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Compositions and methods for the detection of zinc

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Compositions and methods for the detection of zinc


The invention relates generally to compositions and methods for the detection of zinc. In particular, compositions and methods are provided to detect changes in cellular zinc concentration and to correlate them to cellular phenomena.

Browse recent Northwestern University patents - Evanston, IL, US
Inventors: Teresa K. Woodruff, Thomas V. O'Halloran, Alison M. Kim, Emily Que, Betty Kong, Miranda Bernhardt
USPTO Applicaton #: #20120271100 - Class: 600 33 (USPTO) - 10/25/12 - Class 600 
Surgery > Reproduction And Fertilization Techniques



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The Patent Description & Claims data below is from USPTO Patent Application 20120271100, Compositions and methods for the detection of zinc.

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CROSS-REFERENCE TO RELATED APPLICATIONS

The present invention claims the benefit of U.S. Provisional Patent Application Ser. No. 61/475,472, filed Apr. 14, 2011, which is incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant Nos. HD021921 and GM038784 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

The invention relates generally to compositions and methods for the detection of zinc. In particular, compositions and methods are provided to detect physiological changes in zinc concentration (e.g., extracellular, intracellular, etc.) and to correlate them to cellular phenomena.

BACKGROUND OF THE INVENTION

The biological functions of metal ions have traditionally been thought to be limited to structural and catalytic roles within proteins. However, metal ions are also known to play signaling roles at the cellular level. For example, the alkaline earth metal calcium is one metal in which biological signaling roles are particularly well-established. Periodic elevations in the intracellular concentration of free calcium ions, also known as calcium transients or oscillations, are readily detected using fluorescent probes and are known to drive a number of biological processes (Berridge et al. (2003) Nat Rev Mol Cell Biol 4, 517-29.; herein incorporated by reference in its entirety). This is perhaps best illustrated in the egg, where repetitive calcium transients are among the earliest observable events after fertilization (Lawrence et al. (1997) Development 124, 233-41.; herein incorporated by reference in its entirety). Several parameters of these calcium transients, including total number, frequency, and amplitude influence which downstream developmental events are initiated (Ozil et al. (2006) Dev Biol 300, 534-44.; Ducibella et al. (2002) Dev Biol 250, 280-91.; Toth et al. (2006) Reproduction 131, 27-34.; herein incorporated by reference in their entireties). These cellular processes, which include cortical granule (CG) exocytosis and cell cycle progression can be initiated in the absence of sperm by stimulatory agents that induce parthenogenesis (Kline & Kline. (1992) Dev Biol 149, 80-9.; Tahara et al. (1996) Am J Physiol 270, C1354-61.; Liu & Maller. (2005) Curr Biol 15, 1458-68.; Madgwick et al. (2006) J Cell Biol 174, 791-801.; Battaglia & Gaddum-Rosse. (1987) Gamete Res 18, 141-52.; Ozil. (1990) Development 109, 117-27.; Zhang et al. (2005) Hum Reprod 20, 3053-61.; Tingen (2010) Science 330, 453; herein incorporated by reference in their entireties). Interestingly, the normal pattern of calcium oscillations is disrupted in eggs matured under conditions which limit the availability of the transition metal zinc (Kim et al. (2010) Nat Chem Biol 6, 674-81.; herein incorporated by reference in its entirety), suggesting that the physiologies of these metals are somehow connected in the egg.

In a departure from its well-established role as an enzymatic cofactor or structure stabilizing agent, fluctuations in the total concentration of intracellular zinc have recently been shown to contribute to the proper cell cycle regulation in maturing oocytes. Intracellular zinc levels increase by more than fifty percent and over 1010 ions per cell are accrued during the final stage of oocyte maturation, also known as meiotic maturation (Kim et al. (2010) Zinc availability regulates exit from meiosis in maturing mammalian oocytes, Nat Chem Biol 6, 674-81.; herein incorporated by reference in its entirety). This significant cellular metal accumulation event occurs over a remarkably short time interval and is a physiological imperative, as insufficient accumulation of zinc leads to a premature meiotic arrest at telophase I instead of metaphase II (Kim et al. (2010) Nat Chem Biol 6, 674-81.). This zinc-dependent meiotic checkpoint arises, in part, because zinc-insufficient eggs fail to reestablish maturation promoting factor (MPF) activity (Bernhardt et al. (2010) Biol Reprod, published ahead of print Nov. 10, 2010.; herein incorporated by reference in its entirety), which is necessary for eggs to set up and maintain meiotic arrest at metaphase II.

SUMMARY

OF THE INVENTION

In some embodiments, the present invention provides a Zn-responsive probe comprising: (a) a Zn-binding group configured to coordinate one or more Zn ions; (b) a signaling moiety configured to emit a detectable signal; and (c) an attachment group, wherein said attachment group is a chemically reactive with one or more functional groups. In some embodiments, the detectable signal of the signaling moiety is altered by coordination of one or more Zn ions by the Zn-binding moiety. In some embodiments, the signaling moiety comprises a fluorophore. In some embodiments, the fluorescence intensity, emission spectra, and/or excitation spectra of the signaling moiety is altered by coordination of one or more Zn ions by the Zn-binding moiety. In some embodiments, the interaction of the attachment group with a chemically reactive functional group results in covalent or stable non-covalent attachment of the Zn-responsive probe to the functional group. In some embodiments, the Zn-binding group is attached to the signaling group, and the signaling group is attached to the attachment group. In some embodiments, the one or more attachments are though a linker group. In some embodiments, the functional group is attached to a surface.

In some embodiments, the present invention provides a method of non-invasively detecting Zn release and/or uptake by a cell or cells comprising: (a) contacting a sample comprising a cell or cells with a Zn-responsive probe (e.g., a Zn-responsive probe attached to a solid surface); (b) detecting the signal emitted by the signaling moiety over time; and (c) correlating signal emitted with Zn concentration (e.g., intracellular or extracellular). In some embodiments, the sample comprises one or more oocytes (e.g., human oocytes (e.g., for use in in vitro fertilization), non-human oocyctes (e.g., oocytes of a non-human primate, domestic animal (e.g., feline, canine, etc.), agricultural animal (e.g., bovine, porcine, etc.)). In some embodiments, the present invention further comprises (d) removing the sample from the Zn-responsive probe (e.g, separating the cell and the Zn-responsive probe). In some embodiments, removing the sample from the Zn-responsive probe (or the Zn-responsive probe from the sample) is facilitated by the attachment of the probe to a solid surface.

In some embodiments, the present invention provides a method of detecting the fertilization of an oocyte comprising: (a) contacting a sample comprising an metaphase-II-arrested oocyte with a Zn-responsive probe (e.g., a Zn-responsive probe attached to a solid surface); (b) contacting the sample with sperm; (c) detecting a significant increase in extracellular Zn concentration based on a change in the signal of the Zn-responsive probe; and (d) correlating the significant increase in extracellular Zn concentration to fertilization of the oocyte by the sperm. In some embodiments, methods further comprise (f) removing the oocyte from the Zn-responsive probe; and (g) implanting the fertilized oocyte into a subject. In some embodiments, the Zn-responsive probe is attached to a surface. In some embodiments, removing the oocyte from the Zn-responsive probe is facilitated by the attachment of the probe to a surface. In some embodiments, attachment of the Zn-responsive probe to a surface prevents uptake of the probe into the oocyte.

In some embodiments, the present invention provides a method of evaluating the quality of an oocyte for fertilization comprising: (a) contacting a surface with a sample comprising the oocyte, wherein said surface displays a plurality of Zn-responsive probes (e.g., Zn-responsive probes are attached to the surface); (b) detecting the signal emitted by the Zn-responsive probes over time; (c) correlating the signal emitted with extracellular Zn concentration, wherein a decrease in extracellular Zn is indicative of an oocyte that is ready for fertilization. In some embodiments, the oocyte is in prophase-1-arrest upon contacting with the surface. In some embodiments, methods further comprise (d) removing the oocyte from the Zn-responsive probe. In some embodiments, removing the oocyte from the Zn-responsive probe is facilitated by the attachment of the probe to a surface. In some embodiments, attachment of the Zn-responsive probe to a surface prevents uptake of the probe into the oocyte.

In some embodiments, the present invention provides compositions comprising a solid surface displaying one or more Zn-responsive probes, wherein said Zn-responsive probes comprise: (a) a Zn-binding group configured to coordinate one or more Zn ions; and (b) a signaling moiety configured to emit a detectable signal. In some embodiments, the detectable signal of the signaling moiety is altered by coordination of one or more Zn ions by the Zn-binding moiety. In some embodiments, the signaling moiety comprises a fluorophore. In some embodiments, the fluorescence intensity, emission spectrum, and/or excitation spectrum of the signaling moiety is altered by coordination of one or more Zn ions by the Zn-binding moiety. In some embodiments, the composition further comprises a linker. In some embodiments, the linker connects the Zn-binding group with the signaling moiety. In some embodiments, the linker connects the Zn-binding group and/or the signaling moiety to the solid surface. In some embodiments, the Zn-responsive probes are attached to the solid surface through the interaction of a attachment group on the Zn-responsive probe and an anchor moiety on the solid surface. In some embodiments, the solid surface is selected from the group comprising: a plate, bead, well, slide, biopolymer, cell surface, and tube.

In some embodiments, the present invention provides a method for detection of Zn in a sample (e.g., by any suitable method). In some embodiments, a change in Zn concentration is detected. In some embodiment, Zn is detected by a Zn-responsive probe. In some embodiments, Zn is detected without a probe. In some embodiments, detection of Zn is correlated to a biological function or process (e.g., oocyte fertilization). In some embodiments, a Zn-responsive probe is attached to a solid surface (e.g., bead, plate, slide, well, etc.). In some embodiments, attachment of a Zn-responsive probe to a solid surface facilitates removal of the probe from a sample without contaminating the sample with Zn-responsive probe. In some embodiments, attachment of a Zn-responsive probe to a solid surface facilitates removal of the sample from a surface (e.g., (tube, well, slide, etc.) without contaminating the sample with Zn-responsive probe. In some embodiments, attachment of the Zn-responsive probe to a surface prevents uptake of the probe into the sample.

BRIEF DESCRIPTION OF THE DRAWINGS

The description provided herein is better understood when read in conjunction with the accompanying drawings, which are included by way of example and not by way of limitation.

FIG. 1 shows images depicting how changes in extracellular zinc concentration are readily monitored with FluoZin-3 during in vitro fertilization. Rapid, repetitive increases in fluorescence intensity were detected (a) by ROI analysis (denoted by white boxes in b-d). These spikes in fluorescent intensity involve the coordinated release of zinc from the cell in a phenomenon defined as a “zinc spark.” Each zinc spark can be distinguished in the time-lapse series (b, 00:25:12) against background fluorescence (representative example in c, 01:32:56). Successful fertilization was confirmed by the extrusion of a second polar body (d). Zinc sparks were also noted during strontium chloride-induced parthenogenesis (e, 00:02:56).

FIG. 2 shows zinc sparks observed in eggs from two different non-human primate species, Macaca mulatta (a, b) and Macaca fascicularis (c, d). In both cases, a single calcium transient was induced by ionomycin. Intracellular calcium was monitored with Calcium Green-1 AM, and extracellular zinc with FluoZin-3. The first panel begins at 00:04:35 in c and 00:06:37 in d. Each subsequent panel represents an image acquired 4 s following the previous panel. Time is expressed as hh:mm:ss, wherein 00:00:00 represents the start of image acquisition. Scale bar=40 μm in c and d.

FIG. 3 shows that zinc sparks are polarized and are immediately preceded by intracellular calcium transients. Shortly following activation, zinc sparks occur around the egg cortex with the exception of a zinc spark-free region (a, 00:21:04, arrowhead). This spark-free region corresponds to the region containing the meiotic spindle, where the second polar body is extruded (a, 01:20:00, arrowhead). Egg activation was confirmed by simultaneously monitoring intracellular calcium oscillations with Calcium Green-1 AM every 4 s (b). Intracellular calcium increases immediately before a zinc spark, as evident when images were collected at a faster acquisition rate of every 100 ms in an independent experiment (c). Time is expressed as hh:mm:ss, wherein 00:00:00 represents the start of image acquisition. In all cases, extracellular zinc was detected with FluoZin-3.

FIG. 4 shows that zinc sparks did not occur in the absence of calcium transients. The membrane permeable derivative of FluoZin-3 (FluoZin-3 AM) did not detect intracellular oscillations in zinc (a), suggesting that those detected by Calcium Green-1 AM were specific to calcium. Insets are brightfield images taken at the beginning (left, first polar body denoted as PB1) and end (right) of the acquisition period. In each case, the presence of the second polar body (PB2) at the conclusion of imaging was observed, confirming successful egg activation and development. Zinc sparks were absent in the absence of an activating agent such as strontium chloride (b), or in eggs treated with the calcium-selective chelator BAPTA AM (c). Representative images illustrate that BAPTA AM-treated eggs neither extrude a second polar body (b, 2 hrs postactivation, or hpf) nor form a pronucleus (c, 6 hpf). Intracellular calcium was detected by Calcium Green-1 AM (black line) and extracellular zinc was detected by FluoZin-3 (gray line) in b and c. Scale bar=75 μm in d and e.

FIG. 5 shows images depicting cortically polarized zinc ions in the mouse egg. Total zinc, as detected by synchrotron-based x-ray fluorescence (a, b), is uniquely polarized in the unfertilized egg (a; i-iii represent replicates). This distribution is absent in the other essential transition elements, such as iron (b; i-iii represent replicates). The range of each group of images is given units of μg/cm2. Labile (chelator-accessible) zinc, as detected by confocal microscopy (c-h), also has a hemispherical distribution in the live egg, as detected by two chemically distinct zinc fluorophores: zinquin ethyl ester (c-e) and FluoZin-3 AM (f-h). Co-staining with a DNA marker (Syto 64 in d, Hoechst 33342 in g) revealed that zinc was concentrated at the vegetal pole away from the meiotic spindle.

FIG. 6 shows total zinc content of the egg gradually decreases upon fertilization. Eggs and embryos were analyzed by XFM for their transition metal content. The mean zinc quota was highest in the in vivo ovulated (IVO) MII egg and trended towards a decrease following fertilization (see 2 and 6 hpf), reaching its lowest mean abundance in the two-cell embryo. The iron and copper quotas were an order of magnitude less than zinc at all time points examined.

FIG. 7 shows intracellular zinc decreases in the activated egg. Corresponding to the onset of the zinc sparks (FluoZin-3, gray line), there is a gradual decrease in intracellular labile zinc that is detectable with a membrane-permeable zinc fluorophore (FluoZin-3 AM, black line). Successful egg activation and development was confirmed using simultaneous brightfield imaging, which revealed extrusion of a second polar body (PB2). The first (0 min, left) and final (30 min, right) brightfield images are shown.

FIG. 8 shows optical sections from areas near the meiotic spindle (as detected by DNA markers Syto 64 and Hoechst 33342), selected from the complete confocal Z-series and projected to show the cortical localization of zinc-enriched vesicles as detected by two independent fluorophores, zinquin ethyl ester and FluoZin-3 AM. Merged images are also shown for clarity.

FIG. 9 shows exocytosis was inhibited by treating eggs with cytochalasin B prior to activation with strontium chloride. Cytochalasin B did not affect the calcium oscillations (a) but blocked all but the first zinc spark in most cases (b).

FIG. 10 shows dose response of unfertilized eggs to increasing concentrations of zinc pyrithione (ZnPT). Unfertilized eggs were treated with 10, 20, or 50 μM ZnPT for 15 min. A clear cytotoxic phenotype was only seen at 50 μM concentration. Thus, the effects of pyrithione are significantly below the threshold concentration (i.e., 500% lower) and do not involve cytotoxicity, as pyrithione was used at a minimum concentration (10 μM) and for a shorter duration (10 min).

FIG. 11 shows sustained elevation of intracellular zinc availability following egg activation leads to reestablishment of metaphase arrest. Eggs were activated with strontium chloride (SrCl2) then treated with zinc pyrithione (ZnPT) 1.5 hours later (a). At 6 h post-activation, control eggs form pronuclei (b, arrowheads) whereas the majority of eggs treated with ZnPT do not (c). When visualized by fluorescence, control eggs display decondensed DNA organized within a defined nucleus (d). F-actin is homogeneous around the egg's cortex (e) and α-tubulin remains organized as a spindle midbody remnant (f), which is visible between the egg and the second polar body (g, merged image). In contrast, ZnPT-treated eggs display condensed chromosomes (h) adjacent to an area of concentrated, cortical F-actin (i). α-tubulin is organized in a metaphase-like configuration (j) around the chromosomes (k, merged image). This layout mirrors the subcellular arrangement in unfertilized eggs (l-o), which also display condensed chromosomes (l) overlaid with an actin cap (m), surrounded by a metaphase spindle (n). Scale bar=80 μm (b, c) or 25 μm (d-o).

FIG. 12 shows perturbation of intracellular zinc availability causes activation of the egg. Following 8 hours of culture, control eggs maintain a metaphase II spindle (a, sp) with individual chromosomes aligned at the metaphase plate (b). In contrast, eggs exposed to 10 μM TPEN for the same period artificially activate as indicated by the formation of an intact pronucleus (c, pn) with decondensed chromatin surrounding a nucleolus (d). The number of eggs displaying a pronucleus is significantly higher in the TPEN-treated group (e).

FIG. 13 shows the chemical structure of an exemplary Zn-responsive probe of the present invention (A, A zinc-chelating ligand containing five potential zinc binding groups; B, BODIPY fluorophore; C, ethanol ether based linker chain; D, amine group that enables attachment of this fluorophore to carboxylate-modified surfaces via amide-coupling chemistries).

FIG. 14 shows a synthesis scheme for an exemplary Zn-responsive probe of the present invention.

DETAILED DESCRIPTION

The invention relates generally to compositions and methods for the detection of zinc (Zn). In particular, compositions and methods are provided to detect changes in extracellular and/or intracellular zinc concentration and correlate them to cellular phenomena. Experiments conducted during development of embodiments of the present invention demonstrated the in vivo detection of changes in cellular (e.g., extracellular, intracellular) zinc levels and correlation of those levels to cellular events. In some embodiments, detection of changes in cellular (e.g., extracellular, intracellular) zinc levels provides a marker for the occurrence of cellular events (e.g., fertilization). In some embodiments, the present invention provides reagents (e.g., probes) for the in vivo and/or in vitro measurement of Zn levels or changes thereof. In some embodiments, the present invention provides methods for detection, measurement, and/or identification of changes in cellular Zn levels. In some embodiments, the present invention provides compositions and methods for detecting changes in Zn levels (e.g., intracellular, extracellular, non-cellular) and correlating such changes to cellular phenomena (e.g., cell cycle: phase, pausing, resumption, defects, etc.).

In some embodiments, the present invention provides one or more chemical probes comprising, consisting of, or consisting essentially of: A) a metal binding group (e.g., Zn-binding group, general metal chelator, etc.); B) a signaling moiety (e.g., fluorophore) that will exhibit a change in detectable signal (e.g., fluorescence properties) in response to metal ion binding to the metal binding group; C) a linker group; an attachment group that will allow for attachment of the probe to surfaces (e.g., plate, bead, well, slide, biopolynerm cell surface, etc) via coupling chemistries (e.g., amide coupling, thiol/maleimide chemistry, click chemistry, etc.).

I. Zn-Responsive Probes

In some embodiments, the present invention provides probes and other reagents for use in detecting Zn concentrations, levels, presence, or changes thereof. In some embodiments the present invention provides Zn-responsive chemical probes. In some embodiments, the probes and reagents provided herein provide intracellular, extracellular, or non-cellular detection and/or quantification of Zn, and/or changes in the presence or concentration of Zn, either in vivo or in vitro. In some embodiments, the probes described herein are capable of (1) detecting the presence of one or more Zn ions and/or Zn-containing compositions (e.g., by binding to Zn), and (2) signaling the detection of Zn ions and/or Zn-containing compositions (e.g., optically). In some embodiments, probes comprise one or more of: a Zn-binding group (ZB), a signaling moiety (S) (e.g., fluorophore), one or more linkers (Lx), and an attachment group (A). In some embodiments, a Zn-responsive probe comprises a Zn-binding group attached to a signaling moiety, optionally through a linker. In some embodiments, a Zn-binding group and signaling moiety are directly attached. In some embodiments, a Zn binding group and/or signaling moiety are covalently connected to an attachment group (e.g., through a linker, directly, etc.). In some embodiments, a Zn-responsive probe comprises a general structure selected from the group comprising:

A-L-S-ZB  (1)

A-L-ZB-S  (2)

A-S-ZB  (3)

A-S-L-ZB  (4)

A-ZB-L-S  (5)

A-L1-S-L2-ZB  (6)

A-L1-ZB-L2-S  (7)

In some embodiments, a Zn-responsive probe comprises a Zn-binding group and a signaling moiety. In some embodiments, the Zn-binding group comprises a chemical functionality to bind one or more Zn ions present in its local environment. In some embodiments, upon binding a Zn ion by the Zn-binding group, a structural, conformational, chemical, physical, or other change in the Zn-responsive probe causes a detectable change in the signal from the signaling moiety (e.g., shift in emission maximum, shift in excitation maximum, change in intensity, etc.). In some embodiments, detection or quantification of the signal from the signaling moiety provides a qualitative and/or quantitative means for detecting and/or measuring the presence or amount of Zn present in the probe\'s local environment. In some embodiments, detection or quantification of changes in the signal from the signaling moiety provides a qualitative and/or quantitative means for detecting and/or measuring changes in the presence or amount of Zn present in the probe\'s local environment.

In some embodiments, a Zn-responsive probe comprises a Zn binding group, and attachment group, and a signaling moiety. In such embodiments, the Zn binding group and signaling moiety function to bind Zn and provide a signal indicative of the binding event, as described in the preceding paragraph. In some embodiments, the attachment group is a chemical moiety capable of covalently or non-covalently interacting with another chemical moiety known as the anchor moiety. In some embodiments, interaction of the attachment group with the anchor moiety results in stable attachment of the Zn-responsive probe to the anchor moiety. In some embodiments an object or surface (e.g., plate, well, bead, slide, etc.) displays one or more anchor moieties. In some embodiments, interaction of the attachment groups of one or more Zn-responsive probes with one or more anchor groups displayed on a surface results in one or more Zn-responsive probes being displayed on the surface or object.

In some embodiments, a Zn-responsive probe comprises one or more structural or functional features described in U.S. Pat. No. 7,105,680; herein incorporated by reference in its entirety.

A. Zn-Binding Groups

In some embodiments, a Zn-responsive probe comprises a metal-binding group. In some embodiments, a metal binding group comprises a Zn-binding group. In some embodiments, a Zn-responsive probe comprises more than one Zn-binding groups (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.). In some embodiments, a Zn-binding group is a chemical moiety capable of stably interacting with one or more Zn ions. In some embodiments, a Zn-binding group is capable of interacting with one or more Zn ions, while covalently attached to the other functional elements of the Zn-responsive probe. In some embodiments, a Zn-binding group interacts with a Zn ion through covalent and/or non-covalent binding. In some embodiments, a Zn-binding group coordinates and/or partially coordinates a Zn ion. In some embodiments, a Zn-binding group is capable of coordinating a single Zn ion. In some embodiments, a Zn-binding group is capable of coordinating more than one Zn ions at a time (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 . . . 20 . . . 50 . . . 100 . . . 1000, etc.). In some embodiments, coordination of a Zn ion results in a chemical, magnetic, electric, physical, and/or structural change in the Zn-binding group, the signaling moiety, the connection between the Zn-binding group and the signaling moiety, and/or the Zn-responsive probe. In some embodiments, the type, degree, and/or magnitude of the change are dependent or responsive to the number of Zn ions coordinated (e.g., more coordinated Zn ions results in greater change). In some embodiments the denticity and/or identity of Zn-binding groups is adjusted to tune metal binding affinity and selectivity.

In some embodiments, the present invention is not limited to any particular type or class of Zn-binding groups. In some embodiments, a Zn-binding group comprises a functional group capable of transiently or stably binding, coordinating, and/or chelating one or more Zn ions (e.g., free or in another complex). In some embodiments, a Zn-binding group is Zn specific. In some embodiments, a Zn-binding group preferentially binds Zn over other metal ions. In some embodiments, a Zn-binding group is a general metal-binding moiety. Chemical moieties that find use as Zn-binding groups, or within Zn-binding groups, of the present invention include, but are not limited to, e.g., diethyldithiocarbamate (DEDTC) and ethylenediaminetetra-acetic acid (EDTA), 1,10-phenanthroline, pyridyl-containing compounds, amine-containing compounds (e.g., tertiary amines), histidine containing compounds, sulfonamide-containing coimpounds, etc. In some embodiments, a Zn-binding group has at least one functional group selected from polyalkylene oxide, hydroxylated group, or a group having at least one amine, ammonium salt, carboxylate, sulfanyl, sulfinyl, sulfonyl, phosphate, phosphonate, phosphate, tertiary amine, pyridyl group; or combinations thereof. In some embodiments, Zn-binding groups comprise one or more sites for attachment to other functional groups within the Zn-responsive probe (e.g., attachment group, signaling moiety, linker, another Zn-binding group, etc.).

B. Signaling Moiety

In some embodiments, a signaling moiety is a detectable chemical moiety. In some embodiments, a signaling moiety is an optically detectable chemical moiety (e.g., fluorophore, chromophore, etc.). In some embodiments, a signaling moiety comprises a fluorescent dye or fluorophore. In some embodiments, a signaling moiety finds utility as a fluorophore for detection using one or more of optical spectroscopy, fluorescence spectroscopy, confocal spectroscopy, confocal fluorescence spectroscopy, two-photon excitation (TPE) fluorescence microscopy, etc.

In some embodiments, a signaling moiety is configured within a Zn-responsive probe such that coordination of one or more zinc ions by the Zn-binding group results in a detectable change in the signal from the signaling moiety. In some embodiments, a detectable change in signal comprises a change (e.g., increase or decrease) in signal (e.g., fluorescence) intensity. In some embodiments a detectable change (e.g., increase or decrease) in signal (e.g., fluorescence) intensity is readily detectable by a skilled artisan using the compositions and methods of the present invention (e.g., 1.1-fold . . . 1.2-fold . . . 1.5-fold . . . 2-fold . . . 5-fold . . . 10-fold . . . 20-fold . . . 50-fold . . . 100-fold . . . 200-fold . . . 500-fold . . . 1000-fold, etc.). In some embodiments, a detectable change in signal comprises a change (e.g., increase or decrease) in the excitation maximum. In some embodiments, a detectable change in signal comprises a change in the excitation spectrum. In some embodiments, a detectable change in signal comprises a change (e.g., increase or decrease) in the emission maximum. In some embodiments, a detectable change in signal comprises a change in the emission spectrum.

The present invention is not limited to any particular signaling moiety. In some embodiments, the signaling moiety is a fluorophore. The present invention is not limited to any particular fluorophore. Fluorophores and/or fluorescent labels that find use as or within signaling moieties of the present invention include, but are not limited to, e.g., fluorescein and rhodamine dyes, such as fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (commonly known by the abbreviations FAM and F), 6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein (JOE or J), N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA or T), 6-carboxy-X-rhodamine (ROX or R), 5-carboxyrhodamine-6G (R6G5 or G5), 6-carboxyrhodamine-6G (R6G6 or G6), and rhodamine 110; cyanine dyes, e.g., Cy3, Cy5 and Cy7 dyes; alexa dyes, e.g., alexa fluor 555, alexa fluor 594; coumarins, e.g., umbelliferone; benzimide dyes, e.g., Hoechst 33258; phenanthridine dyes, e.g., Texas Red; ethidium dyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes; polymethine dyes, e.g., cyanine dyes such as Cy3, Cy5, etc; BODIPY dyes and quinoline dyes; and derivities thereof. Suitable fluorescent labels include any of the variety of fluorescent labels disclosed in United States Patent Application Publication No. 20010009762, the disclosure of which is incorporated herein by reference. In some embodiments, signaling moieties comprise one or more sites for attachment to other functional groups within the Zn-responsive probe (e.g., attachment group, another signaling moiety, linker, Zn-binding group, etc.).

C. Linker

In some embodiments, present invention provides one or more linkers, linking moieties, linking groups, or linker regions. In some embodiments, a linker connects two or more functional groups of a Zn-responsive probe (e.g., signaling moiety, Zn-binding group, attachment group, etc.). In some embodiments, a linker comprises 1-1000 atoms (e.g., 1-10, 1-100, etc.). In some embodiments, a linker connects a signaling moiety to a Zn-binding group. In some embodiments, a linker connects a signaling moiety to an attachment group. In some embodiments, a linker connects an attachment group to a Zn-binding group. In some embodiments, one functional groups of a Zn-responsive probe (e.g., signaling moiety, Zn-binding group, attachment group, etc.) is connected to more than one other functional groups of a Zn-responsive probe (e.g., signaling moiety, Zn-binding group, attachment group, etc.) by multiple linkers. In some embodiments, a linker is branched for connection of three or more functional groups of a Zn-responsive probe (e.g., signaling moiety, Zn-binding group, attachment group, etc.).

The present invention is not limited to any particular linker group. Indeed, a variety of linker groups are contemplated, suitable linkers could comprise, but are not limited to, alkyl groups, ether, polyether, alkyl amide linker, a peptide linker, a modified peptide linker, a Poly(ethylene glycol) (PEG) linker, a streptavidin-biotin or avidin-biotin linker, polyaminoacids (eg. polylysine), functionalized PEG, polysaccharides, glycosaminoglycans, dendritic polymers (WO93/06868 and by Tomalia et al. in Angew. Chem. Int. Ed. Engl. 29:138-175 (1990), herein incorporated by reference in their entireties), PEG-chelant polymers (WO94/08629, WO94/09056 and WO96/26754, herein incorporated by reference in their entireties), oligonucleotide linker, phospholipid derivatives, alkenyl chains, alkynyl chains, disulfide, or a combination thereof.

In some embodiments the linker comprises a single chain connecting one functional groups of a Zn-responsive probe (e.g., signaling moiety, Zn-binding group, attachment group, etc.) to another functional group of a Zn-responsive probe (e.g., signaling moiety, Zn-binding group, attachment group, etc.). In some embodiments, there are multiple linkers connecting multiple Zn-binding groups to a single signaling moiety. In some embodiments, a linker may connect multiple Zn-binding groups to each other. In some embodiments, a linker may connect multiple signaling moieties to each other. In some embodiments, a linker may be branched. In some embodiments, the linker may be flexible, or rigid.

In some embodiments, the linker of the present invention is cleavable or selectively cleavable. In some embodiments, the linker is cleavable under at least one set of conditions, while not being substantially cleaved (e.g. approximately 50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater remains uncleaved) under another set (or other sets) of conditions. In some embodiments, the linker is susceptible to cleavage under specific conditions relating to pH, temperature, oxidation, reduction, UV exposure, exposure to radical oxygen species, chemical exposure, light exposure (e.g. photo-cleavable), etc. In some embodiments, the linker region is photocleavable. That is, upon exposure to a certain wavelength of light, the linker region is cleaved, allowing release of the connected functional groups (e.g., Zn-binding group, etc.). This embodiment has particular use in developmental biology fields (cell maturation, neuronal development, etc.), where the ability to follow the fates of particular cells is desirable. In some embodiments, a preferred class of photocleavable linkers are the O-nitrobenzylic compounds, which can be synthetically incorporated via an ether, thioether, ester (including phosphate esters), amine or similar linkage to a heteroatom (particularly oxygen, nitrogen or sulfur). Also of use are benzoin-based photocleavable linkers. A wide variety of suitable photocleavable moieties is outlined in the Molecular Probes Catalog, supra. In some embodiments, the linker is susceptible to enzymatic cleavage (e.g. proteolysis). In some embodiments of the present invention, functional groups (e.g., signaling moiety and Zn-binding group) are linked, via a cleavable linker. The present invention is not limited to any particular linker group. In some embodiments, the cleavable linker region contains a peptide portion. In some embodiments, the peptide portion of the cleavable linker region is cleavable. In some embodiments, the peptide portion of the cleavable linker region is enzymatically cleavable. In some embodiments the cleavable linker contains a specific proteolytic site. In some embodiments, in addition to the peptide portion of the cleavable linker region, an additional linker portion is contemplated. Indeed, a variety of additional linker groups are contemplated, suitable linkers could comprise, but are not limited to, alkyl groups, ether, polyether, alkyl amide linker, a peptide linker, a modified peptide linker, a Poly(ethylene glycol) (PEG) linker, a streptavidin-biotin or avidin-biotin linker, polyaminoacids (eg. polylysine), functionalised PEG, polysaccharides, glycosaminoglycans, dendritic polymers (WO93/06868 and by Tomalia et al. in Angew. Chem. Int. Ed. Engl. 29:138-175 (1990), herein incorporated by reference in their entireties), PEG-chelant polymers (WO94/08629, WO94/09056 and WO96/26754, herein incorporated by reference in their entireties), oligonucleotide linker, phospholipid derivatives, alkenyl chains, alkynyl chains, disulfide, or a combination thereof.

D. Attachment Group

In some embodiments, an attachment group is configured for covalent and/or non-covalent binding/interaction with a surface (e.g., well, plate, bead, slide, etc.). In some embodiments, an attachment group is configured for covalent and/or non-covalent binding/interaction with an anchor moiety. In some embodiments, an attachment group and anchor moiety comprise an attachment pair. In some embodiments, an attachment group and anchor moiety are chemically or physically complimentary to allow a stable interaction (e.g., covalent or non-covalent) between them. In some embodiments, an attachment group is part of a Zn-responsive probe. In some embodiments, an anchor moiety is a functional group attached to a surface or object (e.g., plate, bead, well, slide, etc.). In some embodiments, the interaction of the members of an attachment pair provides a mean for attaching a Zn-responsive probe to a surface or object (e.g., plate, bead, well, slide, etc.). In some embodiments, the attachment pair interaction is reversible. In some embodiments, the attachment pair interaction is irreversible. In some embodiments, the interaction of the attachment pair is stable enough to provide stable attachment of the Zn-responsive element to a surface or object (e.g., plate, bead, well, slide, etc.) for at least the duration of Zn detection (e.g., experiment, evaluation, assay, treatment, diagnosis, etc.). The present invention is not limited by the type or class of attachment group. In some embodiments, attachment groups comprise, e.g., one or more chemical groups including, but not limited to: aldehyde, hydroxysuccinimidyl, hydrazide, thiol, triflate, tresylate, azirdine, oxirane, orthopyridyl disulphide, vinylsulfone, iodoacetamide or a maleimide, a group suitable for Click chemistry, etc.

In some embodiments, an attachment pair (e.g., attachment group and anchor moiety) provide chemical means for attaching a Zn-responsive probe to a surface (e.g., plate, bead, well, slide, etc.). The present invention is not limited by the types of attachment pairs, and any suitable attachment pair that allows for attachment of a Zn-responsive probe to a anchor-decorated surface may find use with the present invention (e.g., carboxylate and amine; thiol and maleimide; biotin and streptavidin; azide and phosphine, etc.).

In some embodiments, an attachment pair (e.g., attachment group and anchor moiety) provide chemical means for attaching a Zn-responsive probe to a surface (e.g., plate, bead, well, slide, etc.). The present invention is not limited by the types of surfaces, and any suitable surface or object may find use with the present invention, for example: beads, nanobeads, microparticles, microscope slide, microfluidics chamber, the interior of a reaction tube, a well (e.g., of a 96-well plate, of a 384-well plate, etc.), microtiter plate, etc.

In some embodiments, attachment of one or more Zn-responsive probes to a surface allows a sample to be added to the Zn-responsive probes, assayed for the presence, amount, or a change in Zn concentration, and then the sample is removed from the surface without contaminating the sample. In some embodiments, non-invasive sample assaying is provided by stably anchored Zn-responsive probes. In some embodiments, attachment of one or more Zn-responsive probes to a bead allows the Zn-responsive probes to be added to a sample; the sample is assayed for the presence, amount, or a change in Zn concentration; and then the bead-bound Zn-responsive probes are removed from the sample without contamination. In some embodiments, attachment of one or more Zn-responsive probes to a surface allows a sample to be added to the Zn-responsive probes without contamination of the sample with the probes. In some embodiments, attachment of one or more Zn-responsive probes to a surface allows cells (e.g., oocytes) to be added to the Zn-responsive probes without entry of the probes into the cells (e.g., preventing cell toxicity or disruption of cellular functions).

II. Methods/Applications

In some embodiments, the present invention provides methods for detection, measurement, identification, and/or quantification of Zn ions and/or Zn-containing compounds or compositions. In some embodiments, the present invention provides methods for detection, measurement, identification, and/or quantification of Zn concentration. In some embodiments, the present invention provides methods for detection, measurement, identification, and/or quantification of changes in Zn concentration. In some embodiments, the present invention provides methods for correlating biological and/or cellular events, changes, processes, and/or phenomena to Zn concentration or changes thereof (e.g., extracellular Zn concentration, intracellular Zn concentration, etc.). In some embodiments, any suitable methods of detecting and/or quantifying the presence and/or concentration of Zn ions, or changes thereof, find use in the present invention. In some embodiments, Zn ions are detected and or quantified through the use of Zn-sensitive probes, e.g., those described herein.

In some embodiments, the present invention provides detection or quantification of changes in intracellular and/or extracellular Zn concentration and correlates those changes to changes in the cell cycle (e.g., stall, pause, arrest, resumption). In some embodiments, the compositions and methods herein provide correlation of Zn concentration (e.g., extracellular Zn concentration) to the cell cycle phase of an oocyte (e.g., maturing oocyte) and/or is progression through the maturation process. In some embodiments, methods provided herein detect and/or quantify Zn uptake or release from cells. In some embodiments, methods correlate Zn uptake or release from cells with cellular activity (e.g., cell cycle activities (e.g., moving from one phase to another, arrest of cell cycle, resumption of cell cycle)).



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stats Patent Info
Application #
US 20120271100 A1
Publish Date
10/25/2012
Document #
13442453
File Date
04/09/2012
USPTO Class
600 33
Other USPTO Classes
435/71, 546 13
International Class
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Drawings
17


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