components are from different species. In some embodiments, the biosensor polypeptides of the invention include polypeptides that have 70%, 80%, 90%, 95%, and 99% sequence homology with SEQ ID NO: 6-10 and 12-17.

In some embodiments, the biosensor of the invention includes a polypeptide that directly or indirectly produces a reporter molecule. In some embodiments, the biosensor of the invention includes a polypeptide that is the reporter molecule (e.g., an optical reporter). In some embodiments, the polypeptide is an adenylate cyclase. In some embodiments, the reporter molecule is cAMP. In some embodiments, the reporter(s) produced by the biosensor has a dynamic range of six to seven orders of magnitude, and the biosensor coupled to the reporter can detect binding of odorants and other molecules in a range of 0.15 parts per billion to about 420,000 parts per billion, or 10−9M to about 10−3M. In some embodiments, the window of detection is six to seven orders of magnitude within the range of 10 M to 10−12M. In some embodiments, the biosensor of the invention has a dynamic range of nine to ten orders of magnitude. In some embodiments, the biosensor of the invention can detect binding of odorants and other molecules in a range of 10−11M to about 10−2M. In some embodiments, the window of detection is nine to ten orders of magnitude within the range of 10 M to 10−12M. In some embodiments, the biosensor of the invention has a dynamic range of three to five orders of magnitude. In some embodiments, the window of detection is three to five orders of magnitude within the range of 10 M to 10−12M. In some embodiments, the biosensor of the invention has a dynamic range of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 orders of magnitude.

The invention also relates to nucleic acids encoding the biosensors of the invention. In an embodiment, the nucleic acids of the invention include nucleic acids that hybridize under stringent hybridization conditions to nucleic acids encoding one of the biosensor polypeptides of the invention. In an embodiment, the biosensor polypeptides of the invention include the polypeptides encoded by nucleic acids that hybridize under stringent hybridization conditions to nucleic acids encoding the biosensor polypeptides describe above. In an embodiment, the nucleic acids of the invention encode a polypeptide of one of SEQ ID NOS: 6-10 and 12-17, or are a nucleic acid that hybridizes under stringent hybridization conditions to a nucleic acid encoding a polypeptide of one of SEQ ID NOS: 6-10 and 12-17. In an embodiment, the nucleic acids of the invention encode a polypeptide that has 70%, 80%, 90%, 95%, and 99% sequence identity with one of SEQ ID NOS: 6-10 and 12-17.

The invention relates to the biosensor polypeptides and biosensor nucleic acids contained within host cells. In some embodiments, the host cells are eukaryotic cells. In some embodiments, the host cell is a fungal cell, animal cell, plant cell, or algae cell. In some embodiments, the fungal cell is selected fromSaccharomyces, Pichia, Aspergillus, Chrysosporium, orTrichoderma. In some embodiments, the fungal cell isSaccharomyces cerevisiae, Pichia pastoris, Aspergillus niger, Aspergillus oryzae, Chrysosporium lucknowense, orTrichoderma reesei. In some embodiments, the host cell is a mammalian cell line derived from Chinese hamster cells, Human kidney cells, Monkey kidney cells, Human cervical cancer cells, or Mouse myeloma cells. In some embodiments the host cell is a human cell. In some embodiments, the host cell is a murine cell. In some embodiments, the host cell is a canine cell.

The invention also relates to the use of host cells containing the biosensor polypeptide and biosensor nucleic acids of the invention. In some embodiments, the invention relates to the use of membrane fractions with the biosensor made from host cells with the biosensor. In some embodiments, a reference receptor-reporter is included in the host cell or membrane fraction to allow relative, real-time measurements to be made on the biosensor of the invention. In some embodiments, real time measurements are used to measure the interaction of an odorant with at least one Olfactory Receptor. In some embodiments, real time measurements are used to measure the interaction of an odorant at a plurality of different Olfactory Receptors. In some embodiments, real time measurements are used to measure the interaction of a plurality of different odorants at the same or different Olfactory Receptors. In some embodiments, real time measurements are made and compared to a reference to provide comparative numbers for the interaction of an odorant at Olfactory Receptors. In some embodiments, real time measurements are made and compared to a reference to provide comparative numbers for the interaction of different odorants at the same or different Olfactory Receptors. In some embodiments, quantitating versus a reference will produce aromagraphs for molecules, mixtures, and/or compositions that can be compared and contrasted. In some embodiments, the reporter monitored in real time is an optical reporter. In some embodiments, the reporter monitored in real time is a nonoptical reporter. In some embodiments, the reference is a G-protein coupled receptor with a known affinity for a known ligand. In some embodiments, the reference receptor has an activity range of 1, 2, 3, 4, 5, 6, 7, 8, or 9 orders of magnitude. In some embodiments, the reference receptor has an activity range of 2-4 orders of magnitude and 4-5 different reference receptors are used to cover the activity range of Olfactory Receptors from 10 M to 10−12M. In some embodiments, the reference receptor-ligand pair is selected to have an affinity that is similar to the affinity of the tested Olfactory Receptor-odorant pair. In some embodiments, the concentration of the tested odorant is varied in a series of wells over a range of 3, 4, 5, 6, 7, 8, 9, or 10 orders of magnitude and are compared to a reference. In some embodiments, the concentration of the tested odorant is varied in a series of wells over a range of 3 to 10 orders of magnitude and are compared to a reference. In some embodiments, the Olfactory Receptors are identified as having strong, intermediate, or weak interactions with the tested odorant. In some embodiments, the reference receptor is associated with a reporter that is different from the reporter associated with the Olfactory Receptor(s). In some embodiments, the different reporters are optical reporters. In some embodiments, the different optical reporters are monitored in real time in the same reaction.

In some embodiments, the biosensors of the invention are used to identify odorants in a mixture or composition that contribute to the scent, odor, smell, aroma, or taste of the mixture or composition. In some embodiments, the biosensor of the invention is used to characterize the scent, odor, smell, aroma, or taste of a composition, mixture, or a plurality of mixtures and/or compositions. In some embodiments, the biosensors are used to make a formulation or recipe for a composition or mixture that has a characteristic scent, odor, smell, aroma, or taste. In this embodiment, a component of a composition or a mixture may be replaced by a different component(s) without a loss of the characteristic scent, odor, smell, aroma, or taste by using the biosensor to find replacement component(s) that have similar interactions at the Olfactory Receptors as the replaced component. In some embodiments, the replacement component is from a natural source (replacing a non-natural component(s)). In some embodiments, the replacement component is considered to be healthy and replaces a component considered to be unhealthy. In some embodiments, the replacement component costs less. In some embodiments, the replacement component is easier to manufacture or has better properties. In some embodiments, the replacement component substitutes for an ingredient that has been banned or otherwise becomes expensive, unavailable, or hard to acquire. In some embodiments, the replacement component substitutes for two or more ingredients in the composition. In some embodiments, the composition is completely reverse engineered and a set of components that produce the composition and its characteristic scent, smell, odor, aroma, and/or taste is designed.

In some embodiments, the biosensors of the invention are used to make libraries of molecules with known aromagraphs that can be used to build or maintain designer or desired scents, odors, smells, aromas, or tastes. In some embodiments, molecules from a library with or without known aromagraphs can be used to build mixtures or compositions that mimic the aromagraphs of desired known mixtures or compositions. In some embodiments, molecules from a library can be used to build mixtures or compositions that meet an aromagraph specification. In some embodiments, the aromagraph is for a known mixture, composition or molecule. In some embodiments, the aromagraph is for a designed or theoretical scent, smell, odor, aroma, and/or taste. In some embodiments, the aromagraph is for a desired modification of a known mixture, composition, or molecule. In some embodiments, the molecules from the library can also be used to remediate malodors.

In some embodiments, the biosensors of the invention are used to standardize scents, smells, odors, aromas, and/or taste. For example, aromagraphs can be used to quantify and characterize different scents, smells, odors, aromas, and/or tastes creating a common language and description for the comparing and contrasting compositions, mixtures, and/or molecules. In some embodiments, the biosensors of the invention are used to quantify the interaction of a scent, smell, odor, aroma, and/or taste with the OR repertoire. These quantized interactions can be used to describe a scent, smell, odor, aroma, and/or taste. For example, hundreds of vanilla flavoring or vanilla extract products advertise that they provide vanilla flavor (or smell, scent, aroma). The biosensors of the invention will quantify the interaction of these vanilla products with the OR repertoire and permit standardization of these products on a functional basis. A core set of OR interactions will define the vanilla response, with many minor or side OR interactions producing the differences between these vanilla products. Standardization also allows the quantitative description of scent, smell, aroma, odor, and/or taste for purposes of branding, trademarks, and/or copyrights. Standardization also allows the quantitative description of new scents, smells, aromas, odors, and/or tastes.

In some embodiments, the biosensors of the invention are used to remediate a malodor. In this embodiment, the biosensor can be used to find a molecule(s) that can mask a malodor (e.g., from sports equipment, clothing or shoes, or products that depend on scent/smell). In some embodiments, the masking molecule binds to but does not activate (in the same way) the Olfactory Receptors which are causing the malodor scent. In this embodiment, the masking molecule can block the Olfactory Receptor(s) that perceive the malodor. In some embodiments, the masking molecule activates other Olfactory Receptors that in combination with the Olfactory Receptors activated by the malodor changes the perception of the malodor to a positive or null perception.

In some embodiments, the biosensors of the invention are used to create a composition or mixture with a desired scent, smell, odor, aroma, and/or taste. In some embodiments, the biosensors of the invention are used to make a composition or mixture that suppresses appetite. In this embodiment, the mixture or composition that suppresses appetite is placed in a device that creates an aerosol for delivering the appetite suppressant to the subject. In some embodiments, the biosensors of the invention are used to adjust the scent, smell, odor, aroma, and/or taste of a product to suit the palates of subjects in different geographic locations and/or different demographic groups. In some embodiments, a desired scent, smell, odor, and/or aroma is released from solid that contains the components for the desired scent, smell, odor, and/or aroma. In some embodiments, air freshener delivery systems are used to provide the desired scent, smell, odor, and/or aroma (e.g., glade plug-ins or other air freshener products).

In some embodiments, the biosensors of the invention are used to characterize aromascapes or odorscapes by identifying the components of an aromascape or odorscape that cause a desired perception by some subjects. In some embodiments, the biosensors of the invention are used to build aromascapes or odorscapes that produce a desired response from certain subjects. In some embodiments, certain subjects will find the aromascape or odorscape appealing, repellent, arousing, relaxing, or other desired state for producing a desired behavior. In some embodiments, the aromascape or odorscape is used to brand a product or service.

In some embodiments, the quantified scent, smell, odor, aroma, and/or taste is used to identify the scent, smell, odor, aroma, and/or taste. In some embodiments, the quantified scent, smell, odor, aroma, and/or taste is used to uniquely identify the scent, smell, odor, aroma, and/or taste. In some embodiments, the quantified scent, smell, odor, aroma, and/or taste is used to communicate about the scent, smell, odor, aroma, and/or taste. In some embodiments, the quantified scent, smell, odor, aroma, and/or taste is used in a transaction for the scent, smell, odor, aroma, and/or taste. In some embodiments, quantified scent, smell, odor, aroma, and/or taste is used to describe a product or service. In some embodiments, quantified scent, smell, odor, aroma, and/or taste is used to market a product or service. In some embodiments, quantified scent, smell, odor, aroma, and/or taste is used to sell a product or service. In some embodiments, quantified scent, smell, odor, aroma, and/or taste is used to buy a product or service.

In some embodiments, an aromagraph is used to identify the scent, smell, odor, aroma, and/or taste. In some embodiments, an aromagraph is used to uniquely identify the scent, smell, odor, aroma, and/or taste. In some embodiments, an aromagraph is used to communicate about the scent, smell, odor, aroma, and/or taste. In some embodiments, an aromagraph is used in a transaction for the scent, smell, odor, aroma, and/or taste. In some embodiments, an aromagraph is used to describe a product or service. In some embodiments, an aromagraph is used to market a product or service. In some embodiments, an aromagraph is used to sell a product or service. In some embodiments, an aromagraph is used to buy a product or service.

