Neurotransmitter biosensors are disclosed, including YbeJ-based glutamate binding biosensors, comprising a neurotransmitter binding domain conjugated to donor and fluorescent moieties that permit detection and measurement of Fluorescence Resonance Energy Transfer upon binding neurotransmitter. Such biosensors are useful for the detection of neurotransmitter concentrations in vivo and in culture.

Description of the Invention

SUMMARY OF INVENTION

The present invention provides neurotransmitter biosensors for detecting and measuring changes in neurotransmitter concentrations. In particular, the invention provides an isolated nucleic acid encoding a glutamate binding fluorescent indicator (FLIP-E) comprising a glutamate binding protein moiety from Escherichia coli YbeJ wherein the glutamate binding protein moiety is genetically fused to a donor fluorescent protein moiety and an acceptor fluorescent protein moiety, wherein fluorescence resonance energy transfer (FRET) between the donor moiety and the acceptor moiety is altered when the donor moiety is excited and glutamate binds to the glutamate binding protein moiety. Vectors, including expression vectors, and host cells comprising the inventive nucleic acids are also provided, as well as biosensor proteins encoded by the nucleic acids. Such nucleic acids, vectors, host cells and proteins may be used in methods of detecting changes in neurotransmitter levels and particularly extracellular glutamate levels in neuron samples, and in methods of identifying compounds that modulate glutamate excitotoxicity.

DETAILED DESCRIPTION OF INVENTION

The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed inventions, or that any publication specifically or implicitly referenced is prior art.

Other objects, advantages and features of the present invention become apparent to one skilled in the art upon reviewing the specification and the drawings provided herein. Thus, further objects and advantages of the present invention will be clear from the description that follows.

Biosensors

The present invention provides neurotransmitter biosensors for detecting and measuring changes in neurotransmitter concentrations using Fluorescence Resonance Energy Transfer (FRET). The three major categories of substances that act as neurotransmitters are (1) amino acids (primarily glutamic acid or glutamate, GABA, aspartic acid & glycine), (2) peptides (vasopressin, somatostatin, neurotensin, etc.) and (3) monoamines (norepinephrine, dopamine & serotonin) plus acetylcholine. In particular, the invention provides glutamate binding fluorescent indicators, particularly indicators comprising a glutamate binding protein moiety from the Escherichia coli glutamate/aspartate receptor, YbeJ. Additional neurotransmitter biosensors for the neurotransmitters listed above may also be prepared using the constructs and methods provided herein.

Thus, the invention provides isolated nucleic acids encoding neurotransmitter binding fluorescent indicators. One embodiment, among others, is an isolated nucleic acid which encodes a glutamate binding fluorescent indicator, the indicator comprising: a glutamate binding protein moiety, a donor fluorescent protein moiety genetically fused to the glutamate binding protein moiety, and an acceptor fluorescent protein moiety genetically fused to the glutamate binding protein moiety, wherein FRET between the donor moiety and the acceptor moiety is altered when the donor moiety is excited and glutamate binds to the glutamate binding protein moiety. A preferred glutamate binding protein moiety is a glutamate binding protein moiety from Escherichia coli YbeJ.

YbeJ is also known in the art as YzzK and GltI, and its DNA sequence (SEQ ID No. 1) and protein sequence (YbeJ, protein accession no NP.sub.--415188, SEQ ID No. 2) are known. Any portion of the YbeJ DNA sequence which encodes a glutamate binding region may be used in the nucleic acids of the present invention. For instance, one region that is suitable for use in the nucleic acids of the present invention is provided by SEQ ID No. 3, which encodes a truncated glutamate-aspartate binding protein sequence (SEQ ID No. 4), encoding mature protein without signal peptide. Naturally occurring homologues from other bacterial species may also be used, for instance the PA5082 gene from Pseudomonas aeruginosa, whose gene product is 70% similar to the YbeJ protein from E. coli. Glutamate binding portions of YbeJ or any of its homologues may be cloned into the vectors described herein and screened for activity according to the disclosed assays.

Naturally occurring species variants of YbeJ may also be used, in addition to artificially engineered variants comprising site-specific mutations, deletions or insertions that maintain measurable glutamate binding function. Variant nucleic acid sequences suitable for use in the nucleic acid constructs of the present invention will preferably have at least 70, 75, 80, 85, 90, 95, or 99% similarity or identity to the gene sequence for YbeJ. Suitable variant nucleic acid sequences may also hybridize to the gene for YbeJ under highly stringent hybridization conditions. High stringency conditions are known in the art; see for example Maniatis et al., Molecular Cloning: A Laboratory Manual, 2d Edition, 1989, and Short Protocols in Molecular Biology, ed. Ausubel, et al., both of which are hereby incorporated by reference. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). Generally, stringent conditions are selected to be about 5-10.degree. C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0M sodium ion; typically about 0.01 to 1.0M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30.degree. C. for short probes (e.g. 10 to 50 nucleotides) and at least about 60.degree. C. for long probes (e.g. greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.

Preferred artificial variants of the present invention may exhibit increased or decreased affinity for glutamate, in order to expand the range of concentration that can be measured by YbeJ-based and other glutamate nanosensors. Preferred artificial variants, among others, include glutamate binding regions comprising the mutations A207G, A207P, A207K A207M, A207S, A207C, A207R, A207V, A207L, A207Q, A207T, A207F, A207Y, A207N, A207W, A207H, A207D, and/or S95W. Additional artificial variants showing decreased or increased binding affinity for glutamate may be constructed by random or site-directed mutagenesis and other known mutagenesis techniques, and cloned into the vectors described herein and screened for activity according to the disclosed assays.

The isolated nucleic acids of the invention may incorporate any suitable donor and acceptor fluorescent protein moieties that are capable in combination of serving as donor and acceptor moieties in FRET. Preferred donor and acceptor moieties are selected from the group consisting of GFP (green fluorescent protein), CFP (cyan fluorescent protein), BFP (blue fluorescent protein), YFP (yellow fluorescent protein), and enhanced variants thereof, with a particularly preferred embodiment provided by the donor/acceptor pair CFP/YFP-Venus, a variant of YFP with improved pH tolerance and maturation time (Nagai, T., Ibata, K., Park, E. S., Kubota, M., Mikoshiba, K., and Miyawaki, A. (2002) A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20, 87-90). An alternative is the MiCy/mKO pair with higher pH stability and a larger spectral separation (Karasawa S, Araki T, Nagai T, Mizuno H, Miyawaki A. Cyan-emitting and orange-emitting fluorescent proteins as a donor/acceptor pair for fluorescence resonance energy transfer. Biochem J. 2004 381:307-12). Criteria to consider when selecting donor and acceptor fluorescent moieties are known in the art, for instance as disclosed in U.S. Pat. No. 6,197,928, which is herein incorporated by reference in its entirety.

Also suitable as either a donor or acceptor is native DsRed from a Discosoma species, an ortholog of DsRed from another genus, or a variant of a native DsRed with optimized properties (e.g. a K83M variant or DsRed2 (available from Clontech)). As used herein, the term "variant" is intended to refer to polypeptides with at least about 70%, more preferably at least 75% identity, including at least 80%, 90%, 95% or greater identity to native fluorescent molecules. Many such variants are known in the art, or can be readily prepared by random or directed mutagenesis of native fluorescent molecules (see, for example, Fradkov et al., FEBS Lett. 479:127-130 (2000)).

When the fluorophores of the biosensor contain stretches of similar or related sequence(s), the present inventors have recently discovered that gene silencing may adversely affect expression of the biosensor in certain cells and particularly whole organisms. In such instances, it is possible to modify the fluorophore coding sequences at one or more degenerate or wobble positions of the codons of each fluorophore, such that the nucleic acid sequences of the fluorophores are modified but not the encoded amino acid sequences. Alternative, one or more conservative substitutions that do not adversely affect the function of the fluorophores may also be incorporated. See PCT application [Ser. No. 12/083,197, "Methods of Reducing Repeat-Induced Silencing of Transgene Expression and Improved Fluorescent Biosensors], which is herein incorporated by reference in its entirety.

The invention further provides vectors containing isolated nucleic acid molecules encoding neurotransmitter biosensor polypeptides. Exemplary vectors include vectors derived from a virus, such as a bacteriophage, a baculovirus or a retrovirus, and vectors derived from bacteria or a combination of bacterial sequences and sequences from other organisms, such as a cosmid or a plasmid. Such vectors include expression vectors containing expression control sequences operatively linked to the nucleic acid sequence coding for the neurotransmitter biosensor. Vectors may be adapted for function in a prokaryotic cell, such as E. coli or other bacteria, or a eukaryotic cell, including yeast and animal cells. For instance, the vectors of the invention will generally contain elements such as an origin of replication compatible with the intended host cells, one or more selectable markers compatible with the intended host cells and one or more multiple cloning sites. The choice of particular elements to include in a vector will depend on factors such as the intended host cells, the insert size, whether regulated expression of the inserted sequence is desired, i.e., for instance through the use of an inducible or regulatable promoter, the desired copy number of the vector, the desired selection system, and the like. The factors involved in ensuring compatibility between a host cell and a vector for different applications are well known in the art.

Preferred vectors for use in the present invention will permit cloning of the neurotransmitter binding domain or receptor between nucleic acids encoding donor and acceptor fluorescent molecules, resulting in expression of a chimeric or fusion protein comprising the neurotransmitter binding domain genetically fused to donor and acceptor fluorescent molecules. Exemplary vectors include the bacterial pRSET-FLIP derivatives disclosed in Fehr et al. (2002) (Visualization of maltose uptake in living yeast cells by fluorescent nanosensors. Proc. Natl. Acad. Sci. USA 99, 9846-9851), which is herein incorporated by reference in its entirety. Alternatively, the neurotransmitter binding domain of interest may be first fused to fluorescent donor and acceptor coding sequences and then cloned into an appropriate vector, as described in U.S. Pat. No. 6,596,499, which is herein incorporated by reference in its entirety.

The chimeric nucleic acids of the present invention are preferably constructed such that the donor and acceptor fluorescent moiety coding sequences are fused to separate termini of the neurotransmitter binding domain in a manner such that changes in FRET between donor and acceptor may be detected upon neurotransmitter binding. Alternatively, either or both of the donor fluorophore and/or said acceptor fluorophore moieties may be fused to the ligand binding protein moiety at an internal site of said ligand binding protein moiety. Such fusions are described in provisional application No. 60/658,141, which is herein incorporated by reference. Preferably, the donor and acceptor moieties are not fused in tandem, although the donor and acceptor moieties may be contained on the same protein domain or lobe. A domain is a portion of a protein that performs a particular function and is typically at least about 40 to about 50 amino acids in length. There may be several protein domains contained in a single protein.

Fluorescent domains can optionally be separated from the neurotransmitter binding domain by one or more flexible linker sequences. Such linker moieties are preferably between about 1 and 50 amino acid residues in length, and more preferably between about 1 and 30 amino acid residues. Linker moieties and their applications are well known in the art and described, for example, in U.S. Pat. Nos. 5,998,204 and 5,981,200, and Newton et al., Biochemistry 35:545-553 (1996). Alternatively, shortened versions of the fluorophores or the binding protein may be used.

For instance, the present inventors have also found that removing sequences connecting the core protein structure of the binding domain and the fluorophore, i.e., by removing linker sequences and/or by deleting amino acids from the ends of the analyte binding moiety and/or the fluorophores, closer coupling of fluorophores is achieved leading to higher ratio changes. Preferably, deletions are made by deleting at least one, or at least two, or at least three, or at least four, or at least five, or at least eight, or at least ten, or at least fifteen nucleotides in a nucleic acid construct encoding a FRET biosensor that are located in the regions encoding the linker, or fluorophore, or ligand binding domains. Deletions in different regions may be combined in a single construct to create more than one region demonstrating increased rigidity. Amino acids may also be added or mutated to increase rigidity of the biosensor and improve sensitivity. For instance, by introducing a kink by adding a proline residue or other suitable amino acid. Improved sensitivity may be measured by the ratio change in FRET fluorescence upon ligand binding, and preferably increases by at least a factor of 2 as a result of said deletion(s). See provisional application No. 60/658,141, which is herein incorporated by reference in its entirety.

The invention also includes host cells transfected with a vector or an expression vector of the invention, including prokaryotic cells, such as E. coli or other bacteria, or eukaryotic cells, such as yeast cells or animal cells. In another aspect, the invention features a transgenic non-human animal having a phenotype characterized by expression of the nucleic acid sequence coding for the expression of the neurotransmitter biosensor. The phenotype is conferred by a transgene contained in the somatic and germ cells of the animal, which may be produced by (a) introducing a transgene into a zygote of an animal, the transgene comprising a DNA construct encoding the neurotransmitter biosensor; (b) transplanting the zygote into a pseudopregnant animal; (c) allowing the zygote to develop to term; and (d) identifying at least one transgenic offspring containing the transgene. The step of introducing of the transgene into the embryo can be by introducing an embryonic stem cell containing the transgene into the embryo, or infecting the embryo with a retrovirus containing the transgene. Preferred transgenic animals will express the encoded neurotransmitter biosensor in the brain. Transgenic animals of the invention include transgenic C. elegans and transgenic mice and other animals.

The present invention also encompasses isolated neurotransmitter biosensor molecules having the properties described herein, particularly YbeJ-based glutamate binding fluorescent indicators. Such polypeptides may be recombinantly expressed using the nucleic acid constructs described herein, or produced by chemically coupling some or all of the component domains. The expressed polypeptides can optionally be produced in and/or isolated from a transcription-translation system or from a recombinant cell, by biochemical and/or immunological purification methods known in the art. The polypeptides of the invention can be introduced into a lipid bilayer, such as a cellular membrane extract, or an artificial lipid bilayer (e.g. a liposome vesicle) or nanoparticle.

Methods of Detecting Levels of Neurotransmitters

The nucleic acids and proteins of the present invention are useful for detecting and measuring changes in the levels of neurotransmitters in the brain or nervous system of an animal, particularly changes in the level of extracellular glutamate, which can be a signal of a disorder or disease associated with glutamate excitotoxicity. In one embodiment, the invention comprises a method of detecting changes in the level of extracellular glutamate in a sample of neurons, comprising (a) providing a cell expressing a nucleic acid encoding a glutamate binding biosensor as described herein and a sample of neurons; and (b) detecting a change in FRET between a donor fluorescent protein moiety and an acceptor fluorescent protein moiety, each covalently attached to the glutamate binding domain, wherein a change in FRET between said donor moiety and said acceptor moiety indicates a change in the level of extracellular glutamate in the sample of neurons. Alternatively, the protein may be produced in a heterologous host, e.g. in bacteria, purified and injected into organs directly or into the intercellular spaces. The protein or derivatives thereof may also be coupled to particles including quantum dots and introduced into cells or compartments.

FRET may be measured using a variety of techniques known in the art. For instance, the step of determining FRET may comprise measuring light emitted from the acceptor fluorescent protein moiety. Alternatively, the step of determining FRET may comprise measuring light emitted from the donor fluorescent protein moiety, measuring light emitted from the acceptor fluorescent protein moiety, and calculating a ratio of the light emitted from the donor fluorescent protein moiety and the light emitted from the acceptor fluorescent protein moiety. The step of determining FRET may also comprise measuring the excited state lifetime of the donor moiety or anisotropy changes (Squire A, Verveer P J, Rocks O, Bastiaens P I. J Struct Biol. July 2004; 147(1):62-9. Red-edge anisotropy microscopy enables dynamic imaging of homo-FRET between green fluorescent proteins in cells.). Such methods are known in the art and described generally in U.S. Pat. No. 6,197,928, which is herein incorporated by reference in its entirety.

The amount of glutamate or other neurotransmitter in a sample of neurons can be determined by determining the degree of FRET. First the FLIP-E sensor must be introduced into the sample. Changes in neurotransmitter concentration can be determined by monitoring FRET changes at time intervals. The amount of neurotransmitter in the sample can be quantified for example by using a calibration curve established by titration in vivo.

The neuron sample to be analyzed by the methods of the invention may be contained in vivo, for instance in the measurement of glutamate efflux on the surface of hippocampal neurons, or in vitro, wherein glutamate efflux is measured in neuronal cell culture. Alternatively, a fluid extract from the brain or one or more synaptic spaces may be used as a sample from which extracellular neurotransmitter is detected or measured. Such measurements may be used to detect extracellular glutamate associated with traumatic injury to said neurons, or as a possible indicator of a neurological disorder associated with glutamate excitotoxicity, including stroke, epilepsy, Huntington disease, AIDS dementia complex, and amyotrophic lateral sclerosis, among others.

Methods for detecting neurotransmitter levels as disclosed herein may be used to screen and identify compounds that may be used to modulate neurotransmitter concentrations and particularly compounds useful for modulating glutamate excitotoxicity. In one embodiment, among others, the invention comprises a method of identifying a compound that modulates glutamate excitotoxicity comprising (a) contacting a cell expressing a glutamate biosensor as disclosed herein and a sample of neurons with one or more test compounds, and (b) determining FRET between said donor fluorescent domain and said acceptor fluorescent domain following said contacting, wherein increased or decreased FRET following said contacting indicates that said test compound is a compound that modulates glutamate excitotoxicity. The term "modulate" means that such compounds may increase or decrease glutamate excitotoxicity. Compounds that increase glutamate levels are targets for therapeutic intervention and treatment of disorders associated with glutamate excitotoxicity, as described above. Compounds that decrease glutamate levels may be developed into therapeutic products for the treatment of disorders associated with glutamate excitotoxicity.

The targeting of the sensor to the outer leaflet of the plasma membrane is only one embodiment of the potential applications. It demonstrates that the nanosensor can be targeted to a specific compartment. Alternatively, other targeting sequences may be used to express the sensors in other compartments such as vesicles, ER, vacuole, etc.

Expression systems comprise not only rat neurons, but also human cell lines, animal cells and organs, fungi and plant cells. The sensors can also be used to monitor levels of glutamate in fungal and plant organisms where glutamate serves as an important nitrogen compound, but potentially also a signaling molecule. Expression in bacteria may be used to monitor glutamate levels at sites of infection or in compartments in which the bacteria reside or are introduced.

Specifically, bacteria or fungi expressing the sensors may serve as biosensors or as tools to identify new pesticides using a similar scheme as outlined for drug screening above.

Additional Utilities

The biosensors of the present invention can also be expressed on the surface of animal cells to determine the function of neurons. For example, in C. elegans, many of the neurons present have not been assigned a specific function. Expression of the biosensors on the surface permits visualization of neuron activity in living worms in response to stimuli, permitting assignment of function and analysis of neuronal networks. Similarly, the introduction of multiphoton probes into the brain of living mice or rats, permits imaging these processes. Finally, expression in specific neurons or glia will allow the study of phenomena such as stroke or Alzheimers Disease and the effect of such disorders on glutamate levels inside neuronal cells or on their surface. Moreover, the effect of medication on localized brain areas or neuronal networks can be studied in vivo.

Finally, it is possible to use the sensors as tools to modify glutamate fluxes by introducing them as artificial glutamate scavengers, for instance presented on membrane or artificial lipid complexes, and thus to manipulate brain or neuron function.
 

Claim 1 of 18 Claims

1. An isolated nucleic acid which encodes a glutamate binding fluorescent indicator, the indicator comprising: a glutamate binding protein moiety from Escherichia coli YbeJ wherein the glutamate binding protein moiety is truncated; a donor fluorescent protein moiety covalently coupled to the glutamate binding protein moiety; and an acceptor fluorescent protein moiety covalently coupled to the glutamate binding protein moiety; wherein the donor and the acceptor moieties are on the same lobe of the folded glutamate binding fluorescent indicator, and wherein fluorescence resonance energy transfer (FRET) between the donor moiety and the acceptor moiety is altered when the donor moiety is excited and glutamate binds to the glutamate binding protein moiety.

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