Engineered hormone receptors for monitoring hormones

Abstract

An example engineered hormone receptor includes a ligand binding domain (LBD) that specifically binds a hormone and a tag inserted between a first segment of the LBD and a second segment of the LBD. The first segment includes a 1st to 11th alpha helix of the LBD and the second segment includes a 12th alpha helix of the LBD. A first linker is inserted between the tag and the 11th alpha helix in the first segment of the LBD. A second linker is inserted between the tag and the 12th alpha helix in the second segment of the LBD.

Description

ENGINEERED HORMONE RECEPTORS FOR MONITORING HORMONESSTATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0001] This invention was made with government support under Grant No. 5R01 GM 139850-04, awarded by the National Institutes of Health. The government has certain rights in the invention.CROSS-REFERENCE TO RELATED APPLICATION

[0002] This application claims priority to U.S. Provisional App. No. 63 / 707,672, which was filed on October 15, 2024 and is incorporated by reference herein in its entirety.BACKGROUND

[0003] Steroid hormones control physiological functions such as metabolism, inflammation, immune responses, blood pressure, and sexual characteristics. Their main mode of action is binding to hormone-specific receptors inside specific cells. Upon activation, the nuclear hormone receptors enter the cell nucleus to regulate gene expression in the targeted cells. There are five main types of steroid hormone receptors. Estrogen receptors (ER), include two subtypes, estrogen receptor alpha (ERa or ERa) and estrogen receptor beta (ER or “ERb”), and bind to the hormone estrogen and estrogen- like compounds. Progesterone receptors (PR) bind the hormone progesterone, associated with various reproductive processes, including menstruation, pregnancy, and mammary gland development. Androgen receptors (AR) bind androgens such as testosterone and dihydrotestosterone and control the development and function of male reproductive organs and muscle growth. Glucocorticoid receptors (GCR) bind glucocorticoids like cortisol and regulate gene expression in response to stress and metabolic signals Their main downstream effect is controlling immune response, metabolism, and stress adaptation. Mineralocorticoid receptors (MCR) bind mineralocorticoids such as aldosterone. Their main downstream function is to control ion transport in epithelial cells of the kidney and other tissues, which regulates electrolyte balance and blood pressure.

[0004] Additionally, thyroid hormone receptors (THB) have a similar structure and function as the steroid hormone receptors. Thyroid hormones, such as thyroxine (T4) and triiodothyronine (T3), are derived from the amino acid tyrosine and are primarily produced by the thyroid gland. They contain iodine atoms and are lipophilic. Upon binding to THB they activate gene expression in target cells that are involved in growth, development, and metabolic regulation.BRIEF DESCRIPTION OF THE DRAWINGS

[0005] FIG. 1 illustrates an example environment for detecting the presence and / or concentration of a hormone in a sample using an engineered hormone receptor.

[0006] FIG. 2A illustrates the composition of an estradiol sensor containing the ERa ligand binding domain (LBD) and an inserted circularly permutated green fluorescent protein (cpGFP) between LBD helices 11 and 12. The linker 1 and 2 on the n and c-terminal sides of cpGFP denote variable short amino acid sequences.

[0007] FIG. 2B illustrates an AlphaFold 2 predicted 3D structure of the genetically encoded fluorescent estrogen sensor (also referred to as eNOVA herein), including ERa LBD and cpGFP, as depicted in FIG. 2A.

[0008] FIG. 3 illustrates the fluorescent signal of the estrogen sensor eNOVA described in SEQ ID NO: 22 ERa_cpGFP with linkerl: AG and linker 2: GPS. The sensor was expressed in mammalian human embryonic kidney 293 cell cultures and excited by 490nm light. The emission light was measured at 510 nm using an epi-fluorescence microscope and sCMOS camera at 20x magnification. The sensor was excited using estradiol (E2), estetrol, tamoxifen, fulvestrant,butylated hydroxyanisole (BHA), and erythrosine at 10 pM each which evoked sensor signals at different intensities. The signal is reported as a percent change of fluorescence intensity of the emission light (i.e. brightness) after ligand application compared to baseline (before bath application of ligand).

[0009] FIG. 4 illustrates the composition of an estradiol sensor eNOVA containing the ERa LBD and an inserted cpGFP between LBD helices 11 and 12. The linkers 1 and 2, and 3 denote variable short amino acid sequences. This variant contains an additional mCherry red fluorescent protein on the n-terminal end.

[0010] FIG. 5 illustrates the composition of an estradiol sensor containing the ERa LBD and an inserted cpGFP between LBD helices 11 and 12. The linkers 1 and 2, and 3 denote variable short amino acid sequences. This variant contains an additional nuclear exclusion sequence (NES, SEQ ID NO: 19) on the n-terminal side.

[0011] FIG. 6 illustrates the composition of an estradiol sensor containing the ERa LBD and an inserted cpGFP between LBD helices 11 and 12. The linkers 1 and 2, and 3 denote variable short amino acid sequences. This variant contains an additional co-factor (listed in SEQ ID NOs: 20 and 57-86) on the c-terminal end.

[0012] FIG. 7 illustrates the composition of an estradiol sensor containing the ERa LBD and an inserted cpGFP between LBD helices 11 and 12 Linkers 1 and 2 denote short, variable amino acid sequences. This variant n-terminal IGK and HA sequence and c-terminal PDGFR sequence (listed in SEQ ID NOs: 14-16).

[0013] FIG. 8A illustrates the composition of estrogen receptor variants with the fluorescent protein mNeonGreen inserted between alpha Helices 11 and 12 of ERa. The variants here are shown with and without additional n and c- terminal amino acid sequences as described herein and elsewhere. For example, an additional nuclear exclusion sequence (NES, SEQ ID NO: 19) on the n-terminal side or n-terminal IGK and HA sequence and c-terminal PDGFR sequence (listed in SEQ ID NOs: 14-16) can be added. The linkers 1 and 2, and 3 denote variable short amino acid sequences

[0014] FIG. 8B illustrates an AlphaFold2 predicted 3D structure of the fluorescent sensor, including ERa LBD and mNeoGreen (SEQ ID NO: 21) as described herein.

[0015] FIG. 8C illustrates fluorescent signal of the ERa_mNeonGreen sensors with linkers GGS-EAH-GGSGGS-MYF (SEQ ID NO: 38).

[0016] FIG. 8D illustrates a response of ERajnNeonGreen with linkers GGS-EAH-GGSGGS-IYF (SEQ ID NO: 39) in response to 100 nM, 1 pM, 10 pM, and 50 pM estradiol application. The sensors were expressed in mammalian human embryonic kidney 293 cell cultures and excited by 490nm light. The emission light was measured at >510 nm using an epi-fluorescence microscope and sCMOS camera at 20x magnification. The sensor was excited with estradiol at different concentrations, eliciting sensor signals of varying intensities The signal is reported as a percent change of fluorescence intensity of the emission light after ligand application compared to baseline (before bath application of ligand).

[0017] FIG. 9 illustrates an Alpha-Fold2 predicted 3d structure of the estrogen receptor variants with the protein tag cpHaloTag containing the fluorescent dye JF635. cpHaloTag is inserted between alpha Helices 11 and 12 of ERa. The LXXLL peptide refers to the addition of a co-factor in the design.

[0018] FIG. 10A illustrates the composition of a thyroid hormone sensor containing the thyroid hormone receptor a (THB) LBD and an inserted cpGFP between LBD helices 11 and 12. The linker 1 and 2 on the n and c-terminal sides of cpGFP denote variable short amino acid sequences.

[0019] FIG. 10B illustrates fluorescent signal of the thyroid hormone sensors described in SEQ ID NOs: 49-56 Linkerl : variable as indicated (G, LSSLE, SLE, SSLE) and linker 2: GPS. The sensors were expressed in mammalian human embryonic kidney 293 cell cultures and excited by 490nm light. The emission light was measured at 510 nm using an epifluorescence microscope and sCMOS camera at 20x magnification. The sensor was excited using thyroid hormone receptor agonist T3 at 10 piM each, which evoked sensor signals at different intensities. The signal is reported as a percent change of fluorescence intensity of the emission light after ligand application compared to baseline (before bath application of ligand).

[0020] FIG. 10C illustrates the fluorescent signal of the thyroid hormone sensors described in SEQ ID NOs: 49-56. Linker 2: variable as indicated (LP, LPD, LPDQL, LPDQ) and linker 1 : AG. The sensors were expressed in mammalian human embryonic kidney 293 cell cultures and excited by 490nm light The emission light was measured at 510 nm using an epi-fluorescence microscope and sCMOS camera at20x magnification. The sensor was excited using thyroid hormone receptor agonist T3 at 10 piM each, which evoked sensor signals at different intensities. The signal is reported as a percent change of fluorescence intensity of the emission light after ligand application compared to baseline (before bath application of ligand).

[0021] FIG. 11 illustrates the composition of an androgen, glucocorticoid, and mineralocorticoid sensors containing the androgen receptor (AR), glucocorticoid receptor (GCR), and mineralocorticoid receptor (MCR) LBDs and an inserted cpGFP between LBD helices 11 and 12. The linker 1 and 2 on the n and c-terminal sides of cpGFP denote variable short amino acid sequences

[0022] FIG. 12A-12C illustrates the engineered estrogen sensors (eNOVA (FIG 12A) and eNOVA 2.0 (FIG. 12B)) realtime fluorescence in HEK293 cells upon 100 nM estradiol stimulation. FIG. 12C: eNOVA2.0 is specific to estradiol (E2) and has a smaller response to estetrol (E4). It is not responsive to testosterone (TEST) or progesterone (PROG).

[0023] FIG. 13 illustrates validation of eNOVA in HEK293 cells. Excitation (dashed) and emission (solid) spectra reflect biophysical properties of the underlying cpGFP reporter with and without estradiol present (+E2, -E2).

[0024] FIGs. 14A-14C illustrate preliminary validation of eNOVA in HEK293 cells. A. Ratiometric response (normalized emission ratio 510nm / 605nm) from estrogen sensor fused to mCherry, a red fluorescent non-estrogen sensitive reference. B eNOVA fused to hormone inert mCherry (mC) was stimulated by 10 pM, 10 nM estradiol, and no estradiol (tyrodes). Green and red fluorescence were measured by single snapshots before and 15 minutes after stimulation. C. Continuous (strong) excitation of mC_eNOVA reveals differential bleaching rates (blue) for each emission channel (green, red).

[0025] FIGs. 15A-15C illustrate preliminary validation of eNOVA in HEK293 cells. FIG. 15A. eNOVA was stimulated by 1 pM estradiol. The fluorescence response was measured under 488 nm and 405 nm excitation. At 488 nm excitation the cpGFP fluorescence increases upon E2 exposure. Under 405 nm excitation, the cpGFP fluorescence decreases. FIG. 15B. Zoomed in 405 nm trace from (FIG. 15A.) FIG 150. Ratiometric response (normalized response ratio at 488nm / 405nm excitation.

[0026] FIGs. 16A-16D illustrate Preliminary in vivo characterization of the ESRIcpGFP (aka eNOVA) biosensor. FIG. 16A. AAV-DIO-ESR1 cpGFP was injected into the preoptic area (POA) of adult 25g Esr1-Cre+ / - transgenic female mice. FIG. 16B. 4 weeks post-injection, freely moving mice were recorded for 30 mins. Photometry signal was Butterworth low- pass filtered at 10 Hz, then downsampled by a factor of 10 before calculating change in fluorescence. See example traces (n=3 mice). FIG. 16C. On a separate day, after 30 min of baseline recording, mice were injected (i.p.) with estradiol (E2)and recorded for 150 min A robust change detected ESRIcpGFP fluorescence, which decayed over a period of two hours post injection. FIG. 16D. The average change in amplitude from pre-E2 to post-E2 was analyzed (n = 5 mice). ESR1- sensor fluorescence is significantly changed from baseline 1h post-E2 (repeated measures one-way ANOVA: time point effect - F(1 98,7.92)=4.938, p = 0.0407, R2 = 0.5525; Dunnett’s multiple comparison test to baseline - E2, p = 0.787. +1h, p = 0.0256. +2h, p = 0.6809. * p < 0.05 Error bars = SEM.

[0027] FIG. 17 illustrates FLI M measurements of eNOVA, H2O2 sensor oROS and (Berndt lab) calcium sensor Tq-CA- FLITS (van der Linden FH et al., 2021) before (off) and upon stimulation with specific ligands. eNOVA FLIM contrast will be enhanced by incorporating fluorophores with larger quantum yields.

[0028] FIG. 18 illustrates subcellular targeting of estrogen sensor ESR1-cpGFP-AGGPS in HEK293 cells. Fused peptide sequences locate the sensor at the inner plasma membrane (CAAX), actin (LifeAct), or outside the nucleus (NES: Nuclear Exclusion Signal). All sensors maintain signaling function upon estradiol (E2) application. Trafficking into cell nuclei has not been observed.

[0029] FIG. 19A-19B illustrate the expression of eNOVA in human primary fibrochondrocytes (FIG 19A) and human mesenchymal stem cells (FIG. 19B).

[0030] FIG. 20 illustrates an engineered thyroid hormone sensor upon optimizing the N- and C-terminal linker between cpGFP and the ligand-binding domain.

[0031] FIG. 21 illustrates preliminary validation of novel glucocorticoid sensor prototype in HEK293 cells. The Glucocorticoid receptor was combined with cpGFP. Shown is fluorescence response to 0.7 mM cortisol (dashed line).

[0032] Many of the drawings submitted herein are better understood in color. Applicant considers the color versions of the drawings as part of the original submission and reserves the right to present color images of the drawings in later proceedings.DETAILED DESCRIPTION

[0033] Various implementations of the present disclosure relate to steroid and thyroid hormone sensors that can be used to report the presence of corresponding hormones by emitting light (e.g., fluorescent light), which can be detected by a photodetector. Various engineered hormone receptors described herein include novel polypeptides that enable realtime monitoring of steroid hormone levels in samples, as well as within living cells, tissues, and organisms In various implementations, an example engineered hormone receptor integrates a light-emitting tag (e.g., a light emitting protein, such as cpGFP), between the 11th and 12th alpha helix of the LBD of the hormone receptor. When the engineered hormone receptor binds to a hormone, the tag may emit light that can be measured to detect the presence and / or concentration of the hormone in a sample.

[0034] The fluorescent hormone sensors described herein hold significant potential for broader translational applications, including for instance in clinical diagnostics, environmental testing, and drug discovery. Currently, liquid chromatography-tandem mass spectrometry (LC-MS / MS) is the gold standard for quantifying hormone concentrations in biological fluids such as plasma, serum, and tissue homogenates. However, LC-MS / MS platforms require specialized infrastructure, costly equipment, expert personnel, and labor-intensive sample preparation To address these limitations, the described sensors can be used as part of simplified, field-deployable systems that employ purified hormone sensor(s) in combination with compact, tabletop fluorometers or fluorescence lifetime measurement devices. In this configuration, recombinant hormone sensors can be mixed with biological samples (e.g., blood, saliva, or cerebrospinal fluid), and theresulting hormone-induced fluorescence change can be quantified by fluorescence intensity, fluorescence lifetime, or ratiometrically, such as by comparing emission intensity, for example, under 405 nm, and 488 nm, or 488 nm and 560 nm excitation. By referencing calibration curves obtained under standardized conditions (such as known hormone concentrations in reference samples), the fluorescence signal or fluorescent lifetime from different engineered sensors can be directly converted into hormone concentrations. This portable biosensing platform will ultimately serve as an accessible, real-time alternative to LC-MS / MS for hormone diagnostics in point-of-care and other point-of-analysis settings, as well as in diagnostics and other laboratory settings.

[0035] Additionally, the sensors described herein can readily be adapted for environmental monitoring and toxicological screening. Hormone sensors expressed in living cells or incorporated into cell-free assay formats using purified proteins (e.g as described with respect to FIG. 1 and elsewhere herein) could be used to screen for endocrine-disrupting chemicals (EDCs: environmental contaminants that bind to estrogen, androgen, or corticosteroid receptors and disrupt hormonal signaling) (see, e.g., U.S. Patent No. 9,040,248). These compounds, found in plastics, industrial byproducts, and some pharmaceuticals, have been implicated in reproductive disorders, metabolic diseases, neurodevelopmental abnormalities, and hormone-sensitive cancers. A sensor-based assay employing the herein-described engineered fluorescent hormone sensors could rapidly detect EDCs in water, soil, or food samples, and could be used to evaluate unknown compounds for unintended interactions with hormone receptors. Furthermore, these systems may be used in high-throughput screening platforms to assess the binding activity of new drugs or chemicals on hormone signaling pathways, supporting safer drug development and regulatory assessment.

[0036] Another application of the herein-described engineered fluorescent hormone sensors is use in human-induced (patient derived) pluripotent stem cell (iPSC)-derived models, including neurons, glia, and brain organoids These systems offer a human-specific platform to study hormone signaling under physiological and pathological conditions, while avoiding some of the translational limitations of animal models.

[0037] For example, disorders such as premenstrual dysphoric disorder (PMDD), postpartum depression, and perimenopausal depression have been linked not to absolute hormone levels, but to abnormal cellular sensitivity to hormone fluctuations. Patient-derived iPSC models that express the herein-described engineered fluorescent hormone sensors will enable real-time monitoring of intracellular estrogen or cortisol signaling dynamics in response to controlled hormonal stimuli. It is believed this may enable detection of dysregulated receptor activity, abnormal feedback loops, altered hormone kinetics, or abnormal cellular hormone levels in at-risk individuals.

[0038] Sensors introduced into iPSC-derived neurons or organoids from male and female donors / subjects I patients could be used to test how individuals with different genetic backgrounds respond to hormonal drugs, antidepressants, environmental hormone mimics, or other applied stimuli. Such studies may enable sex-specific prediction of treatment responses or adverse effects.

[0039] For conditions such as major depression, post-traumatic stress disorder (PTSD), or cognitive decline, estrogen and glucocorticoids are major modulators of neural plasticity, stress resilience, and mood regulation. Sensor-expressing human stem cell-derived neurons could be used to dissect how dysregulated hormone signaling contributes to disease mechanisms and provide a platform for screening therapeutic interventions that recover physiological hormone interactions.

[0040] FIG. 1 illustrates an example environment 100 for detecting the presence and / or concentration of a hormone 102 in a sample 104 using an engineered hormone receptor 106. The engineered hormone receptor 106 may also be referred to a “hormone sensor'1or “NOVA” in some implementations.

[0041] The hormone 102 includes a polypeptide (e.g., a protein) utilized for signaling in a multicellular organism. In some cases, the hormone 102 is derived from a human or other animal. In some examples, the hormone 102 is artificially produced. The hormone 102 may include a steroid hormone. In some implementations, the hormone includes an estrogen, such as estrone, estradiol, or estriol. In some cases, the hormone 102 includes a thyroid hormone, such as thyroxine or triiodothyronine In some instances, the hormone 102 includes a progesterone, such as a natural progesterone (e.g., produced by the ovaries of a subject) or asynthetic progestin. In some examples, the hormone 102 includes an androgen, such as testosterone, dihydrotestosterone, androstenedione, or a synthetic anabolic steroid. In some cases, the hormone 102 includes a glucocorticoid, such as cortisol, hydrocortisone (synthetic cortisol), and corticosterone (non-human cortisol) In some instances, the hormone 102 includes a mineralocorticoid, such as aldosterone.

[0042] The sample 104 includes a fluid sample, in various implementations. In some cases, the sample 104 is a biological sample obtained from a subject (e.g., a human). For instance, the sample 104 may include urine, serum, plasma, cerebrospinal fluid, sputum, stool, lymphatic fluid, blood, mucus, semen, or saliva.

[0043] In some implementations, the sample 104 is obtained from an environmental source, such as a sewer sample, a food sample, or the like. In some cases, the sample 104 is obtained by contacting a sample collection apparatus (e.g., a swab) with a surface to be tested, and suspending residue from the surface in a suspending solution (e.g., a saline solution)

[0044] The engineered hormone receptor 106, in some examples, includes a polypeptide. In some cases, the engineered hormone receptor 106 is a single polypeptide.

[0045] In various implementations of the present disclosure, the engineered hormone receptor 106 includes a LBD 108 that specifically binds to the hormone 102. The LBD 108, in various cases, does not significantly bind to other molecules or materials beyond the hormone 102. For example, even if the sample 104 contains multiple hormones, the LBD 108 of the engineered hormone receptor 106 specifically binds to the hormone 102 and not to other hormones in the sample 104. Table 1 provides examples of various sequences of amino acids that can be included in the LBD 108:Table 1.0046] A tag 110 is inserted into the LBD 108 of the hormone receptor 106. When the LBD 108 of the engineered hormone receptor 106 binds to the hormone 102 in the sample 104, the tag 110 emits at least a portion of a detection signal 112. In various cases, the presence and / or concentration of the hormone 102 in the sample 104 can be derived based on one or more detected characteristics (e.g., intensity, timing, fluorescence lifetime, and spectral characteristics) of the detection signal 112.

[0047] In various implementations, the detection signal 112 includes one or more photons. The photon(s) are released based on fluorescence and / or luminescence of the tag 110. In various cases, the photons in the detection signal 112 have a wavelength in a range of 240 nm to 2,000 nm. In some implementations, the tag 110 is fluorescent. In some examples, binding of the hormone 102 to the LBD 108, for instance, induces a conformational change of the tag 110 (e.g., a fluorescent protein) that alters a quality of its fluorescence (e.g. brightness, absorbtion, quantum yield, spectral shift, lifetime, etc.). In some cases, the tag 110 emits the detection signal 112 in response to receiving an excitation signal 115including photons having a smaller wavelength, higher frequency, and / or higher energy level than the photons in the detection signal 112 released when the hormone 102 is bound to the LBD 108. For instance, the excitation signal 115 includes photons having a wavelength in a range of 240 to 2,000 nm.

[0048] In some implementations, the tag 110 includes at least one fluorescent protein. For example, the fluorescent protein can include a green fluorescent protein (GFP) parent and / or a genetic derivative thereof. For example, the fluorescent protein can include a red fluorescent protein (RFP) parent and / or a genetic derivative thereof. In implementations, the fluorescent protein can include split variant fluorescent proteins and / or circularly permuted fluorescent proteins. Examples of fluorescent proteins that can be included in the tag 110 include blue fluorescent proteins (e.g eBFP, eBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire); cyan fluorescent proteins (e.g. eCFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan, mTurquoise); green fluorescent proteins (e.g. GFP, GFP-2, tagGFP, turboGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green (mAzamigreen)), CopGFP, AceGFP, avGFP, ZsGreenl, Oregon Green™ (Thermo Fisher Scientific)); Luciferase; orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato); red fluorescent proteins (mKate, mKate2, mPlum, DsRed monomer, mCherry, mRuby, mRFP1 , DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611 , mRaspberry, mStrawberry, Jred, Texas Red™ (Thermo Fisher Scientific)); far red fluorescent proteins (e.g., mPlum and mNeptune); yellow fluorescent proteins (e.g., YFP, eYFP, Citrine, SYFP2, Venus, YPet, PhiYFP, ZsYellowl); circular permutation of these fluorescent proteins (denoted by a cp prefix, e.g. cpGFP); and tandem conjugates. Implementations of the present disclosure include one or more fluorescent proteins such as GFP, cpGFP, mNeonGreen, HaloTag, mRuby, mApple, mScarlet, mCherry, mClover3, mRuby3, Turquoise2, Citrine, EYFP, and tagRFP.

[0049] Table 2 provides examples of various sequences of fluorescent proteins that can be included within the tag 110:Table 2.0050] In some cases, the tag 110 includes a fluorescence resonance energy transfer (FRET) tag, in which a donor chromophore in an electronic excited state may transfer energy to an accepter chromophore. In some cases, the FRET tag emits detectable photons when the chromophores are brought within a threshold distance of one another, which may occur when the LBD 108 is bound to the hormone 102 Examples of chromophore pairings that embody FRET tags include mClover3 / mRuby3, Turquoise2 / Citrine, or EYFP / tagRFP.

[0051] In some examples, the targeted hormone 102 is an engineered hormone tracer that includes a positron emission tomography (PET) tracer. Examples of hormone PET tracers include 18F-Fluoroestradiol. In some cases, the tracer is configured to emit one or more positrons.

[0052] The tag 110 is inserted between a first segment 113 and a second segment 114 of the LBD 108. For instance, the LBD 108 includes the first segment 113 and the second segment 114, which are separated by the tag 110. The first segment 113 may extend from an N terminus of the LBD 108 and / or the second segment 114 may extend from a C terminus of the LBD 108. In some implementations, the location of the tag 110 within the LBD 108 impacts the functionality (e.g , binding selectivity, detection signal 112 characteristics, etc.) of the engineered hormone receptor 106. That is, relative composition of the first segment 113 and the second segment 114 in the LBD 108 can impact the functionality of the engineered hormone receptor 106.

[0053] In particular implementations, the first segment 113 and the second segment 114 are selected based on the secondary structure of the LBD 108 The LBD 108 may include multiple alpha helices that spontaneously form due to hydrogen bonds between amino acids in the LBD 108. For instance, the first segment 113 may include a first portion of the alpha helices of the LBD 108 and the second segment 114 may include a second portion of the alpha helices of the LBD. In various implementations of the present disclosure, the LBD 108 includes twelve alpha helices. For instance, the first segment 113 includes the first to eleventh alpha helices in the LBD 108. According to some implementations, the delineation of the LBD 108 into the first segment 113 and the second segment 114 can be adjusted. That is, a position of a breakpoint in the LBD 108 between the first segment 113 and the second segment 114 can be adjusted + / - 10 amino acids. For instance, the first segment 113 may include one to ten of the amino acids of the helix 12, or the second segment 114 may include one to ten amino acids of helices 1-11. The following Table 3 provides examples of various sequences of helices 1-11 , which can include or be included in the first segment 113:Table 3.

[0054] In various cases, the second segment 114 includes the twelfth alpha helix in the LBD 108. The following Table 4 provides examples of various sequences of helix 12, which can include or be included in the second segment 114: Table 4.0055] According to various examples, the tag 110 is bound to the segments of the LBD 108 via multiple linkers. For instance, the tag 110 is bound to the first segment 113 by a first linker 116 and / or the tag 110 is bound to the second segment 114 by a second linker 118. Each of the first linker 116 and the second linker 118 may include one or more amino acids. The sequence and length of each of the first linker 116 and the second linker 118 may impact the functionality of the engineered hormone receptor 106. In various cases, a length of the first linker 116 and / or a length of the second linker118 may be in a range of one to twenty-five amino acids. The following Table 5 provides examples of various sequences of the first linker 116 and the second linker 118:Table 5.

[0056] Table 6 provides examples of various sequences included in the engineered hormone receptor 106:Table 6.

[0057] T able 7, for instance, provides examples of various sequences included in the engineered hormone receptor 106 when the hormone 102 is estradiol and the tag 110 is cpGFP:Table ?.0058] Table 8, for instance, provides examples of various sequences included in localized variants of the engineered hormone receptor 106 when the hormone 102 is estradiol:Table d.0059] T able 9, for instance, provides examples of various sequences included in the engineered hormone receptor 106 when the hormone 102 is a thyroid hormone (T3 (triiodothyronine) and / or T4 (thyroxine)) and the tag 110 is cpGFP: Table d.

[0060] Some examples of the tag 110, in some cases, are connected to the LBD 108 using more than two linkers. For instance, in some cases, four linkers can be utilized, as illustrated in the design shown in FIG. 8A. Table 10, for instance, provides examples of various sequences included in the engineered hormone receptor 106 when the hormone 102 is estradiol, the engineered hormone receptor 106 is derived from estrogen receptor alpha, and the tag 110 is mNeonGreen: Table 10.0061] Additional sequences can be incorporated into the engineered hormone receptor 106. Table 11 lists example additional sequences that may also be included in the engineered hormone receptor 106 (e.g., outside of the LBD 108).Table 11.The term “co-factor,” as used in Table 11 above and throughout this disclosure, may refer to a short peptide sequence that bind to a receptor (e.g., peripheral to where a ligand binds) and regulates its activity. Examples of co-factors include co-activators and repressors. Inclusion of various co-factors described herein within the engineered hormone receptor 106 can enhance its performance (e.g., sensitivity, specificity, signal performance, selectivity, etc.).

[0062] In some cases, the engineered hormone receptor 106 includes an n-terminal fluorescent Nuclear Exclusion Signal (NES, SEQ ID NO: 19) to prevent or reduce the likelihood of the sensor localizing to cell nuclei (FIG. 5, SEQ ID NO: 31). In some cases, the engineered hormone receptor 106 includes a c-terminal co-factor (FIG. 6, listed in SEQ ID NOs: 20, 57-86, and 74-103) to improve sensor characteristics. In some cases, the engineered hormone receptor 106 includes an n-terminal IGK and HA-Flag sequence (FIG 7, SEQ ID NOs: 14 and 15) and a c-terminal PDGFR amino acid sequence (SEQ ID NO: 16) that targets the engineered hormone receptor 106 to the extracellular plasma membrane of cells when expressed (pDisplay variants SEQ ID NO: 32). In some implementations, these components can be included in the engineered hormone receptor 106 individually or in combination. In some cases, the engineered hormone receptor 106 includes an n-terminal LifeAct sequence (SEQ ID NO: 18), which localizes the engineered hormone receptor 106 to the actin filament of cells (SEQ ID NOs: 33-35). In some cases, the engineered hormone receptor 106 contains a CAAX amino acid sequence (SEQ ID NO: 17), which localizes the engineered hormone receptor 106 to the intracellular side of the plasma membrane (SEQ ID NO: 36). Variants described in this paragraph were utilized in Experimental Example 7, described below.

[0063] FIG. 1 additionally illustrates a platform for utilizing the engineered hormone receptor 106 to detect the presence and / or concentration of the hormone 102 in the sample 104. As illustrated, copies of the engineered hormone receptor 106 are included in a cartridge 120. The cartridge 120 includes a fluidic channel 122 configured to receive at least a portion of the sample 104. The channel 122, in some cases, is a microfluidic channel. For instance, a total volume of the channel 122 may be in a range of 0.1 microliter (piL) to 2,000 piL. At least a portion of the sample 104 can be manually injected into the channel 122 In some cases, the channel 122 includes one or more pumps (e.g., microfluidic pumps, peristaltic pumps, etc.) configured to propel the sample 104 into and through the channel 122.

[0064] The channel 122 includes a sensing region 124 that includes multiple copies of the engineered hormone receptor 106. In various cases, the sensing region 124 is optically exposed to an exterior environment from the cartridge 120. For example, an exterior wall of the cartridge 120 includes a window configured to transmit photons between the sensing region 124 and the exterior environment. In some examples, the window includes one or more lenses configured to focus and / or otherwise direct photons to and from the sensing region 124. The sensing region 124 is configured to receive the excitation signal 115 and / or to emit the detection signal 112.

[0065] In some implementations, the copies of the engineered hormone receptor 106 are immobilized on or otherwise bound to a substrate 125 within the cartridge 120. In some examples, the substrate 125 includes a plate, a well, or a particle linked to the engineered hormone receptor 106. In particular implementations, particles include microparticles, nanoparticles, nanoshells, nanobeads, microbeads, or nanodots. Particles can include, for example, latex beads, polystyrene beads, fluorescent beads, and / or colored beads, and can be made from organic matter and / or inorganic matter. In particular implementations, the engineered hormone receptor 106 as disclosed herein can be linked to a conjugate by any method known in the art. In particular embodiments, the engineered hormone receptor can be modified to allow for site specific conjugation. Such techniques include the use of naturally occurring or engineered cysteine residues, disulfide bridges, poly-histidine sequences, glycoengineering tags, and transglutaminase recognition sequences Engineered hormone receptors can also be modified for site-specific conjugation, see for example, Kim et al., Mol Cancer Ther 2008;7(8)

[0066] In some examples, the substrate 125 includes host cells that have been genetically engineered to express the engineered hormone receptor 106. Examples of the host cells include, for instance, human primary fibrochondrocytes, human mesenchymal stem cells, or bacterial cells (e.g., E.Coli], In some implementations, the host cells are immobilized on an interior wall of the sensing region 124 of the channel 122. The copies of the engineered hormone receptor 106 in the sensing region 124 are configured to be exposed to at least a portion of the sample 104 in the channel 122. The copies of the hormone receptor 106 are further configured to receive the excitation signal 115 and / or to emit the detection signal 112.

[0067] A detection system 126 is configured to identify the presence and / or concentration of the hormone 102 in the sample 104 by detecting and analyzing the detection signal 112. In various implementations, the detection system 126 includes one or more photodetectors 128 configured to receive the detection signal 112 from the sensing region 124. Examples of the photodetector(s) 128 include photodiodes, phototransistors, metal-semiconductor-metal (MSM) photodetectors, charge-coupled devices (CCDs), photomultiplier tubes (PMTs), and the like. The photodetector(s) 128 may include a microscope. In some cases, the photodetector(s) 128 is included in a camera configured to capture an image of the sensing region 124.

[0068] The detection system 126, in some examples, further includes one or more light sources 130 configured to emit the excitation signal 115 toward the sensing region 124 Examples of the light source(s) 130 include lasers, light-emitting diodes (LEDs), incandescent light sources, or the like.

[0069] The detection system 126, in various examples, includes at least one computing device. The detection system 126 includes memory 132 and one or more processors 134. In various implementations, the memory 132 is volatile (such as random access memory (RAM)), non-volatile (such as read only memory (ROM), flash memory, etc.) or some combination of the two. The memory 132 stores instructions that, when executed by the processor(s) 134, causes the detection system 126 to perform various operations. In various examples, the memory 132 stores methods, threads, processes, applications, objects, modules, any other sort of executable instruction, or a combination thereof. In some cases, the memory 132 stores files, databases, or a combination thereof. In some examples, the memory 132 includes, but is not limited to, RAM, ROM, electrically erasable programmable read-only memory (EEPROM), flash memory, or any other memory technology. In some examples, the memory 132 includes one or more of CD-ROMs, digital versatile discs (DVDs), content-addressable memory (CAM), or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other medium which can be used to store the desired information and which can be accessed by the processor(s) 134 and / or the detection system 126. The processor(s) 134 includes a central processing unit (CPU), a graphics processing unit (GPU), both CPU and GPU, or other processing unit or component known in the art.

[0070] In various implementations, the photodetectors) 128 include one or more analog-to-digital converters (ADCs) configured to generate data indicative of the detection signal 112. The data may be in the form of a spectrum representing the wavelength, intensity, and fluorescence lifetime of the photons in the detection signal 112. The processor(s) 134, by executing instructions stored in the memory 132, is configured to determine the presence and / or concentration of the hormone 102 in the sample 104 by analyzing the data indicative of the detection signal 112. For instance, the presence of the hormone 102 can be inferred by determining that the intensity of photons in the detection signal 112 corresponding to an emission wavelength associated with the tag 110 is greater than a threshold intensity. In some implementations, the concentration of the hormone 102 in the sample 104 is reflected by the number of copies of the engineered hormone receptor 106 bound to the hormone 102 and whose tags 110 emit photons. The processor(s) 134 is configured to infer the concentration of the hormone 102 based on the amount (e.g., intensity) of the photons emitted by the tags 110 of the copies of the engineered hormone receptor 106 within the detection signal 112. In various implementations, the memory 132 stores a calibration curve that indicates the relationship between the concentration of hormone 102 in the sample 104 and the detected intensity of photons or their fluorescent lifetime in the detection signal 112. The processor(s) 134 may access and rely upon the calibration curve to estimate the concentration of the hormone 102 in the sample 104. In some implementations, the detection system 126 includes a structure and function similar to devices described in Liu et al., Nature Communications 16, 6913 (2025) in which sensor proteins are unbound.

[0071] The detection system 126 further includes input / output devices 136. The input / output devices 136 collectively function as an interface between a user and the detection system 126, for instance. Input devices may be configured to receive an input from a user and includes at least one of a keypad, a cursor control, a touch-sensitive display, a voice input device (e.g., a microphone), a haptic feedback device (e.g , a gyroscope), or any combination thereof. Output devices include at least one of a display, a speaker, a haptic output device, a printer, or any combination thereof. According tosome cases, the input / output devices 136 receive an input signal from a user, which triggers detection of the detection signal 112, and output information to the user, such as the detected presence and / or concentration of the hormone 102. In some cases, the input / output devices 136 include at least one transceiver configured to receive communication signals from and / or transmit communication signals to at least one external device (not illustrated). For instance, the communication signals may encode the detected presence and / or concentration of the hormone 102.

[0072] In some cases, the engineered hormone receptor 106 includes multiple tags. For instance, the tag 110 may emit first photons in response to the hormone 102 binding to the LBD 108, and a reference tag (not illustrated) in the engineered hormone receptor 106 may emit second photons regardless of whether the hormone 102 is bound to the LBD 108. Any tag structure described herein can also be utilized as the reference tag. In some implementations, the reference tag is bound or otherwise integrated into the LBD 108 and / or a portion of the engineered hormone receptor 106 that is outside of the LBD 108. The first photons and the second photons may be emitted in the detection signal 112, for instance. The inclusion of the reference tag, that is not sensitive to the presence of the hormone 102, can be used to depict expression levels and localization of the copies of the engineered hormone receptor 106.

[0073] In particular examples, the engineered hormone receptor 106 includes an n-terminal reference tag. For instance, the tag 110 may include a cpGFP that emits green photons and the reference tag includes an RFP that emits red photons. Table 12 provides examples of sequences in the engineered hormone receptor 106 with multiple tags (cpGFP and mCherry) when the hormone 102 is estradiol (see also FIG. 4):Table 12.

[0074] The first photons may have a first wavelength, the second photons may have a second wavelength, and the first wavelength may differ from the second wavelength. In some implementations, the processor(s) 134 can more accurately identify the presence and / or concentration of the hormone 102 in the sample 104 by analyzing the data indicative of the detection signal 112 in view of the second wavelength. For example, the portion of the spectrum corresponding to the second wavelength may correspond to a baseline reference signal indicative of the number of copies of the engineered hormone receptor 106 in the sensing region 124 that are capable of contributing to the detection signal 112. In some elements, the concentration of the hormone 102 in the sample 104 is dependent on the ratio of the portion of the spectrum corresponding to the first wavelength to the portion of the spectrum corresponding to the second wavelength.

[0075] In some aspects of the present disclosure, the cartridge 120 can be designed to enable detection of the presence and / or concentration of multiple hormones in the sample 104. For instance, an additional engineered hormone receptor (not illustrated), including an LBD that specifically binds another hormone, may be included within the sensing region 124. In some cases, the LBD of the additional engineered hormone receptor includes a tag that is distinct from the tag 110, and which emits photons having a different wavelength than the tag 110 when the LBD of the additional engineered hormone receptor is bound to the other hormone. The additional engineered hormone receptor may otherwise have a similar structure to that of the engineered hormone receptor 106. In various examples, the processor(s) 134 can simultaneously detect the presence and / or concentration of both the hormone 102 and the additional hormone in thesample 104 by analyzing different portions of the spectra corresponding to the different wavelengths of photons emitted by the tag 110 and the additional tag. Accordingly, multiplexed assays can be performed using various implementations of the present disclosure.

[0076] In various implementations, the cartridge 120 can be reused. For example, the engineered hormone receptor 106 may be designed to release the hormone 102 when a washout solution (not illustrated) is injected through the channel 122.

[0077] The cartridge 120 and / or detection system 126 can be utilized to perform various types of analyses. The cartridge 120 and / or detection system 126, in some cases, may be used a point-of-care diagnostic tool. For instance, the cartridge 120 and the detection system 126 may be located in a clinical environment (e.g., a hospital) and can analyze the sample 104 shortly after it has been obtained from a subject. In some cases, the cartridge 120 and / or detection system 126 can be utilized in a laboratory environment as a research tool to detect the presence and / or concentration of a hormone in a cell culture sample. In some examples, the cartridge and / or detection system can be utilized to perform environmental testing in a wide variety of environments, such as to perform testing for the hormone 102 on a sewage sample in a sewage treatment plant.

[0078] The present disclosure provides methods of detecting hormones inside and outside eukaryotic and prokaryotic cells by emitting fluorescent light corresponding to the level of present hormones. These methods can use the expression of engineered hormone receptors (e.g., the engineered hormone receptor 106) inside and outside of cells In some cases, the cell is a mammalian neuron or cardiomyocyte. In some cases, the cells are human-derived stem cells. In some cases, the cells are in culture or intact tissue or organisms. Methods of detecting the sensor activity include registering the emitted light from the fluorescent sensors using fluorescence microscopy, fluorescent lifetime measurements, fiber photometry, and spectroscopy by means of camera sensors, photodiodes, photoamplifiers, and other light-sensitive instruments.

[0079] Implementations of the present disclosure include nucleic acid molecules encoding engineered hormone receptors described herein. Coding sequences encoding molecules (e.g., engineered hormone receptors) described herein can be obtained from publicly available databases and publications. Coding sequences can further include various sequence polymorphisms, mutations, and / or sequence variants wherein such alterations do not affect the function of the encoded molecule. The term “encode” or “encoding” refers to a property of sequences of nucleic acids, such as a vector, a plasmid, a gene, cDNA, mRNA, to serve as templates for synthesis of other molecules such as proteins.

[0080] The term “gene” may include not only coding sequences but also regulatory regions such as promoters, enhancers, insulators, and / or post-regulatory elements, such as termination regions. The term further can include all introns and other DNA sequences spliced from the mRNA transcript, along with variants resulting from alternative splice sites. The sequences can also include degenerate codons of a reference sequence or sequences that may be introduced to provide codon preference in a specific organism or cell type.

[0081] Promoters can include general promoters, tissue-specific promoters, cell-specific promoters, and / or promoters specific for the cytoplasm. Promoters may include strong promoters, weak promoters, constitutive expression promoters, and / or inducible promoters Inducible promoters direct expression in response to certain conditions, signals or cellular events. For example, the promoter may be an inducible promoter that requires a particular ligand, small molecule, transcription factor or hormone protein in order to effect transcription from the promoter. Particular examples of promotersinclude CMV, SV40, the immediate early promoter, the Hsp68 minimal promoter (proHSP68), and the Rous Sarcoma Virus (RSV) long-terminal repeat (LTR) promoter.

[0082] Various implementations of the present disclosure include methods of manufacturing the engineered hormone receptors described herein. Engineered hormone receptors and other proteins disclosed herein can be produced by recombinant expression. Recombinant polynucleotide constructs typically include an expression control sequence operably linked to the coding sequence of an engineered hormone receptor. Coding sequences can be derived based on the protein sequences disclosed herein. In particular implementations, the expression control sequences are promoter systems in vectors capable of transforming or transfecting prokaryotic and eukaryotic host cells. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and the collection and purification of the engineered hormone receptors.

[0083] Various implementations of the present disclosure include viruses, viral vectors, and vectors for inducing expression of various engineered hormone receptors described herein. The term “vector’’ refers to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule, such as an expression construct. The transferred nucleic acid is generally linked to, e.g., inserted into, the vector nucleic acid molecule. A vector may include sequences that direct autonomous replication in a cell or may include sequences that permit integration into host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors

[0084] Viral vector is widely used to refer to a nucleic acid molecule that includes virus-derived components that facilitate transfer and expression of non-native nucleic acid molecules within a cell. The term adeno-associated viral vector refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, that are primarily derived from AAV. The term "retroviral vector" refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, that are primarily derived from a retrovirus. The term "lenti viral vector" refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, that are primarily derived from a lentivirus, and so on. The term "hybrid vector" refers to a vector including structural and / or functional genetic elements from more than one virus type.

[0085] Adenovirus vectors refer to those constructs containing adenovirus sequences sufficient to (a) support packaging of an artificial expression construct and (b) to express a coding sequence that has been cloned therein in a sense or antisense orientation. A recombinant adenovirus vector includes a genetically engineered form of an adenovirus. Knowledge of the genetic organization of adenovirus, a 36 kb, linear, double-stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kb. In contrast to retrovirus, the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity. Also, adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification.

[0086] Adeno-Associated Virus (AAV) is a parvovirus, discovered as a contamination of adenoviral stocks. It is a ubiquitous virus (antibodies are present in 85% of the US human population) that has not been linked to any disease. It is also classified as a dependovirus, because its replication is dependent on the presence of a helper virus, such as adenovirus. Various serotypes have been isolated, of which AAV-2 is the best characterized. AAV has a single-strandedlinear DNA that is encapsidated into capsid proteins VP1, VP2 and VP3 to form an icosahedral virion of 20 to 24 nm in diameter.

[0087] Other viral vectors may also be employed. For example, vectors derived from viruses such as vaccinia virus, polioviruses and herpes viruses may be employed. They offer several attractive features for various mammalian cells.

[0088] Retroviruses are a common tool for gene delivery. "Retrovirus" refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA into a host genome. Once the virus is integrated into the host genome, it is referred to as a "provirus." The provirus serves as a template for RNA polymerase II and directs the expression of RNA molecules which encode the structural proteins and enzymes needed to produce new viral particles.

[0089] Illustrative retroviruses suitable for use in particular embodiments, include: Moloney murine leukemia virus (M- MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, Murine Stem Cell Virus (MSCV), Rous Sarcoma Virus (RSV), and lentivirus.

[0090] "Lentivirus" refers to a group (or genus) of complex retroviruses. Illustrative lentiviruses include: HIV (human immunodeficiency virus; including HIV type 1 , and HIV type 2); visna-maedi virus (VMV); the caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV). In particular embodiments, HIV based vector backbones (i.e., HIV cisacting sequence elements) can be used.

[0091] In particular implementations, expression of coding sequences in viral vectors is increased by incorporating posttranscriptional regulatory elements, efficient polyadenylation sites, and optionally, transcription termination signals into the vectors. A variety of posttranscriptional regulatory elements can increase expression of a coding nucleic acid. Examples include the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE; Zufferey et al., 1999, J. Virol., 73:2886); the posttranscriptional regulatory element present in hepatitis B virus (HPRE) (Smith et al., Nucleic Acids Res. 26(21 ):4818-4827, 1998); and the like (Liu et al., 1995, Genes Dev., 9:1766).

[0092] Elements directing the efficient termination and polyadenylation of a coding nucleic acid transcripts can increase expression Transcription termination signals are generally found downstream of the polyadenylation signal. In particular embodiments, vectors include a polyadenylation signal 31of a polynucleotide encoding a molecule (e.g., protein) to be expressed. The term "poly(A) site" or"poly(A) sequence" denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase II. Polyadenylation sequences can promote mRNA stability by addition of a poly(A) tail to the 3' end of the coding sequence and thus, contribute to increased translational efficiency.

[0093] In particular embodiments, a viral vector further includes one or more insulator elements. Insulators elements may contribute to protecting viral vector-expressed sequences from integration site effects, which may be mediated by cis-acting elements present in genomic DNA and lead to deregulated expression of transferred sequences (i.e., position effect; see, e.g., Burgess-Beusse et al., PNAS., USA, 99:16433, 2002; and Zhan et al., Hum Genet, 109:471, 2001). In particular embodiments, viral transfer vectors include one or more insulator elements at the 3' LTR and upon integration of the provirus into the host genome, the provirus includes the one or more insulators at both the 5' LTR and 31LTR, by virtue of duplicating the 3' LTR. Suitable insulators for use in particular embodiments include the chicken p-globin insulator(see Chung et al., Cell 74:505, 1993; Chung et al., PNAS USA 94:575, 1997; and Bell et al., Cell 98:387, 1999), SP10 insulator (Abhyankar et al., JBC 282:36143, 2007), or other small CTCF recognition sequences that function as enhancer blocking insulators (Liu et al., Nature Biotechnology, 33:198, 2015).

[0094] Beyond the foregoing description, a wide range of suitable expression vector types will be known to a person of ordinary skill in the art. These can include commercially available expression vectors designed for general recombinant procedures, for example plasmids that contain one or more reporter genes and regulatory elements required for expression of the reporter gene in cells. Numerous vectors are commercially available, e.g., from Invitrogen, Stratagene, Clontech, etc , and are described in numerous associated guides. In particular embodiments, suitable expression vectors include any plasmid, cosmid or phage construct that is capable of supporting expression of encoded genes in mammalian cell, such as pUC or Bluescript plasmid series

[0095] In particular embodiments, mammalian cells are used as a host for expressing engineered hormone receptors and / or nucleotide segments encoding engineered hormone receptors. See Winnacker, From Genes to Clones, (VCH Publishers, NY, 1987). A number of suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art, and include CHO cell lines (e.g , DG44), various COS cell lines, HeLa cells, HEK293 cells, L cells, and non-antibody-producing myelomas including Sp2 / 0 and NS0. In particular embodiments, the cells are nonhuman. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and information processing sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. In particular embodiments, expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, and bovine papillomavirus (see Co et al., J. Immunol 1992, 148:1149).

[0096] Multicellular organisms, such as transgenic animals, expressing engineered hormone receptors, are also incorporated into the present disclosure. A transgenic animal, for instance, may have a genome encoding an engineered hormone receptor In particular embodiments, when a non-integrating vector is utilized, a transgenic animal includes an expression construct expression construct encoding an engineered hormone receptor within one or more of its cells.

[0097] Detailed methods for producing transgenic animals are described in U.S. Pat. No. 4,736,866. Transgenic animals may be of any nonhuman species, and in particular embodiments include nonhuman primates (NHPs), sheep, horses, cattle, pigs, goats, dogs, cats, rabbits, chickens, and rodents such as guinea pigs, hamsters, gerbils, rats, mice, and ferrets.

[0098] In particular embodiments, construction of a transgenic animal results in an organism that has an engineered construct present in all cells in the same genomic integration site. Thus, cell lines derived from such transgenic animals will be consistent in as much as the engineered construct will be in the same genomic integration site in all cells and hence will suffer the same position effect variegation.

[0099] In addition to the methods of delivery described above, the following techniques are also contemplated as alternative methods of delivering expression constructs encoding an engineered hormone receptor to target cells or targeted tissues and organs of an animal, and in particular, to cells, organs, or tissues of a vertebrate mammal: sonophoresis (e.g , ultrasound, as described in U.S. Pat No. 5,656,016); intraosseous injection (U.S. Pat. No. 5,779,708); microchip devices (U.S. Pat. No. 5,797,898); ophthalmic formulations (Bourlais et al., Prog Retin Eye Res, 17(1 ):33-58,1998); transdermal matrices (U.S. Pat. No. 5,770,219 and U.S. Pat. No. 5,783,208); feedback-controlled delivery (U.S. Pat. No. 5,697,899), and any other delivery method available and / or described elsewhere in the disclosure.

[0100] Once expressed, engineered hormone receptors can be purified from eukaryotic and prokaryotic host cells according to standard procedures of the art, including high-performance liquid chromatography (HPLC) purification, column chromatography, gel electrophoresis and the like (see generally, Scopes, Protein Purification (Springer- Verlag, NY, 1982))

[0101] The present disclosure provides examples of a polypeptide that includes the amino acid chain depicted in FIG. 2A (e.g., SEQ ID NO: 22), wherein cpGFP is inserted between the 11thand 12thalpha helices of ERa. A 3D structure of this polypeptide is depicted in FIG. 2B. Various polypeptide structures integrating cpGFP into the LBD of ERa are also illustrated in FIGS 4-7

[0102] The present disclosure provides examples of a polypeptide includes the amino acid chains in FIG 8A and SEQ ID NOs: 38-41, wherein ERa contains the fluorescent protein mNeonGreen between ERa LBD helix 11 and 12, which creates a variant that emits fluorescent light in the presence of estrogen and estrogen-like compounds under <500 nm excitation and >500 nm emission wavelengths In some cases, the fluorescent protein can be replaced by others, such as cpmRuby, cpmApple, cpmScarlet, or the protein domain cpHaloTag (FIG. 9, SEQ ID NO: 12). In some cases, the variant contains an n-terminal IGK, HA amino acid and c-terminal PDGFR amino acid sequence that targets the sensor to the extracellular plasma membrane of cells (SEQ ID NOs: 14-16). In some cases, the variant contains a c-terminal co-factor (SEQ ID NOs: 20 and 50-86) to improve sensor function. In some cases, the variants contain a CAAX amino acid sequence, which localizes the sensor to the intracellular side of the plasma membrane, a NES or LifeAct sequence (SEQ ID NOs: 17-19) or contain an n-terminal mCherry (SEQ ID NO: 13).

[0103] In various examples, a polypeptide includes the fluorescent protein mNeonGreen between ERa LBD helix 11 and 12 (FIGs 8A and 8B and SEQ ID NOs: 38-41). The polypeptide, for instance, emits fluorescent light in the presence of estrogen and estrogen-like compounds under <500 nm excitation and >500 nm emission wavelengths (see FIGs. 8C and 8D). In some cases, the fluorescent protein can be replaced by others, such as cpmRuby, cpmApple, cpmScarlet, or the protein domain cpHaloTag (FIG. 9, SEQ ID NO: 12). In some cases, the polypeptide contains an n-terminal IGK, HA amino acid and c-terminal PDGFR amino acid sequence that targets the sensor to the extracellular plasma membrane of cells (SEQ ID NOs: 14-16). In some cases, the polypeptide contains a c-terminal co-factor (SEQ ID NOs: 20 and 57-86) to improve sensor function. In some cases, the polypeptide includes a CAAX amino acid sequence, which localizes the polypeptide to the intracellular side of the plasma membrane, a NES or LifeAct sequence (SEQ ID NOs: 17-19) or contain an n-terminal mCherry (SEQ ID NO: 13).

[0104] In some examples, a polypeptide includes cpGFP between THB LBD helix 11 and 12, linked by various linker groups (FIG. 10A and SEQ ID NOs: 42-56). FIGS. 10B and 100 illustrate example responses of the polypeptide with different linker variants. These fusion protein variants emit fluorescent light in the presence of thyroid hormone T3 under <500 nm excitation and >500 nm emission wavelengths. In some cases, the polypeptide contains an n-terminal IGK, HA amino acid and c-terminal PDGFR amino acid sequence that targets the polypeptide to the extracellular plasma membrane of cells (SEQ ID NOs: 14-16). In some cases, the polypeptide contains a c-terminal co-factor (SEQ ID NOs: 20 and 57- 86) to improve sensor function. In some cases, the polypeptide contains a CAAX amino acid sequence, which localizes the sensor to the intracellular side of the plasma membrane, a NES or LifeAct sequence (SEQ ID NOs: 17-19). In somecases, the fluorescent protein can be replaced by others, such as cpmRuby, cpmApple, cpmScarlet, or cpHaloTag (SEQ ID NO: 12) or contain an n-terminal mCherry (SEQ ID NO: 13).

[0105] In some examples of the present disclosure, a polypeptide includes cpGFP between helices 11 and 12 of an AR, PR, GCR, THB, or MCR LBD (FIG. 11 and SEQ ID NOs: 7-10). These examples of the engineered hormone receptor 106 may include linkers, including but not exclusive to all linker variants mentioned herein or elsewhere. In some cases, the polypeptide contains an n-terminal IGK, HA amino acid and c-terminal PDGFR amino acid sequence that targets the polypeptide sensor to the extracellular plasma membrane of cells (SEQ ID NOs: 14-16). In some cases, the polypeptide contains a c-terminal co-factor (e.g., any one of SEQ ID NQs:20 and 57-86) to improve sensor function. In some cases, the polypeptide contains a CAAX amino acid sequence, which localizes the sensor to the intracellular side of the plasma membrane, a NES or LifeAct sequence (SEQ ID NOs: 17-19). In some cases, the fluorescent protein can be replaced by others, such as cpmRuby, cpmApple, cpmScarlet, or cpHaloTag (SEQ ID NO: 12) or contain an n-terminal mCherry (SEQ ID NO: 13)

[0106] Various specific examples will now be described with reference to the accompanying Experimental Examples. Particular examples of engineered hormone receptors described herein enable the detection of estradiol and estrogen receptor agonists between 10 nM and 10 M in cells and tissues by an estradiol sensor including of the ligand binding domain of ERa and cpGFP inserted between alpha helices 11 and 12 of the ERa LBD In this example the fluorescent excitation wavelengths are below 500 nm, and the emission wavelengths are above 510 nm. Some examples of engineered hormone receptors described herein enable the detection of estradiol and estrogen receptor agonists between 10 nM and 10 pM in cells and tissues by an estradiol sensor including of the ligand binding domain of the ER and the green fluorescent protein mNeonGreen inserted between alpha helices 11 and 12 of the ER LBD. The fluorescent excitation wavelengths are below 500 nm, and the emission wavelengths are above 510 nm. Particular implementations described herein relate to a polypeptide chain including the LBD of THB and a cpGFP inserted between alpha helices 11 and 12 can emitfluorescent light to report changing hormone levels in samples, cells, tissues, and organisms The intensity of the emitted light corresponds to the thyroid hormone level.EXPERIMENTAL EXAMPLE 1 : FIRST GENERATION ESTROGEN SENSORS

[0107] Emerging evidence suggests that estradiol (E2), a potent agonist of estrogen receptors, plays a modulatory role in various brain functions, including mood regulation, cognitive processes, and reproductive behaviors (Taxier et al., Nat Rev Neurosci, 21 (10):535-550, 2020). Nevertheless, circuit- and cell-type-specific studies of E2 in vivo remain challenging due to the absence of analytical tools capable of precise spatiotemporal detection of E2 in the brain, time-locked with behavior.

[0108] The present disclosure presents a genetically encoded fluorescent indicator (SEQ ID NO: 22) for the real-time detection of estradiol, enabling the study of estradiol dynamics with high spatial specificity and temporal resolution. FIG. 3 illustrates the fluorescent signal of the estrogen sensor eNOVA described in SEQ ID NO: 22 ERa_cpGFP with linkerl : AG and linker 2: GPS. The sensor was expressed in mammalian human embryonic kidney 293 cell cultures and excited by 490nm light. The emission light was measured at 510 nm using an epi-fluorescence microscope and sCMOS camera at 20x magnification. The sensor was excited using estradiol (E2), estetrol, tamoxifen, fulvestrant, butylated hydroxyanisole (BHA), and erythrosine at 10 pM each which evoked sensor signals at different intensities. The signal isreported as a percent change of fluorescence intensity of the emission light (i.e brightness) after ligand application compared to baseline (before bath application of ligand).

[0109] The DNA plasmid encoding the sensor (the genetically encoded fluorescent indicator) was propagated and purified from E.coli cells. The sensor plasmid was expressed in human epithelial kidney (HEK) cells via lipofectamine transfection. During live cell imaging using epi-fluorescence microscopy, the sensor exhibits a 180% dynamic fluorescence increase in response to 10 pM E2 at saturation. The sensor also shows high sensitivity to estradiol, responding to concentrations as low as the nanomolar range. Interestingly, the sensor exhibits affinities to other estradiol agonists similar to those of native estrogen receptors, with distinct response characteristics for each ligand. A similar estradiol response profile was observed when the sensor was expressed in primary rat cortical neurons. To further validate the sensor’s compatibility in the brain, stereotactic injection of an AAV1-DIO vector containing the estradiol sensor into the medial preoptic area of the hippocampus in Cre-ESR1 mice was performed. The animals were then sacrificed for ex vivo brain sectioning followed by 2-photon imaging. The sensor expressed in ESR1 -positive neurons of the targeted brain region exhibited a 100% fluorescence increase in response to a 300 nM bath application of E2.EXPERIMENTAL EXAMPLE 2: SECOND GENERATION ESTROGEN SENSORS

[0110] This Experimental Example compares washout and other characteristics of different example engineered estrogen receptors suitable for detecting estradiol. A first estrogen sensor (“eNOVA,” including SEQ ID NO: 22) exhibits very slow decay times following exposure to and washout of estrogen To accelerate the decay, a single mutation R394A was introduced to generate eNOVA2.0 (SEQ ID NO: 104), which has faster decay after the washout of estradiol (E2) (FIG. 12A-C). The more efficient washout characteristics of eNOVA2.0 indicate that it may be a better candidate for inclusion into a reusable platform for estrogen detection.EXPERIMENTAL EXAMPLE 3: SPECTRAL PROPERTIES OF ESTROGEN SENSORS

[0111] The excitation and emission spectra for eNOVA (+E2, -E2) were measured. The excitation peak wavelength (dashed) was confirmed at ~490nm and emission peak wavelength (solid) was confirmed at -512 nm (FIG. 13).EXPERIMENTAL EXAMPLE 4: RATIOMETRIC MEASUREMENTS OF EXAMPLE ESTROGEN SENSORS

[0112] Two types of ratiometric measurements were conducted with estrogen sensors. mCherry was fused to eNOVA (SEQ ID NO: 29). mCherry is a red fluorescent protein and can be used as an inert reference for long-term measurements of hormones. In use scenarios, the ratio of red to green fluorescence was measured to qualitatively estimate whether the hormone concentration increases or decreases (FIGs. 14A-14C). Ratiometric measurements of eNOVA at 405 nm vs 488 nm excitation for quantitative measures of hormone concentrations are shown in FIG. 15A-15C. Interestingly, it can be seen that when excited by 405 nm, and exposed to E2, eNOVA has a negative response amplitude, in contrast to 488 nm excitation. The ratio between the 488 nm and 405 nm signal reports quantitative values of E2 concentration, which could be exploited in point-of-care devices for measuring hormone concentration in body fluids.EXPERIMENTAL EXAMPLE 5: IN VIVO MEASUREMENTS OF HORMONE SENSORS IN MAMMALIAN BRAIN

[0113] eNOVA (SEQ ID NO: 22) responses were measured in the brains of mice. The sensors were delivered to the pre-optic area (POA) of the hypothalamus via viral (AAV) transduction. The virus serotype was AAV1 and the virus was injected into POA via stereotactic procedures using micro syringes. The expression was Ore recombinase-dependent, meaning that the eNOVA sequence was in a double-floxed inverse orientation (DIO) within the viral DNA. That is, the encoding DNA sequence was inverted and flanked by Ore recognition sites. In this configuration, no functional sensor canbe expressed. Cre recombinase is only present in Estrogen receptor-positive neurons in the POA (Cre-Esr1), recombining the sensor DNA sequence in the forward reading direction, enabling functional protein expression. The mice were transgenic. That is, they were engineered to express Cre-recombinase only in Esr1 -positive neurons. After recombination, sensor expression was driven by the Ef 1 a promoter present in the viral DNA before the sensor encoding DNA segment. Bulk fluorescence changes were measured in the targeted estrogen receptor 1 positive neurons via fiber photometry upon injection of estradiol (E2) The measured responses were time-locked to the injection and robust (FIGs. 16A-16D).EXPERIMENTAL EXAMPLE S: FLUORESCENT LIFETIME IMAGING OF ESTROGEN SENSORS FOR QUANTITATIVE MEASURES OF HORMONE CONCENTRATIONS

[0114] Fluorescence lifetime (FLT) is a biophysical property of every fluorophore. Fluorescent sensors may have different FLTs depending on the concentration of their specific ligand, i.e. the molecule that they are detecting Thus, FLT could be used to quantify hormone concentrations through specifically optimized NOVA sensors eNOVA (SEQ ID NO: 22) was tested and found a small FLT contrast between 1 and 0 piM E2 exposure (FIG. 17) The aim was to increase FLT contrast by using fluorescent reporters with larger quantum yields such as mScarlet, mScarlet3, mTurquoise2, and HaloTag-based sensors. The experiments were conducted in HEK 293 cells after transfecting the sensor encoding plasmids. A fluorescence microscope capable of measuring fluorescence lifetime of the sensor as a biophysical property was used. The cells were stimulated using 1uM estradiol and the fluorescence lifetime was measured before (off) and after stimulation (on). Excitation photons had a wavelength of 488 nm and detected emission photons had a wavelength of 510 nm.EXPERIMENTAL EXAMPLE 7: SUBCELLULAR TARGETING OF HORMONE SENSORS IN MAMMALIAN CELLS

[0115] Hormone concentrations may differ between different subcellular compartments. The engineered hormone receptor designs described herein enable subcellular targeting by adding specific amino acid sequences that traffic them into specific compartments, such as inner plasma membrane (by CAAX), actin (by LifeAct), or outside the nucleus (by NES: Nuclear Exclusion Signal). Using the DNA plasmid encoding these modified variants (as described in

[0062] , including SEQ ID NO: 22 and other components to traffic the sensors to specific subcellular locations), the sensors can be expressed in a variety of cells via lipofectamine transfection and / or viral transduction. Fluorescent sensor responses were measured upon stimulation with 10 pM estradiol (E2) using 490 nm excitation photons and 510 nm emission photons. With these modified sensors, it will be possible to measure spatially defined cellular hormone fluctuations (FIG. 18).EXPERIMENTAL EXAMPLE 8: CELLULAR EXPRESSION OF HORMONE SENSORS

[0116] To prove the translational potential of the hormone sensors for drug screening and precision medicine, eNOVA was expressed in human primary fibrochondrocytes (FIG. 19A) and human mesenchymal stem cells (FIG. 19B) (both sensitive to estrogen signaling). Expression was achieved via plasmid transfection (lipofection) of DNA plasmids encoding the sensor DNA. The expression was driven by the CAG promoter. Exposure to E2 yielded strong responses from the sensor, demonstrating that they are functional in these host cellsEXPERIMENTAL EXAMPLE 9: IMPROVED THYROID HORMONE AND GLUCOCORTICOID HORMONE SENSOR PROTOTYPES

[0117] Additional screening of thyroid sensors (SEQ ID NOs: 42, 105, 106) was conducted with modified linker amino acids between the thyroid receptor LBD and cpGFP and yielded improved response amplitudes upon exposure to T3 hormones (10 iM) compared to an initial prototype (FIG. 20). Based on the principles described herein, a functionalsensor (ATLPQLTPTLVSLLEVIEPEVLYAGYDSSVPDSTWRIMTTLNMLGGRQVIAAVKWAKAIPGFRNLHLDDQMTLL QYSWMFLMAFALGWRSYRQSSANLLCFAPDLIINEQRMTLPCMYDQCKHMLYVSSELHRLQVSYEEYLCMKTLLLLSSV PKDGLKSQELFDEIRMTYIKELGKAIVKREGNSSQNWQRFYQLTKLLDSMHEWENLLNYCFQTFLDKTMSAGNVYIKAD KQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDE LYKGGTGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQ CFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNGPSIEF PEMLAEIITNQIPKYSNGNLLFHQK (SEQ ID NO: 108)) for cortisol, a glucocorticoid, was developed by combining the LBD of the glucocorticoid receptor with cpGFP The sensor responses were measured in mammalian HEK293 cells under cortisol exposure (FIG. 21).EXAMPLE CLAUSES

[0118] The following clauses provide various examples of the present disclosure. However, the scope of the disclosure is not limited to any of the clauses listed herein.1 . An engineered hormone receptor including: a ligand binding domain (LBD) that specifically binds a hormone, the LBD including: a first segment including a 1stto 11thalpha helix of the LBD; and a second segment including a 12thalpha helix of the LBD; a tag; a first linker inserted between the tag and the 11thalpha helix in the first segment of the LBD; and a second linker inserted between the tag and the 12thalpha helix in the second segment of the LBD.2. The engineered hormone receptor of clause 1, wherein the hormone includes an estrogen.3. The engineered hormone receptor of clause 2, wherein the estrogen includes estradiol.4. The engineered hormone receptor of clause 2 or 3, wherein the engineered hormone receptor includes a sequence having at least 70%, 90%, 95%, or 99% sequence identity to one of SEQ ID NOs: 22-28, 31-37, 66, or 104.5. The engineered hormone receptor of any of clauses 2 to 4, wherein the engineered hormone receptor includes a sequence having at least 70%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 226. The engineered hormone receptor of any of clauses 2 to 5, wherein the engineered estrogen receptor includes a sequence having at least 70%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 104.7. The engineered hormone receptor of any of clauses 2 to 6, wherein the engineered estrogen receptor includes a sequence having at least 70%, 90%, 95%, or 99% sequence identity to one of SEQ ID NOs: 38-41 .8. The engineered hormone receptor of any of clauses 2 to 7, wherein the engineered estrogen receptor includes a sequence having at least 70%, 90%, 95%, or 99% sequence identity to one of SEQ ID NOs: 29, 30, or 1079. The engineered hormone receptor of any of clauses 2 to 8, wherein the first segment of the LBD includes a sequence having at least 70%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 2 or 58:10. The engineered hormone receptor of any of clauses 2 to 9, wherein the second segment of the LBD includes a sequence having at least 70%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 3 or 60 .11. The engineered hormone receptor of any of clauses 2 to 10, wherein the first linker consists essentially of AG, G, SAG, or SLE.12. The engineered hormone receptor of any of clauses 2 to 11 , wherein the second linker consists essentially of G, GP, GPS, or SEQ ID NO: 64.13. The engineered hormone receptor of any of clauses 1 to 12, wherein the hormone includes a thyroid hormone.14. The engineered hormone receptor of clause 13, wherein the thyroid hormone includes triiodothyronine (T3).15. The engineered hormone receptor of clause 13 or 14, wherein the engineered hormone receptor includes a sequence having at least 70%, 90%, 95%, or 99% sequence identity to one of SEQ ID NOs: 42-56, 105, or 106.16. The engineered hormone receptorof any of clauses 13 to 15, wherein the first segment of the LBD includes a segment having at least 70%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 5.17. The engineered hormone receptor of any of clauses 13 to 16, wherein the second segment of the LBD includes a segment having at least 70%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6.18. The engineered hormone receptor of any of clauses 13 to 17, wherein the first linker consists essentially of SLE, G, AG, SEQ ID NO: 62, or SEQ ID NO: 63.19. The engineered hormone receptor of any of clauses 13 to 18, wherein the second linker consists essentially of GPS, LP, LPD, SEQ ID NO: 64, or SEQ ID NO: 65.20. The engineered hormone receptor of any of clauses 1 to 19, wherein the hormone includes an androgen, a progesterone, a glucocorticoid, a mineralocorticoid, or an endocrine-disrupting chemical.21. The engineered hormone receptor of any of clauses 1 to 20, wherein the hormone includes a glucocorticoid and includes a sequence having at least 70%, 90%, 95%, or 99% sequence identity to one of SEQ ID NOs: 59 or 61.22. The engineered hormone receptor of any of clauses 1 to 21 , wherein the hormone includes cortisol and includes a sequence having at least 70%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 108.23. The engineered hormone receptor of any of clauses 1 to 22, wherein the first linker has a length in a range of about 1 to about 25 amino acids, and / or wherein the second linker has a length in a range of about 1 to about 25 amino acids.24. The engineered hormone receptor of any of clauses 1 to 23, further including at least one of an IgK leader, an HA flag, a PDGFR transmembrane domain, a CaaX, a LifeAct, dmito, or an NES.25. The engineered hormone receptor of any of clauses 1 to 24, further including a co-factor consisting essentially of one of SEQ ID NOs: 20 and 74-103.26. The engineered hormone receptor of clause 25, including: a first polypeptide including the LBD, the tag, the first linker, and the second linker; and a second polypeptide fused with the first polypeptide, the second polypeptide including the co-factor.27. The engineered hormone receptor of any of clauses 1 to 26, wherein the engineered hormone receptor includes a polypeptide chain including the first segment, the second segment, the tag, the first linker, and the second linker.28. The engineered hormone receptor of any of clauses 1 to 27, wherein the tag emits a photon when the LBD binds to the hormone.29. The engineered hormone receptor of clause 28, the tag being a first tag, the photon being a first photon, the engineered hormone receptor further including: a second tag that emits a second photon without the LBD binding to the hormone, the first photon having a different wavelength than the second photon.30. The engineered hormone receptor of clause 28 or 29, wherein the photon has a wavelength in a range of about 240 nm to about 2,000 nm.31 . The engineered hormone receptor of clause 30, wherein the tag includes at least one fluorescent protein32. The engineered hormone receptor of clause 31, wherein the at least one fluorescent protein includes circularly permutated GFP (cpGFP), mNeonGreen, circularly permuted HaloTag (cpHaloTag), circularly permuted mRuby(cpmRuby), circularly permuted mApple (cpmApple), circularly permuted mScarlet (cpmScarlet), mCherry, split GFP, split HaloTag, split NeonGreen, split mRuby, split mApple, or split mScarlet.33. The engineered hormone receptor of any of clauses 28 to 32, wherein the tag includes a fluorescence resonance energy transfer (FRET) tag including a pair of fluorescent proteins, the pair of fluorescent proteins including at least one of mClover3 / mRuby3, Turquoise2 / Citrine, or EYFP / tagRFP.34. The engineered hormone receptor of any of clauses 28 to 33, wherein the hormone includes a positron emission tomography (PET) tracer35. The engineered hormone receptor of any of clauses 28 to 34, wherein the PET tracer includes 18F-Fluoroestradiol.36. A system, including: the engineered hormone receptor of any of clauses 28 to 35 exposed to a fluid sample; a light source configured to emit excitation light to the engineered hormone receptor; and a photodetector configured to detect the hormone in the fluid sample by detecting a detection signal including the photon from the tag.37. The system of clause 36, further including: a sensing region including the engineered hormone receptor and configured to receive the fluid sample including the hormone.38. The system of clause 37, wherein the sensing region has a volume in range of about 0.1 piL to about 2,000 piL.39. The system of any of clauses 36 to 38, wherein a concentration of the hormone in the fluid sample is in a range of 0.1 pM to about 100 mM.40. The system of any of clauses 36 to 39, further including: a processor configured to: identify data indicative of a spectrum of the detection signal; and determine, by analyzing the data, a concentration of the hormone in the fluid sample.41. A cartridge including a sensing region that includes multiple copies of the engineered hormone receptor of any of clauses 1 to 35.42. A genetically modified cell expressing the engineered hormone receptor of any of clauses 1 to 35.43. The genetically modified cell of clause 42 including a cardiomyocyte, a neuron, human primary fibrochondrocyte, or a human mesenchymal stem cell.44. A nucleic acid molecule encoding the engineered hormone receptor of any of clauses 1 to 3545. A virus including the nucleic acid molecule of clause 44.46. A method of genetically modifying a cell to express the engineered hormone receptor of any of clauses 1 to 35.47. A method of manufacturing the engineered hormone receptor of any of clauses 1 to 35.48. A method, including: detecting, from an engineered hormone receptor in an environment, a signal indicating binding between the engineered hormone receptor and a hormone, the engineered hormone receptor including a tag inserted between an 11thand 12thalpha helix of a LBD that specifically binds the hormone, the tag emitting the signal in response to the hormone binding to the LBD; and determining a presence and / or concentration of the hormone in the environment based on the signal.49. The method of clause 48, wherein the engineered hormone receptor is expressed by a cell in the environment.50. The method of clause 48 or 49, wherein the environment includes a tissue sample, at least a portion of an organism, or a fluid sample.51. The method of any of clauses 48 to 50, wherein the environment includes a living cell, a living tissue, or a living organism.52. The method of any of clauses 48 to 51 , wherein the signal includes a photon53. The method of clause 52, the photon being a first photon, wherein the method further includes: illuminating the engineered hormone receptor with a second photon having a higher energy level than the first photon, thereby causing the engineered hormone receptor to fluoresce54. The method of clause 53, wherein the first photon has a wavelength in a range of about 240 nm to about 2,000 nm.55. The method of any of clauses 48 to 54, wherein the concentration of the of the hormone in the environment is determined to be in a range of about 0.1 pM to about 100 mM.56. The method of any of clauses 48 to 55, the signal being a first signal, the method further including: detecting, from the engineered hormone receptor in the environment, a second signal; and determining a location of the engineered hormone receptor in the environment based on the second signal.57. The method of clause 56, wherein the first signal includes a first photon with a first wavelength, the second signal includes a second photon with a second wavelength, and the first wavelength is different than the second wavelength.58. The method of any of clauses 48 to 57, the engineered hormone receptor being a first engineered hormone receptor, the hormone being a first hormone, the signal being a first signal, the method further including: detecting, from a second engineered hormone receptor in the environment, a second signal indicating binding between the second engineered hormone receptor and a second hormone, the second hormone being different than the first hormone; and determining a presence and / or concentration of the second hormone in the environment based on the second signal.59. The method of clause 58, wherein the first signal includes a first photon having a first wavelength, the second signal includes a second photon having a second wavelength, and the first wavelength is different than the second wavelength.CLOSING PARAGRAPHS

[0119] All references cited are incorporated by reference herein in their entirety

[0120] The features disclosed in the foregoing description, or the following claims, or the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for attaining the disclosed result, as appropriate, may, separately, or in any combination of such features, be used for realizing implementations of the disclosure in diverse forms thereof.

[0121] As will be understood by one of ordinary skill in the art, each implementation disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, or component. Thus, the terms “include” or “including” should be interpreted to recite: "comprise, consist of, or consist essentially of.” The transition term “comprise” or “comprises” means has, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of” excludes any element, step, ingredient or component not specified The transition phrase “consisting essentially of’ limits the scope of the implementation to the specified elements, steps, ingredients or components and to those that do not materially affect the implementation. As used herein, the term “based on” is equivalent to “based at least partly on,” unless otherwise specified.

[0122] Unless otherwise indicated, all numbers expressing quantities, properties, conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinaryrounding techniques. When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ± 13% of the stated value; ± 12% of the stated value; ±11 % of the stated value; ± 10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1 % of the stated value.

[0123] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0124] The terms “a,” “an,” “the” and similar referents used in the context of describing implementations (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate implementations of the disclosure and does not pose a limitation on the scope of the disclosure. No language in the specification should be construed as indicating any non-claimed element essential to the practice of implementations of the disclosure.

[0125] Groupings of alternative elements or implementations disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0126] Variants of the sequences disclosed and referenced herein are also included. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological activity can be found using computer programs well known in the art, such as DNASTAR™ (Madison, Wisconsin) software. Preferably, amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains.

[0127] In a peptide or protein, suitable conservative substitutions of amino acids are known to those of skill in this art and generally can be made without altering a biological activity of a resulting molecule. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224). Naturally occurring amino acids are generally divided into conservative substitution families as follows:Group 1 : Alanine (Ala), Glycine (Gly), Serine (Ser), and Threonine (Thr); Group 2: (acidic): Aspartic acid (Asp), and Glutamic acid (Glu); Group 3: (acidic; also classified as polar, negatively charged residues and their amides): Asparagine (Asn), Glutamine (Gin), Asp, and Glu; Group 4: Gin and Asn; Group 5: (basic; also classified as polar, positively charged residues): Arginine (Arg), Lysine (Lys), and Histidine (His); Group 6 (large aliphatic, nonpolar residues): Isoleucine (lie), Leucine (Leu), Methionine (Met), Valine (Vai) and Cysteine (Cys); Group 7 (uncharged polar): Tyrosine (Tyr), Gly, Asn, Gin, Cys, Ser, and Thr; Group 8 (large aromatic residues): Phenylalanine (Phe), Tryptophan (Trp), and Tyr; Group 9 (nonpolar): Proline (Pro), Ala, Vai, Leu, lie, Phe, Met, and Trp; Group 11 (aliphatic): Gly, Ala, Vai, Leu, and lie; Group 10 (small aliphatic, nonpolar or slightly polar residues): Ala, Ser, Thr, Pro, and Gly; and Group 12 (sulfur-containing): Met and Cys. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.

[0128] In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, J. Mol. Biol. 157(1), 105-32). Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values are: lie (+4.5); Vai (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1 .9); Ala (+1 .8); Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3); Pro (-1 .6); His (-3.2); Glutamate (-3.5); Gin (-3.5); aspartate (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5).

[0129] It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity.

[0130] As detailed in US 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: Arg (+3.0); Lys (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); Ser (+0.3); Asn (+0.2); Gin (+O 2); Gly (0); Thr (-0.4); Pro (-0.5± 1 ); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); Vai (-1 .5); Leu (-1 .8); lie (-1.8); Tyr (-2.3); Phe (-2.5); Trp (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred

[0131] As outlined above, amino acid substitutions may be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. As indicated elsewhere, variants of gene sequences can include codon optimized variants, sequence polymorphisms, splice variants, and / or mutations that do not affect the function of an encoded product to a statistically-si gn ificant degree.

[0132] Variants of the protein, nucleic acid, and gene sequences disclosed herein also include sequences with at least 70% sequence identity, 80% sequence identity, 85% sequence, 90% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity to the protein, nucleic acid, or gene sequences disclosed herein.

[0133] “a / o sequence identity” refers to a relationship between two or more sequences, as determined by comparing the sequences In the art, "identity" also means the degree of sequence relatedness between protein, nucleic acid, or gene sequences as determined by the match between strings of such sequences. "Identity" (often referred to as "similarity")can be readily calculated by known methods, including those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds ) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G , ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Oxford University Press, NY (1992). Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wisconsin). Multiple alignment of the sequences can also be performed using the Clustal method of alignment (Higgins and Sharp CABIOS, 5, 151-153 (1989) with default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Relevant programs also include the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin); BLASTP, BLASTN, BLASTX (Altschul, et al , J. Mol. Biol. 215:403-410 (1990); DNASTAR (DNASTAR, Inc., Madison, Wisconsin); and the FASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York, N.Y.. Within the context of this disclosure it will be understood thatwhere sequence analysis software is used for analysis, the results of the analysis are based on the "default values" of the program referenced. As used herein "default values" will mean any set of values or parameters, which originally load with the software when first initialized.

[0134] Variants also include nucleic acid molecules that hybridizes under stringent hybridization conditions to a sequence disclosed herein and provide the same function as the reference sequence Exemplary stringent hybridization conditions include an overnight incubation at 42 °C in a solution including 50% formamide, 5XSSC (750 mM NaCI, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5XDenhardt's solution, 10% dextran sulfate, and 20 pig / ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1XSSC at 50 °C. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, moderately high stringency conditions include an overnight incubation at 37°C in a solution including 6XSSPE (20XSSPE=3M NaCI; 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 pig / ml salmon sperm blocking DNA; followed by washes at 50 °C with 1XSSPE, 0 1 % SDS In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5XSSC). Variations in the above conditions may be accomplished through the inclusion and / or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility

[0135] "Specifically binds" refers to an association of a binding domain (of, for example, a CAR binding domain or a nanoparticle selected cell targeting ligand) to its cognate binding molecule with an affinity or Ka (i e., an equilibrium association constant of a particular binding interaction with units of 1 / M) equal to or greater than 105M‘1, while not significantly associating with any other molecules or components in a relevant environment sample. “Specifically binds" is also referred to as “binds" herein. Binding domains may be classified as "high affinity" or "low affinity" In particularimplementations, "high affinity" binding domains refer to those binding domains with a Ka of at least 107 M-1, at least 108 M-1, at least 109 M-1 , at least 1010 M-1 , at least 1011 M-1, at least 1012 M-1 , or at least 1013 M-1 In particular implementations, "low affinity" binding domains refer to those binding domains with a Ka of up to 107 M-1, up to 106 M-1, up to 105 M-1. Alternatively, affinity may be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., 10-5 M to 10-13 M). In certain implementations, a binding domain may have "enhanced affinity," which refers to a selected or engineered binding domains with stronger binding to a cognate binding molecule than a wild type (or parent) binding domain. For example, enhanced affinity may be due to a Ka (equilibrium association constant) for the cognate binding molecule that is higher than the reference binding domain or due to a Kd (dissociation constant) for the cognate binding molecule that is less than that of the reference binding domain, or due to an off-rate (Koff) for the cognate binding molecule that is less than that of the reference binding domain. A variety of assays are known for detecting binding domains that specifically bind a particular cognate binding molecule as well as determining binding affinities, such as Western blot, ELISA, and BIACORE® analysis (see also, e.g., Scatchard, et al., 1949, Ann. N Y. Acad. Sci. 51 :660; and US 5,283,173, US 5,468,614, or the equivalent).

[0136] Unless otherwise indicated, the practice of the present disclosure can employ conventional techniques of immunology, molecular biology, microbiology, cell biology and recombinant DNA. These methods are described in the following publications. See, e.g., Sambrook, et al. Molecular Cloning: A Laboratory Manual, 2nd Edition (1989); F. M. Ausubel, et al. eds., Current Protocols in Molecular Biology, (1987); the series Methods IN Enzymology (Academic Press, Inc.); M. MacPherson, et al., PCR: A Practical Approach, IRL Press atOxford University Press (1991); MacPherson et al., eds. PCR 2: Practical Approach, (1995); Harlow and Lane, eds. Antibodies, A Laboratory Manual, (1988); and R. I. Freshney, ed Animal Cell Culture (1987).

[0137] Certain implementations are described herein, including the best mode known to the inventors for carrying out implementations of the disclosure Of course, variations on these described implementations will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for implementations to be practiced otherwise than specifically described herein. Accordingly, the scope of this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by implementations of the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIMSWhat is claimed is:1 . An engineered hormone receptor comprising: a ligand binding domain (LBD) that specifically binds a hormone, the LBD comprising: a first segment comprising a 1stto 11thalpha helix of the LBD; and a second segment comprising a 12thalpha helix of the LBD; a tag; a first linker inserted between the tag and the 11thalpha helix in the first segment of the LBD; and a second linker inserted between the tag and the 12thalpha helix in the second segment of the LBD.

2. The engineered hormone receptor of claim 1 , wherein the hormone comprises an estrogen.

3. The engineered hormone receptor of claim 2, wherein the estrogen comprises estradiol.

4. The engineered hormone receptor of claim 2, wherein the engineered hormone receptor comprises a sequence having at least 90% sequence identity to one of SEQ ID NOs: 22-28, 31-37, 66, or 104.

5. The engineered hormone receptor of claim 2, wherein the engineered hormone receptor comprises a sequence having at least 90% sequence identity to SEQ ID NO:

226. The engineered hormone receptor of claim 2, wherein the engineered estrogen receptor comprises a sequence having at least 90% sequence identity to SEQ ID NO: 104.

7. The engineered hormone receptor of claim 2, wherein the engineered estrogen receptor comprises a sequence having at least 90% sequence identity to one of SEQ ID NOs: 38-41 .

8. The engineered hormone receptor of claim 2, wherein the engineered estrogen receptor comprises a sequence having at least 90% sequence identity to one of SEQ ID NOs: 29, 30, or 107.

9. The engineered hormone receptor of claim 2, wherein the first segment of the LBD comprises a sequence having at least 90% sequence identity to SEQ ID NO: 2 or 58:

10. The engineered hormone receptor of claim 2, wherein the second segment of the LBD comprises a sequence having at least 90% sequence identity to SEQ ID NO: 3 or 60 .

11. The engineered hormone receptor of claim 2, wherein the first linker consists essentially of AG, G, SAG, or SLE.

12. The engineered hormone receptor of claim 2, wherein the second linker consists essentially of G, GP, GPS, or SEQ ID NO: 64.

13. The engineered hormone receptor of claim 1, wherein the hormone comprises a thyroid hormone.

14. The engineered hormone receptor of claim 13, wherein the thyroid hormone comprises triiodothyronine (T3).

15. The engineered hormone receptor of claim 13, wherein the engineered hormone receptor comprises a sequence having at least 90% sequence identity to one of SEQ ID NOs: 42-56, 105, or 106.

16. The engineered hormone receptor of claim 13, wherein the first segment of the LBD comprises a segment having at least 90% sequence identity to SEQ ID NO:

517. The engineered hormone receptor of claim 13, wherein the second segment of the LBD comprises a segment having at least 90% sequence identity to SEQ ID NO:

618. The engineered hormone receptor of claim 13, wherein the first linker consists essentially of SLE, G, AG, SEQ ID NO: 62, or SEQ ID NO: 63.

19. The engineered hormone receptor of claim 13, wherein the second linker consists essentially of GPS, LP, LPD, SEQ ID NO: 64, or SEQ ID NO: 65.

20. The engineered hormone receptor of claim 1 , wherein the hormone comprises an androgen, a progesterone, a glucocorticoid, a mineralocorticoid, or an endocrine-disrupting chemical.

21. The engineered hormone receptor of claim 1 , wherein the hormone comprises a glucocorticoid and comprises a sequence having at least 90% sequence identity to one of SEQ ID NOs: 59 or 61 .

22. The engineered hormone receptor of claim 1 , wherein the hormone comprises cortisol and comprises a sequence having at least 90% sequence identity to SEQ ID NO: 108.

23. The engineered hormone receptor of claim 1 , wherein the first linker has a length in a range of about 1 to about 25 amino acids, and / or wherein the second linker has a length in a range of about 1 to about 25 amino acids.

24. The engineered hormone receptor of claim 1 , further comprising at least one of an IgK leader, an HA flag, a PDGFR transmembrane domain, a CaaX, a LifeAct, dmito, or an NES.

25. The engineered hormone receptor of claim 1 , further comprising a co-factor consisting essentially of one of SEQ ID Nos: 20 and 74-103.

26. The engineered hormone receptor of claim 25, comprising: a first polypeptide comprising the LBD, the tag, the first linker, and the second linker; and a second polypeptide fused with the first polypeptide, the second polypeptide comprising the co-factor.

27. The engineered hormone receptor of claim 1 , wherein the engineered hormone receptor comprises a polypeptide chain comprising the first segment, the second segment, the tag, the first linker, and the second linker.

28. The engineered hormone receptor of claim 1 , wherein the tag emits a photon when the LBD binds to the hormone.

29. The engineered hormone receptor of claim 28, the tag being a first tag, the photon being a first photon, the engineered hormone receptor further comprising: a second tag that emits a second photon without the LBD binding to the hormone, the first photon having a different wavelength than the second photon.

30. The engineered hormone receptor of claim 28, wherein the photon has a wavelength in a range of about 240 nm to about 2,000 nm.

31. The engineered hormone receptor of claim 30, wherein the tag comprises at least one fluorescent protein.

32. The engineered hormone receptor of claim 31 , wherein the at least one fluorescent protein comprises circularly permutated GFP (cpGFP), mNeonGreen, circularly permuted HaloTag (cpHaloTag), circularly permuted mRuby (cpmRuby), circularly permuted mApple (cpmApple), circularly permuted mScarlet (cpmScarlet), mCherry, split GFP, split HaloTag, split NeonGreen, split mRuby, split mApple, or split mScarlet.

33. The engineered hormone receptor of claim 28, wherein the tag comprises a fluorescence resonance energy transfer (FRET) tag comprising a pair of fluorescent proteins, the pair of fluorescent proteins comprising at least one of mClover3 / mRuby3, Turquoise2 / Citrine, or EYFP / tagRFP.

34. The engineered hormone receptor of claim 28, wherein the hormone comprises a positron emission tomography (PET) tracer35. The engineered hormone receptor of claim 28, wherein the PET tracer comprises 18F-Fluoroestradiol.

36. A system, comprising: the engineered hormone receptor of claim 28 exposed to a fluid sample; a light source configured to emit excitation light to the engineered hormone receptor; and a photodetector configured to detect the hormone in the fluid sample by detecting a detection signal comprising the photon from the tag.

37. The system of claim 36, further comprising: a sensing region comprising the engineered hormone receptor and configured to receive the fluid sample comprising the hormone.

38. The system of claim 37, wherein the sensing region has a volume in range of about 0.1 piL to about 2,000 piL.

39. The system of claim 36, wherein a concentration of the hormone in the fluid sample is in a range of 0.1 pM to about 100 mM.

40. The system of claim 36, further comprising: a processor configured to: identify data indicative of a spectrum of the detection signal; and determine, by analyzing the data, a concentration of the hormone in the fluid sample.

41. A cartridge comprising a sensing region that comprises multiple copies of the engineered hormone receptor of claim 1.

42. A genetically modified cell expressing the engineered hormone receptor of claim 1.

43. The genetically modified cell of claim 42 comprising a cardiomyocyte, a neuron, human primary fibrochondrocyte, or a human mesenchymal stem cell.

44. A nucleic acid molecule encoding the engineered hormone receptor of claim 1 .

45. A virus comprising the nucleic acid molecule of claim 44.

46. A method of genetically modifying a cell to express the engineered hormone receptor of claim 1 .

47. A method of manufacturing the engineered hormone receptor of claim 1.

48. A method, comprising: detecting, from an engineered hormone receptor in an environment, a signal indicating binding between the engineered hormone receptor and a hormone, the engineered hormone receptor comprising a tag inserted between an 11thand 12thalpha helix of a LBD that specifically binds the hormone, the tag emitting the signal in response to the hormone binding to the LBD; and determining a presence and / or concentration of the hormone in the environment based on the signal.

49. The method of claim 48, wherein the engineered hormone receptor is expressed by a cell in the environment.

50. The method of claim 48, wherein the environment comprises a tissue sample, at least a portion of an organism, or a fluid sample51. The method of claim 48, wherein the environment comprises a living cell, a living tissue, or a living organism.

52. The method of claim 48, wherein the signal comprises a photon.

53. The method of claim 52, the photon being a first photon, wherein the method further comprises: illuminating the engineered hormone receptor with a second photon having a higher energy level than the first photon, thereby causing the engineered hormone receptor to fluoresce.

54. The method of claim 53, wherein the first photon has a wavelength in a range of about 240 nm to about 2,000 nm.

55. The method of claim 48, wherein the concentration of the of the hormone in the environment is determined to be in a range of about 0.1 pM to about 100 mM.

56. The method of claim 48, the signal being a first signal, the method further comprising: detecting, from the engineered hormone receptor in the environment, a second signal; and determining a location of the engineered hormone receptor in the environment based on the second signal.

57. The method of claim 56, wherein the first signal comprises a first photon with a first wavelength, the second signal comprises a second photon with a second wavelength, and the first wavelength is different than the second wavelength.

58. The method of claim 48, the engineered hormone receptor being a first engineered hormone receptor, the hormone being a first hormone, the signal being a first signal, the method further comprising: detecting, from a second engineered hormone receptor in the environment, a second signal indicating binding between the second engineered hormone receptor and a second hormone, the second hormone being different than the first hormone; and determining a presence and / or concentration of the second hormone in the environment based on the second signal.

59. The method of claim 58, wherein the first signal comprises a first photon having a first wavelength, the second signal comprises a second photon having a second wavelength, and the first wavelength is different than the second wavelength.

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