# Patent

## Title
Imaging individual mRNA molecules using multiple singly labeled probes

## Abstract

A method for probing a target sequence of messenger ribonucleic acid molecules (mRNA's) in a fixed, permeabilized cell, said target sequence including at least 30 non-overlapping probe binding regions of 15-100 nucleotides, comprising immersing said cell in an excess of at least 30 nucleic acid hybridization probes, each singly labeled with the same fluorescent label and each containing a nucleic acid sequence that is complementary to a different probe binding region of said target sequence; washing said fixed cell to remove unbound probes; and detecting fluorescence from said probes.

## Background
RELATIONSHIP TO PRIOR APPLICATIONS
This application is the U.S. National Phase of International Patent Application Ser. No. PCT/US09/56564, filed Sep. 10, 2009, which claims priority to a U.S. Provisional Patent Application Ser. No. 61/191,724, filed on Sep. 10, 2008, both of which are incorporated herein by reference in their entireties.


GOVERNMENT INTERESTS
The invention disclosed herein was made, at least in part, with Government support under Grant Nos. NIH GM-070357 and NIH MH-079197 from the National Institutes of Health. Accordingly, the U.S. Government has certain rights in this invention.


FIELD OF THE INVENTION
This invention generally relates to methods of nucleic acid sequence detection.
BACKGROUND
As it has become increasingly apparent that gene expression in individual cells deviates significantly from the average behavior of cell populations, new methods that provide accurate integer counts of mRNA copy numbers in individual cells are needed. Ideally, such methods should also reveal the intracellular locations of the mRNAs, as mRNA localization is often used by cells to spatially restrict the activity gene.
In situ hybridization, followed by microscopic analysis, is a well-established means of studying gene expression. The first generation of in situ hybridizations was performed with radioactive probes. Early improvements involved linking the probes to enzymes that catalyze chromogenic or fluorogenic reactions. However, because the products of these reactions were small molecules or precipitates that diffuse away from the probe, the location of the target molecules could not be precisely determined. Conversely, probes labeled directly with a few fluorophores maintained spatial resolution, but the sensitivity that can be achieved is relatively poor.
Robert Singer and colleagues developed an in situ hybridization procedure that was not only sensitive enough to permit the detection of single mRNA molecules, but also restricted the signals to close proximity of the targets. They hybridized five oligonucleotide probes simultaneously to each mRNA target, each of which was about 50-nucleotides in length and each of which was labeled with five fluorophore moieties. Although the authors convincingly demonstrated single molecule sensitivity and other groups have successfully used these probes, the system has not been widely adopted. One reason for this is difficulty in the synthesis and purification of heavily labeled oligonucleotides. Usually, flurophore moieties are introduced via primary amino groups that are incorporated into oligonucleotides during their synthesis. When multiple amino groups are introduced into the same oligonucleotide some are lost due to side reactions such as transamidation. Coupling of fluorophores to the remaining amino groups is inefficient and requires several consecutive coupling reactions and it is difficult to purify oligonucleotides in which all designed sites are coupled to fluorophores from those that are partially coupled. Also, when some fluorophores are present in multiple copies on the same oligonucleotide they interact with each other altering the hybridization characteristics of the oligonucleotides and exhibiting severe self-quenching. These problems are obviated if each probe had just a single terminal amino group to serve as the site of attachment.
Another issue with the use of small numbers of heavily labeled probes is that a significant portion of the fluorescence is lost for every probe that does not bind to the target, whereas every non-specific binding event increases the background. This leads to a widened distribution of number of probes bound to each target mRNA. For instance, when using 5 fluorescent probes targeted to a single mRNA, Femino et al estimated that the majority of the fluorescent spots observed had intensities indicating the presence of only 1 or 2 probes. Science 280, 585-590 (1998). This makes it difficult to unambiguously identify those fluorescent spots as mRNA molecules, since it is impossible to determine whether the detection of an individual probe arises from legitimate binding to the target mRNA or non-specific binding. These “thresholding” problems limit the ability of such methods to provide reliable counts of mRNA numbers in individual cells.
Thus there remains a need for improved methods to provide reliable counts of mRNA numbers in individual cells and a need for probes that are easily synthesized and purified.
SUMMARY OF THE INVENTION
This invention provides a method for detecting individual nucleic acid molecules, such as, for example, RNA molecules, e.g., mRNA molecules in fixed, permeabilized cells using a plurality of nucleic acid hybridization probes that are singly fluorescently labeled, as with the same fluorophore. The inventors have surprisingly discovered that if at least 30, preferably 40-60, and very preferably 48 different probes, all labeled with the same fluorophore, are hybridized simultaneously to a target sequence of an mRNA molecule, a fluorescent spot is created that can be detected from the combined fluorescences of the multiple probes. The probes are non-overlapping; that is, the region of the target sequence to which each probe hybridizes is unique (or non-overlapping). Probes in a set of 30 or more for a selected target sequence can be designed to hybridize adjacently to one another or to hybridize non-adjacently, with stretches of the target sequence, from one nucleotide to a hundred nucleotides or more, not complementary to any of the probes. Accordingly, in one aspect, the invention provides a method for probing a target sequence of nucleic acid molecules such as, for example, mRNAs in a fixed, permeabilized cell, said target sequence including at least 30 non-overlapping probe binding regions of 15-100 nucleotides, comprising immersing said cell in an excess of at least 30 nucleic acid hybridization probes, each singly labeled with the same fluorescent label and each containing a nucleic acid sequence that is complementary to a different probe binding region of said target sequence; washing said fixed cell to remove unbound probes; and detecting fluorescence from said probes.
Probes useful in this invention may be DNA, RNA or mixtures of DNA and RNA. They may include non-natural nucleotides, and they may include non-natural internucleotide linkages. Non-natural nucleotides that increase the binding affinity of probes include 2′-O-methyl ribonucleotides. The lengths of probes useful in this invention are 15-40 nucleotides for typical DNA or RNA probes of average binding affinity. Preferred lengths of DNA probes and RNA probes are in the range of 15-30 nucleotides, more preferably 17-25 nucleotides and even more preferably 17-22 nucleotides. The inventors have constructed the probes to be about 20 nucleotides long. If means are included to increase a probe's binding affinity, the probe can be shorter, as short as seven nucleotides, as persons in the art will appreciate. A fluorophore can be attached to a probe at any position, including, without limitation, attaching a fluorophore to one end of a probe, preferably to the 3′ end. The probes may be included in a hybridization solution that contains the multiple probes in excess, commonly in the range of 0.2-1 nanograms per microliter. Sufficient solution is added to cover and wet the cell so that the cell is immersed in the probe-containing solution.
A single cell can be probed simultaneously for multiple mRNA target sequences, either more than one target sequence of one mRNA molecule, or one or more sequences of different mRNA molecules. Additionally, one target sequence of an mRNA molecule can be probed with more than one set of probes, wherein each set is labeled with a distinguishable fluorophore, and the fluorophores are distinguishable. For example, in probing a gene sequence, at least 30 green-labeled probes can be used to probe one portion of the gene sequence as its target sequence, and at least 30 red-labeled probes can be used to probe a different portion of the gene sequence as its target sequence. Using more than one color for each of multiple targets permits use of color-coding schemes in highly multiplexed probing methods according to this invention.
Methods of this invention may include simply looking to see if one or more spots representing a target sequence are present. Methods according to this invention also include counting spots of a given color corresponding to a given mRNA species. When it is desired to detect more than one species of mRNA, different sets of probes labeled with distinct fluorophores can be used in the same hybridization mixture. A gene expression profile for each species of mRNA is constructed by counting spots of different colors.
Spots can be detected utilizing microscopic methods. It is not necessary to use a confocal microscope, as a wide-field fluorescence microscope is sufficient. To distinguish spots that positively reflect a target sequence from dim spots that may reflect background fluorescence or nonspecific binding, methods according to this invention include detection. In one embodiment, the detection comprises filtering images with a three-dimensional linear Laplacian of Gaussian filter and applying a detection threshold. If one plots the number of spots in three dimensions for all thresholds ranging from zero to the maximum pixel intensity in the filtered image, there is a wide plateau, indicative of a region in which the number of spots detected is insensitive to threshold. Thus, the method further comprises plotting the number of spots, determining the boundaries of a plateau region, and selecting the threshold preferably within that region.
In another aspect, this invention includes sets of probes for in situ hybridization that enable detection of individual mRNA molecules in cells. The probes render each molecule so intensely fluorescent that it can be seen as a fine fluorescent spot in fluorescence microscopy.
A computer program can be used to identify and count all the mRNA molecules in the cell from the microscopic image. In situ hybridizations performed with the sets of probes described above allow accurate and simple gene expression analysis, detection of pathogens and pathogenic states such as cancer.
Accordingly, in another aspect, provided is a method of screening for compounds which alter the amount of a subcellular distribution of the target sequence. The method includes incubating a cell with a test compound for a period of time sufficient to elicit a response, detecting the amount of distribution pattern of the target sequence, and comparing this amount or distribution with an amount or distribution of the target mRNA in a control cell which was treated identically, but not incubated with the test compound.
In yet another aspect, the invention provides a computer readable medium, comprising instructions for: obtaining a 3-D stack of 2-D fluorescent images; filtering said 3-D stack using a 3-D filter; counting a total number of 3-D spots in said filtered 3-D stack for each of a plurality of intensity thresholds; obtaining an optimum intensity threshold representative of a plateau region in a plot of said total number of 3-D spots verses the intensity threshold at which said total number was counted; and using the total number of 3-D spots obtained at said optimum threshold as representative of a number of fluorescing particles detected in said 3-D stack.
The invention also provides a kit, generally comprising the set of probes and the computer-readable media as described above.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows simultaneous detection of a unique sequence and a repeated sequence in individual mRNA molecules. FIG. 1A is a schematic depiction of the construct used. The 48 probes used to detect the GFP coding sequence were labeled with Alexa-594 and the four different probes used to detect the tandem repeat in the 3′-UTR were labeled with TMR. FIG. 1B illustrates maximum intensity merges of a pair of z-stack of fluorescent images of CHO cells taken in the Alexa-594 channel (left) and the TMR channel (right) corresponding to GFP coding region probes and UTR probes, respectively. FIG. 1C illustrates false color merge of the images in FIG. 1B enclosed by the red (GFP) and green (UTR) squares, with red circles representing computationally identified GFP mRNA particles, green circles representing UTR particles, and yellow circles representing co-localized particles. All scale bars are 5 μm long.
FIG. 2 shows intensity analysis of colocalized spots. Spot intensities corresponding to the GFP-targeted probes (Alexa 594 channel, y axis) and multimeric UTR-targeted probes (TMR channel, x axis) were computed by taking the maximum intensity in the computationally identified spot region and subtracting the mean intensity of an annular region surrounding the spot. Marginal histograms show the distributions of GFP spot intensities (right) and UTR spot intensities (top).
FIG. 3 shows sensitivity of method when using different numbers of probes. FIG. 3A illustrates spot intensity (defined as maximum intensity within the spot minus the mean background taken in an annular region surrounding the spot) as a function of the number of probes chosen. Intensities for 12 and 24 probes are artifactual in that spots were not readily identifiable in those cases, so spots identified were biased towards being brighter. FIG. 3B illustrates the number of spots (i.e., connected components) found upon thresholding the filtered image plotted as a function of the threshold value, ranging from 0 to the maximum intensity of the filtered image (normalized to 1) for different numbers of probes. The grey bar indicates the threshold used for the analysis in FIG. 3A.
FIG. 4 shows comparison with the mRNA detection method of Femino et al. (Science 1998). FIG. 4A is a schematic depicting the method described in this manuscript with 48 singly labeled probes (left) and the method of Femino et al. in which each 45 bp probe contains five fluorophores each and is targeted to a sequence element that is repeated 32 times in the 3′UTR of the target mRNA expressed from a transgene in Chinese hamster ovary cells. FIG. 3B illustrates a comparison of spot intensities when using 48 singly labeled probes or using a 45 bp probe labeled with five fluorophores. Error bars represent one standard deviation.
FIG. 5 shows computational identification of mRNA spots. FIG. 5A illustrates raw image data (maximum intensity merge) obtained from imaging FKBP5 mRNA particles in A549 cells induced with dexamethasone. FIG. 5B illustrates mage (maximum merge) obtained by running raw data through Laplacian of a Gaussian filter to enhance spots. FIG. 5C illustrates the number of spots (i.e., connected components) found upon thresholding the filtered image from FIG. 5B is plotted as a function of the threshold value, ranging from 0 to the maximum intensity of the filtered image (normalized to 1). FIG. 5D is an image showing the results of using the threshold represented by the grey line in FIG. 5C with each distinct spot assigned a random color. All scale bars are 5 μm long.
FIG. 6 shows simultaneously imaging single molecules of three different mRNAs in mammalian cells. FIG. 6A-FIG. 6C illustrate images showing FLJ11127, Cox-2 and FKBP5 mRNA particles in the same set of A549 cells not treated with dexamethasone. FIG. 6D-FIG. 6F illustrate images showing FLJ11127, Cox-2 and FKBP5 particles in cells treated for 8 hours with 24 nM dexamethasone. FIG. 6G illustrates fold induction for all three genes as measured by FISH and realtime RT-PCR; error bars for FISH were obtained by bootstrapping and those for RTPCR were obtained by repetition as described in the supplementary information. All images are maximum merges of a z-stack of fluorescent images spanning the extent of the cells with nuclear DAPI counterstaining in purple, and all scale bars are 5 μm long.
FIG. 7 shows examination of fluorescent spot bleedthrough. FIG. 7A is an illustration of images of an FLJ11127 mRNA spot labeled with TMR as seen through the TMR, Alexa 594 and Cy5 filter channels. Linescans of fluorescent intensity corresponding to the line through the image are given below, with the different linescans corresponding to measurements taken at increasing z (0.25 μm spacing). The green linescan corresponds to the z-slice shown in the image itself. A similar analysis was performed for a Cox-2 mRNA spot labeled with Alexa 594 (FIG. 7B) and an FKBP5 mRNA particle labeled with Cy5 (FIG. 7C). All linescan intensity measurements had the camera background subtracted but range between 0 and 200 arbitrary fluorescence units.
FIG. 8 shows demonstration that the oxygen-scavenger increases photostability of Cy5. FIG. 8A illustrates the mean of the maximum spot fluorescence for a number of FLJ11127 mRNAs labeled using TMR conjugated probes was plotted as a function of the number of 2 second exposures using a filter specific for TMR. Curves were generated for images taken both with (blue) and without (red) the oxygen scavenging system. A similar analysis was performed for Cox-2 mRNAs labeled using Alexa-594 conjugated probes with 2 second exposures (FIG. 8B) and FKBP5 mRNAs labeled using Cy5 conjugated probes with 2.5 second exposures (FIG. 8C). FIG. 8D illustrates the bleach rate per exposure (in units of fraction of fluorescence lost per exposure) for the TMR, Alexa-594 and Cy5 conjugates probes in (FIG. 8A-FIG. 8C) both with and without the oxygen-scavenging anti-bleach system. The bleach rate was calculated by fitting each individual particle's decay curve to an exponential and taking the mean of the fitted decay constants. The error bars correspond to one standard deviation. A minimum of 6 particles were chosen in each condition.
FIG. 9 shows imaging localized mRNAs in C. elegans and D. melanogaster. FIG. 9A is an illustration of elt-2 mRNA molecules (red) in an early stage embryo ( ̃100 cell stage) from C. elegans; the nuclei have been counterstained with DAPI (blue). FIG. 9B is an illustration of elt-2 mRNA molecules in an L1 larva from C. elegans. Inside the blue box, a single focal plane is shown in which the intestinal track is visible. FIG. 9C illustrates a schematic depiction of dpp and engrailed expression in the imaginal wing discs of third instar larvae from D. melanogaster. FIG. 9D is an image showing the locations of the computationally identified dpp mRNA molecules (light blue circles) and Engrailed expression detected by immunofluorescence (dark blue). FIG. 9E is an image containing enhanced dpp mRNA molecule signals (light blue) and Engrailed protein expression detected by immunofluorescence (dark blue). All images except the boxed portion of FIG. 9B are maximum merges of a z-stack of fluorescent images, and all scale bars are 5 μm long.
FIG. 10 shows imaging single mRNA molecules in yeast and neurons. FIG. 10A and FIG. 10B illustrate STL1 mRNA particles in both unperturbed cells (FIG. 10A) and cells subjected to a 10 minute 0.4M NaCl salt shock, with nuclear DAPI counterstaining in purple (FIG. 10B). FIG. 10C illustrates expression of β-actin (green) and Map2 (red) mRNAs in rat hippocampus neurons in a dissociated neuron culture. FIG. 10D illustrates an enlarged and contrasted image of a segment of a dendrite enclosed by the red box in FIG. 10C. All scale bars are 5 μm long.
FIG. 11A shows a 3′-UTR multimeric sequence (SEQ ID NO: 483) and probes (SEQ ID NOs: 484-487) used in the instant invention.
FIG. 11B shows sequences of β actin (SEQ ID NO: 1) and related probes (SEQ ID NOs: 2-49) used in the instant invention.
FIG. 11C shows sequences of COX2 (SEQ ID NO: 50) and related probes (SEQ ID NOs: 51-98) used in the instant invention.
FIG. 11D shows sequences of d2EGFP (SEQ ID NO: 99) and related probes (SEQ ID NOs: 100-147) used in the instant invention.
FIG. 11E shows sequences of dpp (SEQ ID NO: 148) and related probes (SEQ ID NOs: 149-196) used in the instant invention.
FIG. 11F shows sequences of elt-2 (SEQ ID NO: 197) and related probes (SEQ ID NOs: 198-245) used in the instant invention.
FIG. 11G shows sequences of FKBPS (SEQ ID NO: 246) and related probes (SEQ ID NOs: 247-306) used in the instant invention.
FIG. 11H shows sequences of FLJ11127 (SEQ ID NO: 307) and related probes (SEQ ID NOs: 308-360) used in the instant invention.
FIG. 11I shows sequences of Map2 (SEQ ID NO: 361) and related probes (SEQ ID NOs: 362-433) used in the instant invention.
FIG. 11J shows sequences of STL1(SEQ ID NO: 434) and related probes (SEQ ID NOs: 435-482) used in the instant invention.



DETAILED DESCRIPTION OF THE INVENTION
This invention relates in part to the development of an image analysis algorithm that utilizes a principled thresholding strategy and shows that we can accurately and unambiguously identify and count all the target mRNA molecules present in the cell. The simplicity and robustness of this approach permits reliable detection of three different mRNA species within the same cells. Using a rigorous set of criteria the inventors have demonstrated that the method allows extremely specific single mRNA imaging across a wide spectrum of cell types and model organisms.
The inventors have taken advantage of the availability of 96 well DNA synthesizers to synthesize many different terminally labeled smaller probes for the same target. The obtained results show that when a set of at least 30, preferably at least 40, more preferably, about 48 (half of a 96-well plate that is used for high throughput DNA synthesis) or more singly labeled probes bind to the same mRNA molecule, they render it sufficiently fluorescent that it becomes visible as a diffraction-limited spot in wide-field microscopy. The non-specific sites only associate with one or a few probes, yielding diffused signals, whereas the legitimate targets bind to all or most of the probes yielding a clearly detectable spot for each mRNA molecule.
The inventors have also developed an image analysis algorithm that utilizes a principled thresholding strategy and shows that it is possible to accurately and unambiguously identify and count the all target mRNA molecules present in the cell. The simplicity and robustness of this approach permits reliable detection of three different mRNA species within the same cells. Using a rigorous set of criteria the inventors demonstrate that the method allows extremely specific single mRNA imaging across a wide spectrum of cell types and model organisms.
Thus, 48 or more singly labeled oligonucleotide probes allow the detection of individual mRNA molecules. The mRNA molecules were visualized as diffraction limited spots that can be easily detected in a standard wide-field microscopic set up. The spots were bright enough to be accurately counted with the spot detection image processing algorithm of the instant invention. The inventors obtained quantitative counts of three different species of mRNA molecules within individual cells. Such analysis facilitates accurate multiplex gene expression profiling of even lowly expressed genes across a host of model organisms.
The basis of specificity of the instantly disclosed system is that most or all of the probes bind to the intended target mRNA and yield a particulate signal whereas the non-specific binding sites elsewhere in the cell associate with fewer probe molecules and give a diffused signal that the spot counting algorithm ignores. This highlights a key advantage of the instant method over other in situ hybridization methods that use heavily labeled probes such as dendrimers. If every probe molecule is detectable, each non-specific binding event will result in a false positive and any mRNA to which the probe does not bind will result in a false negative. The likelihood of false negatives and positives decreases, however, as the number of probes is increased, and in general, given a certain efficiency of hybridization, increasing the number of different probes will narrow the distribution of probes bound per molecule. The image analysis according to the instant invention showed that increasing the number of the probes resulted in robust spot detection that does not depend on arbitrarily chosen thresholds. This is crucial for accurately counting the number of mRNAs per cell, which is a key feature of the method of the invention.
In a related point, a potential factor in the design of the probe set is uniformity in hybridization affinities. Since oligonucleotide affinity is largely dominated by its relative GC content, the inventors have created a computer program to design a set of probes with optimally uniform overall GC content. This computer program is publicly available.
From a practical standpoint, the instantly claimed method also yields significant benefits over previous single molecule mRNA FISH method both in terms of time and cost. Due to advances in synthesis, researchers can easily and cheaply purchase large numbers of oligonucleotides with 3′ amine modifiers. These can then be pooled, coupled, and purified en-masse, significantly reducing the labor associated with the multiple couplings and purifications required to generate multiply labeled probe. The resulting simplicity and cost-effectiveness of the instant method will facilitate genomics-scale studies involving the detection of many different mRNAs. Furthermore, the flexibility of the hybridization procedure allows for it to be combined with other standard techniques, such as immunofluorescence.
In another embodiment, the fluorophores can be incorporated into the probes during automated DNA synthesis.
Other methods for quantifying the number of mRNAs in individual cells include single-cell RT-PCR and digital RT-PCR. One problem with these methods is the practical difficulties associated with assembling large numbers of individual reactions that require the use of microfluidic or robotic devices. Moreover, those methods suffer from concerns about stochastic variations in exponential amplification when the target inputs are just a few molecules. Such stochastic behavior complicates the analysis of single cell gene expression, which itself is subjected to stochastic forces. Moreover, these methods do not provide any information about the spatial location of the mRNAs.
Given the simplicity and broad applicability of our single-molecule mRNA detection method, such method is suitable for a variety of studies. By obtaining exact mRNA counts in individual cells, one can make accurate determinations of both expression differences in different conditions and the cell-to-cell variability in gene expression. By yielding quantitative, spatial measurements of individual mRNAs in single cells, this method is valuable in many studies in systems biology, cell biology, neurobiology and developmental biology.
Accordingly, this method may be utilized for multiple assays, including, without limitation a screening assay. In one embodiment, the screening assay determines whether a test compound affects an amount of a distribution of a target sequence of messenger ribonucleic acid molecules (mRNA's) said target sequence including at least 30 non-overlapping probe binding regions of 15-100 nucleotides in a cell. The assay generally comprises the following steps: incubating a cell with a test compound for a period of time sufficient to elicit a response; permeabilizing the cell; immersing said cell in an excess of at least 30 nucleic acid hybridization probes, each singly labeled with the same fluorescent label and each containing a nucleic acid sequence that is complementary to a different probe binding region of said target sequence; washing said fixed cell to remove unbound probes detecting an amount of a distribution of fluorescence from said probes, comparing said amount or said distribution with an amount of a distribution, respectively, obtained from a control cell, treated as described above, but with the exception of being incubated with the test compound.
Suitable test compound candidates include, without limitation, peptide-based compounds (e.g., antibodies or nanobodies), RNA interference agents (i.e., siRNA, shRNA, miRNA etc), and small molecules. All these compounds may be made according to the methods known in the art. For example Naito (US 20080113351) and Khvorova (US 20070031844) provide methods of selecting active RNA interference compounds. Antibodies may also be prepared by known techniques including the use of hybridomas, selection of monoclonal antibodies, use of phage display libraries, antibody humanization and the like.
Small molecule compounds may be selected from screening of the appropriate libraries. In one aspect, small molecule libraries are synthesized according to methods well known and routinely practiced in the art. See, for example, Thompson and Ellman, Chem. Rev. 1996, 96, 555-600, Shipps, et al., Proc. Natl. Acad. Sci. USA, Vol. 94, pp. 11833-11838, October 1997, and Combinatorial Library Design and Evaluation—Principles, Software Tools and Applications in Drug Discovery, Ghose and Viswanadhan (eds), Marcel Dekker 2001. Alternatively, small libraries are obtained from any of a number of sources including, for example, the NIH Molecular Libraries Small Molecule Repository. Alternative sources include AnalytiCon Discovery GmbH (Potsdam, Germany) which makes available MEGAbolite®, pure natural product small molecule libraries and NatDiverseTM, semi-synthetic natural product analogue small molecule libraries; Quantum Pharmaceuticals Ltd. (Moscow, Russian Federation); and Praecis Pharmaceuticals Incorporated (Waltham, Mass.).
In yet another aspect, the invention provides software implementing the thresholding algorithm as described above. Thus, in one embodiment, provided is a computer readable medium, comprising instructions for: obtaining a 3-D stack of 2-D fluorescent images; filtering said 3-D stack using a 3-D filter; counting a total number of 3-D spots in said filtered 3-D stack for each of a plurality of intensity thresholds; obtaining an optimum intensity threshold representative of a plateau region in a plot of said total number of 3-D spots verses the intensity threshold at which said total number was counted; and using the total number of 3-D spots obtained at said optimum threshold as representative of a number of fluorescing particles detected in said 3-D stack.
In one embodiment, the thresholding is accomplished using three dimensional linear Laplacian of Gaussian filter.
In another aspect, a kit is provided. The kit comprises a computer-readable media implementing the thresholding algorithm, as described above, and a set of probes against a pre-selected target sequence. The probes described in connection with the claimed method are also suitable for the instant kit.
Specific embodiments according to the methods of the present invention will now be described in the following examples. The examples are illustrative only, and are not intended to limit the remainder of the disclosure in any way.
EXAMPLES
Example 1
Materials and Methods
The procedures described in this section are applicable to all examples unless indicated otherwise.
Probe Design
The sets of probes were designed to consist of at least 48 oligonucleotides each with lengths varying from 17 to 22 nucleotides long with a 3′-amine modification (FKBP5, FLJ11127, and Map2 mRNAs were probed using 63, 53 and 72 oligonucleotides respectively). Additionally, the GC content of the oligonucleotides was kept close to 45% when possible. The oligonucleotides were pooled and coupled to a fluorophore in a single reaction, after which the uncoupled oligonucleotides and remaining free fluorophores were removed by HPLC purification.
Fluorescence in situ Hybridization
In preparation for FISH, all samples were fixed with 3.7% formaldehyde and permeabilized with ethanol. The hybridization was performed using buffers and conditions similar to those outlined by Femino et al., with the key difference being the stringency of the hybridization, which was lowered by reducing the amount of formamide used to 10%. The concentration of the probe that gave optimal signal was determined empirically.
Imaging and Data Analysis
All images were acquired using a standard wide-field fluorescence microscope. Computer-aided detection and counting of particles was performed with linear filters designed for enhancing particulate signals.
Example 2
Probing Repeated and Unique Sequences Present in the Same mRNA Molecule
Utilizing small oligonucleotide probes labeled with a single fluorophore moiety, the inventors have shown that individual mRNA molecules that were engineered to contain 32-96 tandem copies of a probe-binding sequence can be detected by in situ hybridization. The inventors also demonstrated that the individual spots in the image represent single mRNA molecules, utilizing a number of different approaches, including correlating the average mRNA copy number obtained by directly counting the diffraction-limited spots to a measurement of the number of target molecules obtained by real-time RT-PCR. Thus, if many different probes are utilized, each targeted to a distinct region of a natural mRNA, it would be possible to obtain single-molecule sensitivity without resorting to the use of engineered genes.
For the initial test of this hypothesis, the inventors constructed a doxycycline-controlled gene that produced an mRNA encoding green fluorescent protein and possessed 32 tandemly repeated 80 nucleotide-long sequences in its 3′-UTR; and then this engineered gene was stably integrated into the genome of a Chinese hamster ovary cell line. The mRNA expressed from this gene was probed simultaneously with 48 different oligonucleotides, each complementary to a unique region in the coding sequence, and a set of four oligonucleotides, each having a complementary sequence in the repeated motif (a total of 128 probes bound) (FIG. 1A). Each oligonucleotide in the probe set that was specific for the coding sequence was labeled with a single Alexa-594 fluorophore, and each oligonucleotide in the set specific for the repeat sequence was labeled with a single tetramethylrhodamine (TMR) fluorophore. The use of appropriate filter sets ensured that the fluorescence emitted from TMR fluorophores was not detected in the Alexa-594 channel and vice versa, as described below.
After performing FISH with these probes, the inventors have found that many “particles” with a diameter of about 0.25 micrometers were visible in both the TMR and Alexa-594 channels (FIG. 1B). The particles were identified computationally using an image processing program (described in the next section) that categorizes the particles as being labeled with either the GFP-coding-sequence probes (TMR), the UTR-specific probes (Alexa-594), or both (FIG. 1C). Upon identifying and localizing particles in four fields of view similar to the ones shown in FIG. 1c, a total of 599 particles corresponding to GFP-coding sequence-specific probes and 565 particles corresponding to the UTR-specific probes were counted. Of these particles, 85% of the “UTR particles” co-localized with the “GFP particles,” whereas 81% of the GFP particles co-localized with the UTR particles. The high degree of co-localization between particles detected by the previously established tandem repeat detection method and the particles detected via simultaneous probing with 48 different singly-labeled oligonucleotides demonstrates the validity of using multiple single-labeled probes for the detection of endogenous transcripts. The fraction of particles that did not display co-localization likely correspond to mRNA molecules that lost either their coding sequence or their 3′-UTR in the natural processes of mRNA degradation.
The inventors also analyzed the fluorescent intensity of the co-localized spots in both the TMR and Alexa-594 channel and found that the spot intensities displayed a unimodal distribution (FIG. 2), arguing that the particles detected are not clumps of many mRNAs but rather individual molecules. The spot intensities displayed a strong correlation between the two channels (FIG. 3). Since there is no cross talk between the two channels, this indicates that the variability in spot intensity was not primarily due to random variability in probe hybridization (which would be uncorrelated between different probe sets) but rather other factors, such as mRNA integrity or accessibility, that affect both probes equally.
The inventors also explored how the signal intensity would vary with the number of probes by performing in situ hybridization using either first 12, 24, 36 probes or all 48 probes in the set. For this particular target mRNA, it was found that particles could be detected with fewer numbers of probes, albeit with decreased intensity (FIG. 3A). However, the automatic spot detection algorithm (described in details below) performed particularly well with 48 probes, detecting the same number of spots over a broad range of thresholds (FIG. 3B, see further discussion below). The number of probes required for robust signal is likely to depend on the target sequence, though, as the inventors have obtained clear mRNA signals using as few as 30 probes. When the instant method was compared to the method of Femino et al. by using a 45 bp long oligonucleotide labeled with 5 fluorophores and complementary to a sequence repeated 32 times in the 3′UTR of a gene, potentially yielding 160 fluorophores per mRNA (FIG. 4A), it was found that the signal to background were about the same in both methods (FIG. 4B), indicating that the instantly claimed method is at least as sensitive despite using fewer fluorophores.
Moreover, CHO cells lacking the reporter gene yielded no signals while CHO cells having the reporter gene that was turned off by addition of doxycycline, yielded mRNA particles in only a few cells, indicating that the signals observed were specific.
Example 3
Computational Algorithm for Spot Detection
In order to reliably identify large numbers of mRNA molecules, the inventors developed a semiautomated computational algorithm for finding spots in a three-dimensional stack of fluorescent images. One of the difficulties associated with spot detection is the nonuniform background arising from cellular autofluoresence and low levels of non-specific probe hybridization. To circumvent these issues, the inventors filtered image stacks using a three dimensional linear Laplacian of Gaussian filter designed to enhance spot-like signals of the correct size and shape (FIG. 5A and FIG. 5B) while removing the slowly varying background. In the next step in the algorithm, the inventors applied a threshold to the filtered image in order to define the spots. In order to make a rational choice of threshold, the number of spots in three dimensions for all thresholds ranging from zero to the maximum pixel intensity in the filtered image was counted. When the inventors plotted the number of particles as a function of the threshold, a wide plateau was found, indicating that there is a region over which the number of particles detected is fairly insensitive to the particular threshold chosen (FIG. 5C). When a threshold in this region is chosen, the spots detected correspond very well with those identified by eye, demonstrating the efficacy of the spot detection algorithm (FIG. 5D).
Example 4
Gene Expression Profiling of Three Different mRNA Species
A potential use of the instantly claimed method is the simultaneous detection of single molecules of multiple mRNAs in individual cells. To demonstrate this capability, the inventors designed probes specific to three mRNAs encoding FK506 binding protein 5 (FKBP5), Cox-2 and FLJ11127 in the human carcinoma cell line A549. These probes were coupled to the spectrally distinct fluorophores Cy5, Alexa 594 and TMR, respectively. Upon performing FISH with all three probes simultaneously, individual spots were visible in the three different fluorescence channels (FIG. 6A-FIG. 6F); an intensity analysis showed that fluorescent spots did not bleed through into other channels (FIG. 7).
To demonstrate that the claimed method of mRNA detection was specific and quantitative, the cells were incubated with the cell-permeable glucocorticoid dexamethasone, thus upregulating the expression of FKBP5 and F111127 while mildly downregulating the expression of Cox-2 in this particular cell-line. The inventors found that the mean number of FKBP5 and F111127 mRNAs measured by combining FISH with the instantly disclosed spot detection algorithm increased while the mean number of Cox-2 mRNAs decreased (compare FIG. 6A-FIG. 6C to FIG. 6D-FIG. 6F). These numbers corresponded well to RT-PCR measurements of the fold induction and repression of these genes performed on the same samples, demonstrating that the fluorescent spots are the appropriate mRNAs and that a majority of the mRNA molecules (FIG. 6G) was detected using the instantly claimed methods. Moreover, this further demonstrates the effectiveness of the spot detection method for accurate gene expression quantification.
One technical challenge that arose in imaging multiple mRNAs simultaneously was fluorophore photolability, particularly in the case of Cy5. In order to image all of the mRNA molecules within a single cell, 10 to 30 “z-section” images for each visual field were acquired, utilizing a one-to-three second exposure for each image and a high numerical aperture objective. Only TMR and (to a lesser extent) Alexa-594 could withstand this intense and relatively prolonged exposure to light; Cy5, for instance, proved extremely photolabile under these conditions (FIG. 8). To overcome this problem, the inventors employed a special mounting medium in which fluorophores are much more photostable. This method was adapted from Yildiz et al. with minor modifications. In this medium, a mixture of catalase, glucose oxidase, and glucose enzymatically removes molecular oxygen from the medium, thereby inhibiting oxygen-dependent, light-initiated pathways that destroy fluorophores. The use of these enzymes lead to a dramatic 10-fold enhancement of Cy5 photostability while not adversely affecting the imaging of TMR and Alexa-594, thus facilitating the acquisition of multiple z-sections when performing three color imaging.
Example 5
mRNA Detection in Model Organisms and Cell Types
One of the canonical uses for in situ hybridization has been for the detection of mRNA localization during development. The inventors tested the instantly claimed method for efficacy in two commonly studied developmental systems: the nematode, Caenorhabditis elegans, and the fruit fly, Drosophila melanogaster. In the nematode, the inventors constructed probes to detect mRNA molecules from the gene elt-2, a transcription factor that is expressed only in the nematode gut, and only after the nematode embryo has developed to the 45-cell stage. After hybridization of the probe set to both embryos and larvae, it was found that elt-2 mRNA molecules were present only within the gut region (FIG. 9A) of both the embryos and the larvae (FIG. 9B). However, consistent with the known timing of the onset of expression, elt-2 mRNAs were only detected in the gut of embryos older than the 45-cell stage, again highlighting the specificity of the instantly claimed method. Furthermore, at those early stages, only a few transcripts were detected, showing that this method is sensitive enough to detect even small numbers of transcripts in complex tissues.
In the fruit fly, one of the most well-studied examples of the localization of gene expression occurs in wing imaginal disc development. The wing discs of fruit fly larvae display a remarkable set of gene expression patterns, one of which is the formation of a stripe of expression of the gene dpp in response to gradients of the proteins Hedgehog and Engrailed. In particular, Engrailed, which negatively regulates dpp mRNA synthesis, is high in the posterior compartment of the wing disc and low in the anterior compartment of the wing disc. Similarly, Hedgehog, which positively regulates dpp mRNA synthesis, is high in the posterior compartment of the wing disc and low in the anterior compartment of the wing disc. However, there is a region between the posterior and the anterior where the levels of Hedgehog is high enough to activate dpp but not high enough to activate engrailed, resulting in the synthesis of dpp mRNA in a narrow stripe (FIG. 9C).
To check whether this narrow stripe of dpp mRNA synthesis can be imaged, the inventors constructed a set of singly labeled probes against dpp mRNA and performed in situ hybridization on imaginal wing discs isolated from third-instar larvae. Moreover, this in situ procedure was combined with immunofluorescence against Engrailed protein (shown in blue). FIG. 9D shows a full image, in which the locations of the algorithmically identified mRNA molecules are presented as blue circles; and FIG. 9E shows an enlarged portion of the image with enhanced mRNA signals. The images show that mRNA molecules were found only at the anterior edge of the area of Engrailed expression, again confirming the specificity of detection.
The inventors also tested the instantly claimed method in Saccharomyces cerevisae by designing a set of probe to target transcripts from the gene STU. STL1 is one among a number of yeast genes whose expression is significantly up-regulated by the addition of salt to the growth medium. It was found that non-shocked cells contain virtually no STL1 mRNA molecules (FIG. 10A), while cells subjected to a ten minute 0.4 M salt shock possessed a large numbers of STL1 mRNA molecules (FIG. 10B).
Another cell type in which mRNA localization is commonly studied is neurons. To show efficacy of the instantly claimed method in that system the inventors imaged β-actin mRNA and Map2 mRNA in cultured hippocampal neurons. FIG. 10C shows that a β-actin probe set (labeled with TMR) and a differently colored Map2 probe set (labeled with Alexa-594) can be used to image and distinguish their targets with a single molecule resolution. A fraction of these mRNAs migrate to distant reaches of dendrites (FIG. 10D). Particle counts indicated that 14% of the 791 β-actin mRNA molecules were located in dendrites, whereas 37% of the 140 Map2 mRNA molecules were located in the dendrites, which is similar to the previously reported distributions.
All publications cited in the specification, both patent publications and non-patent publications, are indicative of the level of skill of those skilled in the art to which this invention pertains. All these publications are herein fully incorporated by reference to the same extent as if each individual publication were specifically and individually indicated as being incorporated by reference.
References
1. Kaufmann, B. B. & van Oudenaarden, A. Stochastic gene expression: from single molecules to the proteome. Curr Opin Genet Dev 17, 107-112 (2007).
2. St Johnston, D. Moving messages: the intracellular localization of mRNAs. Nat Rev Mol Cell Biol 6, 363-375 (2005).
3. Gall, J. G. Differential synthesis of the genes for ribosomal RNA during amphibian oogenesis. Proc Natl Acad Sci USA 60, 553-560 (1968).
4. Levsky, J. M. & Singer, R. H. Fluorescence in situ hybridization: past, present and future. J Cell Sci 116, 2833-2838 (2003).
5. Tautz, D. & Pfeifle, C. A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98, 81-85 (1989).
6. Raap, A. K. et al. Ultra-sensitive FISH using peroxidase-mediated deposition of biotin- or fluorochrome tyramides. Hum Mol Genet 4, 529-534 (1995).
7. Femino, A. M., Fay, F. S., Fogarty, K. & Singer, R. H. Visualization of single RNA transcripts in situ. Science 280, 585-590 (1998).
8. Tsokas, P. et al. Local protein synthesis mediates a rapid increase in dendritic elongation factor 1A after induction of late long-term potentiation. J Neurosci 25, 5833-5843 (2005).
9. Maamar, H., Raj, A. & Dubnau, D. Noise in gene expression determines cell fate in Bacillus subtilis. Science 317, 526-529 (2007).
10. Femino, A. M., Fogarty, K., Lifshitz, L. M., Carrington, W. & Singer, R. H. Visualization of single molecules of mRNA in situ. Methods Enzymol 361, 245-304 (2003).
11. Randolph, J. B. & Waggoner, A. S. Stability, specificity and fluorescence brightness of multiply-labeled fluorescent DNA probes. Nucleic Acids Res 25, 2923-2929 (1997).
12. Sindelar, L. E. & Jaklevic, J. M. High-throughput DNA synthesis in a multichannel format. Nucleic Acids Res 23, 982-987 (1995).
13. Vargas, D. Y., Raj, A., Marras, S. A., Kramer, F. R. & Tyagi, S. Mechanism of mRNA transport in the nucleus. Proc Natl Acad Sci USA 102, 17008-17013 (2005).
14. Raj, A., Peskin, C. S., Tranchina, D., Vargas, D. Y. & Tyagi, S. Stochastic mRNA synthesis in mammalian cells. PLoS Biol 4, e309 (2006).
15. Garneau, N. L., Wilusz, J. & Wilusz, C. J. The highways and byways of mRNA decay. Nat Rev Mol Cell Biol 8, 113-126 (2007).
16. Gonzalez, R. C., Woods, R. E. & Eddins, S. L. Digital Image Processing Using Matlab. (Pearson Prentice Hall, Upper Saddle River, N.J.; 2004).
17. Wang, J. C. et al. Chromatin immunoprecipitation (ChIP) scanning identifies primary glucocorticoid receptor target genes. Proc Natl Acad Sci USA 101, 15603-15608 (2004).
18. Yildiz, A. et al. Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization. Science 300, 2061-2065 (2003).
19. Benson, D. M., Bryan, J., Plant, A. L., Gotto, A. M., Jr. & Smith, L. C. Digital imaging fluorescence microscopy: spatial heterogeneity of photobleaching rate constants in individual cells. J Cell Biol 100, 1309-1323 (1985).
20. Lecuyer, E. et al. Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function. Cell 131, 174-187 (2007).
21. Fukushige, T., Hawkins, M. G. & McGhee, J. D. The GATA-factor elt-2 is essential for formation of the Caenorhabditis elegans intestine. Dev Biol 198, 286-302 (1998).
22. Sanicola, M., Sekelsky, J., Elson, S. & Gelbart, W. M. Drawing a stripe in Drosophila imaginal disks: negative regulation of decapentaplegic and patched expression by engrailed. Genetics 139, 745-756 (1995).
23. Rep, M., Krantz, M., Thevelein, J. M. & Hohmann, S. The transcriptional response of Saccharomyces cerevisiae to osmotic shock. Hot1p and Msn2p/Msn4p are required for the induction of subsets of high osmolarity glycerol pathwaydependent genes. J Biol Chem 275, 8290-8300 (2000).
24. Tiruchinapalli, D. M. et al. Activity-dependent trafficking and dynamic localization of zipcode binding protein 1 and beta-actin mRNA in dendrites and spines of hippocampal neurons. J Neurosci 23, 3251-3261 (2003).
25. Blichenberg, A. et al. Identification of a cis-acting dendritic targeting element in MAP2 mRNAs. J Neurosci 19, 8818-8829 (1999).
26. Warren, L., Bryder, D., Weissman, I. L. & Quake, S. R. Transcription factor profiling in individual hematopoietic progenitors by digital RT-PCR. Proc Natl Acad Sci USA 103, 17807-17812 (2006).

## Claims

1. A method for detecting individual RNA molecules containing a first target sequence in a fixed and permeabilized cell, said first target sequence including at least 12 different non-overlapping probe-binding regions 7-40 nucleotides long, comprising
providing a first set of hybridization probes consisting of DNA, RNA or a mixture of DNA and RNA free of non-natural nucleotides that increase binding affinity of probes, that are complementary to the probe-binding regions of said first target sequence and that are singly labeled with the same first fluorophore, said first set containing at least twelve different non-overlapping hybridization probes that have sequences complementary to the at least 12 different probe-binding regions of said first target sequence respectively;
fixing and permeabilizing the cell;
probing said molecules in the fixed, permeabilized cell with an excess of the first set of hybridization probes, whereby individual RNA molecules in the cell are rendered sufficiently fluorescent to be seen as spots of said first fluorophore in fluorescence microscopy;
washing said cell to remove unbound probes; and
detecting said spots of the first fluorophore by fluorescence microscopy, wherein the first set of hybridization probes are 7-30 nucleotides in length.

2. The method of claim 1 wherein the RNA molecules are mRNA molecules.

3. The method of claim 1 wherein the step of detection includes counting said spots.

4. The method of claim 1 wherein said first target sequence includes at least 30 probe-binding regions and said first set of probes includes at least 30 different probes.

5. The method of claim 4 wherein the RNA molecules contain a second target sequence having at least 30 different non-overlapping probe-binding regions 7-40 nucleotides long, wherein the step of providing includes providing a second set of hybridization probes that are complementary to the probe-binding regions of the second target sequence, are 7-30 nucleotides in length, and are each singly labeled with the same second fluorophore distinguishable from said first fluorophore, said second set containing at least 30 different non-overlapping hybridization probes that have sequences complementary to the at least 30 different probe-binding regions of the second target sequence, respectively; wherein the step of probing includes probing said molecules with an excess of the second set of hybridization probes, whereby individual RNA molecules in the cell are rendered sufficiently fluorescent to be seen as spots in fluorescence microscopy; and wherein the step of detecting includes detecting spots of said second fluorophore by fluorescence microscopy.

6. The method of claim 1 wherein said first target sequence includes 40-60 different probe-binding regions and said first set of probes consists of 40-60 different probes.

7. The method of claim 2 wherein said first target sequence is a coding sequence.

8. The method of claim 1 wherein the step of detecting includes imaging to show said spots of the first fluorophore, processing the image to enhance the spots, and detecting the spots utilizing a selected intensity threshold at which the number of spots is insensitive to threshold value.

9. The method of claim 8 wherein the selected intensity threshold is obtained by filtering the images, counting spots for each of a plurality of intensity thresholds, determining a plateau region for the total number of spots as a function of intensity threshold, at which the total number of spots is relatively insensitive to the threshold, and selecting the intensity threshold from within the plateau region.

10. The method of claim 9 wherein filtering the images comprises filtering a 3-D stack of 2-D images.

11. The method of claim 10 wherein filtering utilizes a three-dimensional linear Laplacian of Gaussian filter.

12. The method of claim 8 wherein detecting is quantitative.

13. The method of claim 1 wherein the RNA molecules contain a second target sequence having at least 12 different non-overlapping probe-binding regions 7-40 nucleotides long, wherein the step of providing includes providing a second set of hybridization probes that are complementary to the probe-binding regions of the second target sequence, are 7-30 nucleotides in length, and are each singly labeled with the same second fluorophore distinguishable from the first fluorophore, said second set containing at least twelve different non-overlapping hybridization probes that have sequences complementary to the at least 12 different probe-binding regions of the second target sequence, respectively; wherein the step of probing includes probing said molecules with an excess of the second set of hybridization probes, whereby individual RNA molecules in the cell are rendered sufficiently fluorescent to be seen as spots in fluorescence microscopy; and wherein the step of detecting includes detecting said spots of said second fluorophore by fluorescence microscopy.

14. A method for determining whether a test compound affects an amount of distribution in a cell of a first RNA target sequence having at least 12 different non-overlapping probe-binding regions 7-40 nucleotides long, comprising
incubating the cell with the test compound for a time sufficient to elicit a response;
fixing and permeabilizing the cell;
immersing said permeabilized cell in a hybridization solution containing an excess of a first set of at least twelve different non-overlapping hybridization probes consisting of DNA, RNA or a mixture of DNA and RNA free of non-natural nucleotides, that are 7-30 nucleotides long, that have sequences complementary to said first target sequence, and that are each singly labeled with the same first fluorophore, said at least twelve different non-overlapping hybridization probes are complementary to the at least 12 different probe-binding regions of the first target sequence, respectively, whereby individual RNA molecules in the cell are rendered sufficiently fluorescent to be seen as spots in fluorescence microscopy;
washing said cell to remove unbound probes;
detecting an amount of a distribution of said spots of the first fluorophore; and
comparing said amount or said distribution with that obtained from a control cell similarly treated but without the test compound.

15. The method according to claim 14 wherein said first target sequence includes at least 30 different probe-binding regions and said first set of probes includes at least 30 different probes.

16. The method according to claim 14 wherein the first target sequence is an mRNA sequence.

17. The method of claim 14 wherein the step of detecting includes imaging to show said spots of the first fluorophore, processing the image to enhance the spots, and analyzing the enhanced spots utilizing an intensity threshold at which the number of spots is insensitive to threshold value.

18. The method of claim 17 wherein the step of detection includes counting said spots corresponding to single molecules of the RNA to obtain a gene expression profile.