Methods and compositions relating to targeted analysis

Abstract

The technology described herein is directed to a method, system and composition for analyzing, detecting and / or visualizing target molecules.Described herein is a composition and system comprising an oligonucleotide tag with a barcode cassette.Also described herein is a method for analyzing target molecules, comprising: contacting sample with at least one oligonucleotide tag, contacting sample with at least two readout molecules, and detecting the relative spatial order of the readout molecules. TIFF2022543562000002.tif65138

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 USC §119(e) of U.S. Provisional Application No. 62 / 880,216, filed July 30, 2019, the contents of which are incorporated herein by reference in their entirety.

[0002] Government support This invention was made with Government support under Grant Nos. GM123289 and HG008525 awarded by the National Institutes of Health. The United States Government has certain rights in the invention.

[0003] Sequence Listing This application contains a Sequence Listing that has been submitted in ASCII format via EFS-Web and is incorporated by reference in its entirety. The ASCII copy, created on July 27, 2020, is named 002806-094580_SL.txt and is 1,055 bytes in size.

[0004] Technical Field The technology described herein relates to methods, systems, and compositions for analyzing, detecting, and / or visualizing target molecules. [Background technology]

[0005] background For the visualization of targets at the cellular and subcellular level, fluorophores linked to detection molecules (e.g., antibodies, oligos, synthetic nucleic acids, etc.) are usually used. Due to the overall spectral limitations of fluorescence microscopy, only 4-5 targets can be visualized simultaneously. However, some applications require the simultaneous visualization of more than five targets, for example in the case of complex systems containing more than five components, or large target molecules such as chromosomes. Therefore, new methods that can simultaneously detect many targets, or many regions of one target, are required. Summary of the Invention

[0006] overview The technology described herein is directed to a method, system and composition for analyzing, detecting and / or visualizing target molecules, which provides the ability to visualize more targets at once than conventional technology can.As an example, when using fluorescent labels to detect target biomolecules, the method uses detection probes with four colors to localize to targets in a group; the probes then assemble with each different target in the group to form a barcoded color sequence.Using four colors with existing technology would only allow detection of four different targets.The disclosed method can simultaneously detect 16 targets using only two-bit barcodes.

[0007] As disclosed herein, analysis of target molecules can be performed using OligoCASSEQ technology that includes oligonucleotide tags (e.g., Oligopaints). Readout molecules can be used non-enzymatically to detect the identity of a particular oligonucleotide tag (e.g., Oligopaint).

[0008] In one aspect, described herein is a method of analyzing at least one target molecule in a sample, the method comprising the steps of: (a) contacting the sample with at least one oligonucleotide tag, each oligonucleotide tag comprising (i) a recognition domain that specifically binds to the target molecule to be analyzed, and (ii) at least one street comprising at least one cassette, each cassette comprising (1) a barcode region comprising at least one nucleotide flanked on at least one side by an anchor region, wherein the street of each oligonucleotide tag is unique compared to the streets of other oligonucleotide tags of step (a) in at least: A. the spatial order of the cassettes within the street is different; or B. the sequence of the barcode region is different from the barcode regions of other oligonucleotide tags of step (a). (b) contacting the sample with at least two readout molecules, each readout molecule comprising (i) an oligonucleotide that specifically hybridizes to the at least one oligonucleotide tag cassette used in step (a) and (ii) a detection molecule, wherein the at least two readout molecules collectively constitute at least two distinguishable detection molecules; and (c) detecting the relative spatial order of the detection molecules hybridized to the at least one oligonucleotide tag, wherein at least one oligonucleotide tag is hybridized to at least one target molecule, whereby the relative spatial order of the detection molecules enables identification of which oligonucleotide tag is hybridized to the target molecule at that position.

[0009] In some embodiments of any of the above aspects, the barcode region comprises between 1 and 10 nucleotides.

[0010] In some embodiments of any of the above aspects, the street comprises at least three cassettes.

[0011] In some embodiments of any of the above aspects, the barcode region is flanked on both sides by anchor regions.

[0012] In some embodiments of any of the above aspects, the anchor region of all of the oligonucleotide tags is constant.

[0013] In some embodiments of any of the above aspects, specific hybridization of the readout molecule to the cassette is determined by the identity of the barcode region.

[0014] In some embodiments of any of the above aspects, the detection molecule is a fluorophore.

[0015] In some embodiments of any of the above aspects, the detecting step is carried out using fluorescence microscopy.

[0016] In some embodiments of any of the above aspects, the detection molecule comprises biotin, an amine, a metal, an anchoring molecule, or an acrydite.

[0017] In some embodiments of any of the above aspects, the detecting step is performed with at least single-cell resolution.

[0018] In some embodiments of any of the above aspects, step (b) comprises contacting the sample with at least four readout molecules.

[0019] In some embodiments of any of the above aspects, step (b) comprises contacting the sample with a group of readout molecules that collectively constitute at least three distinguishable detection molecules.

[0020] In some embodiments of any of the above aspects, step (b) comprises contacting the sample with a group of readout molecules that collectively constitute at least four distinguishable detection molecules.

[0021] In some embodiments of any of the above aspects, at least two target molecules are analyzed simultaneously.

[0022] In some embodiments of any of the above aspects, at least three target molecules are analyzed simultaneously.

[0023] In some embodiments of any of the above aspects, at least 10 target molecules are analyzed simultaneously.

[0024] In some embodiments of any of the above aspects, at least 20 target molecules are analyzed simultaneously.

[0025] In some embodiments of any of the above aspects, the target molecule is a nucleic acid, a polypeptide, a cell surface molecule, or an inorganic entity.

[0026] In some embodiments of any of the above aspects, the target molecule is DNA or mRNA.In some embodiments of any of the above aspects, the sample is a cell, a cell culture, or a tissue sample.

[0027] In another aspect, described herein is a system for analyzing at least one target molecule in a sample, the system comprising: (a) a detector capable of detecting at least two detectable molecules; (b) at least one oligonucleotide tag, each oligonucleotide tag comprising (i) a recognition domain that specifically binds to the target molecule to be analyzed, and (ii) a street comprising at least one cassette, each cassette comprising (1) a barcode region comprising at least one nucleotide flanked on at least one side by an anchor region, wherein the street of each oligonucleotide tag is unique compared to the streets of other oligonucleotide tags of (b) in at least: A. the spatial order of the cassettes within the street is different; or B. the sequence of the barcode region is different from the barcode regions of other oligonucleotide tags of (b); and (c) at least two readout molecules, each readout molecule comprising (i) an oligonucleotide that specifically hybridizes to a cassette of at least one oligonucleotide tag used in (b) and (ii) a detection molecule, wherein the at least two readout molecules collectively constitute at least two distinguishable detection molecules; Here, a sample is contacted with at least one oligonucleotide tag and at least two readout molecules, and the relative spatial order of detection molecules hybridized to the at least one oligonucleotide tag is detected; where at least one oligonucleotide tag is hybridized to at least one target molecule, whereby the relative spatial order of the detection molecules makes it possible to identify which oligonucleotide tag is hybridized to the target molecule at that position. [Brief description of the drawings]

[0028] [Figure 1A]Figure 1A-1H are a series of images and schematic diagrams showing multiplexed genome tracing using OligoCASSEQ. Figure 1A is a schematic diagram showing Oligopaint modified for OligoCASSEQ. Genomic homologous regions hybridize with genomic DNA and are flanked by non-genomic homologous regions, main street and back street. These streets contain universal priming regions that allow amplification of Oligopaint libraries. The streets also contain sequencing cassettes (e.g., cassette 1, cassette 2, cassette 3, and cassette 4) that are used for multiplexing. Although four cassettes are depicted here, there is no limit to the total number of cassettes. [Figure 1B] FIG. 1B is a schematic diagram showing the sequencing cassette from FIG. 1A. The barcode region contains a variable region (X). Each bit (X) can be coded by one of four nucleotides (e.g., A, C, T, or G). A barcode of 5 nucleotides (nt) is shown here, but the length of the barcode can vary. Increasing the length of the barcode increases multiplexing. The barcode is interrogated by a specific readout oligo (see, e.g., FIG. 1C). Each probe contains a different barcode that allows multiplexing. On either side of the barcode region are anchor regions (e.g., 5 nt is shown here, but the length of the barcode region can vary) that are constant across all probes. The anchor regions provide stable hybridization and allow for barcode differentiation. [Figure 1C] Figure 1C is a schematic diagram showing examples of readout oligos for two positions within a cassette. Each pool of readout oligos is used to interrogate a specific barcode position. The fluorescently labeled readout oligos specifically base pair with unique bases (e.g., A, C, G, and T) at a particular position and mix nucleotides at non-interrogated bases ("N"). The anchor ("Anch") nucleotide sequence is cassette specific (e.g., cassette 1 shares a particular anchor, cassette 2 shares a different anchor). [Figure 1D] FIG. 1D is a schematic showing an example workflow of an OligoCASSEQ experiment for reading out cassette positions 1 and 2. From left to right, the Oligopaint probe first hybridizes to genomic DNA. The readout oligo of a particular pool hybridizes to sequence barcode position 1 on cassette 1 ("A" on cassette). The red "T" readout oligo binds to the cassette with perfect complementarity. After hybridization, an image is taken and the readout oligo is washed away with 60% formamide in 2XSSCT. Position 2 on cassette 1 can then be interrogated by hybridizing the cassette 1 oligo at position 2. Other positions and cassettes are interrogated in the same manner. [Figure 1E] Figures 1E-1H are a series of images showing the localization of OligoCASSEQ at five loci along human chromosome 2 (Chr.2). Figure 1E is a schematic showing the Chr.2 loci used in the validation experiment, nucleotide barcodes, and color barcodes (see, for example, Figures 1F-1H). [Figure 1F] Figure 1F is an image showing micrographs of OligoCASSEQ interrogation of two barcode locations (see, for example, top and middle rows) followed by re-interrogation of the first location in the same nucleus (see, for example, bottom row). The schematic above each micrograph shows the color code of the specific locus at the different barcode locations. Note that when using both red and cyan labeled probes, the red and cyan colors overlap because the microscope filter sets were not optimal. However, when red and cyan overlap, the cyan will be brighter than the red. The micrographs are maximum intensity z-projections taken from multiple z-slices. The cells are PGP1-F cells. [Figure 1G] Figure 1G is an image showing barcode identification. After sequencing of at least two positions, the OligoCASSEQ focus can be decoded. [Figure 1H]Figure 1H is an image showing that OligoCASSEQ enables chromosome tracing, since the genomic location of every focus can be identified. The lines are two-dimensional (2D) traces based on the decoded foci. The arrows indicate the base of the chromosome (e.g., the locus closest to the telomere on the long arm q). OligoCASSEQ can also be used for super-resolution microscopy by using readout probes containing compatible fluorophores (e.g., see Figure 1C). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Detailed Description Aspects of the technology described herein include methods of analyzing target molecules using OligoCASSEQ. As described herein, OligoCASSEQ refers to methods, systems, and / or compositions that include oligonucleotide tags (e.g., Oligopaints). The disclosed methods allow for the simultaneous visualization of at least two targets or at least two regions of a target. Furthermore, by using barcoded cassettes, the disclosed methods are capable of simultaneously visualizing more than four targets, more than can currently be detected with conventional Oligopaints.

[0030] These methods have advantages over alternative methods: the disclosed methods do not require enzymes such as ligases or polymerases that are required by alternative methods; the disclosed methods also require shorter oligos and fewer oligos than are required by alternative techniques; and finally, the disclosed methods are simpler and less expensive than alternative methods.

[0031] Thus, in one aspect, described herein is a method of analyzing at least one target molecule in a sample, the method comprising the steps of: (a) contacting the sample with at least one oligonucleotide tag (e.g., an Oligopaint), each oligonucleotide tag comprising (i) a recognition domain that specifically binds to the target molecule to be analyzed, and (ii) at least one street comprising at least one cassette, each cassette comprising (1) a barcode region comprising at least one nucleotide flanked on at least one side by an anchor region, wherein the street of each oligonucleotide tag is unique compared to the streets of other oligonucleotide tags of step (a) in that at least the spatial order of the cassettes within the street is different; (b) contacting the sample with at least one oligonucleotide tag (e.g., an Oligopaint), each oligonucleotide tag comprising (i) a recognition domain that specifically binds to the target molecule to be analyzed, and (ii) at least one street comprising at least one cassette, each cassette comprising (1) a barcode region comprising at least one nucleotide flanked on at least one side by an anchor region, wherein the street of each oligonucleotide tag is unique compared to the streets of other oligonucleotide tags of step (a) in that at least the spatial order of the cassettes within the street is different; and (c) detecting a relative spatial order of the detection molecules hybridized to the at least one oligonucleotide tag, wherein at least one oligonucleotide tag is hybridized to at least one target molecule, whereby the relative spatial order of the detection molecules enables identification of which oligonucleotide tag is hybridized to the target molecule at that position.

[0032] In other words, the OligoCASSEQ method can include contacting a sample with an oligonucleotide tag (e.g., an Oligopaint), contacting the sample with at least two readout molecules, and determining the spatial order of the barcoded cassettes.

[0033] In another aspect, described herein is a system for analyzing at least one target molecule in a sample, the system comprising: (a) a detector capable of detecting at least two detectable molecules; (b) at least one oligonucleotide tag (e.g., an Oligopaint), each oligonucleotide tag comprising (i) a recognition domain that specifically binds to the target molecule to be analyzed, and (ii) a street comprising at least one cassette, each cassette comprising a barcode region comprising at least one nucleotide flanked on at least one side by an anchor region, wherein at least the spatial order of the cassettes within a street is different, or or B. an oligonucleotide tag, wherein each oligonucleotide tag street is unique relative to the other oligonucleotide tag streets of (b) in that the sequence of the barcode region differs from the barcode region of the other oligonucleotide tags of (b); and (c) at least two readout molecules, each readout molecule comprising (i) an oligonucleotide that specifically hybridizes to at least one oligonucleotide tag cassette used in (b) and (ii) a detection molecule, wherein the at least two readout molecules collectively constitute at least two distinguishable detection molecules. In some embodiments of any of the above aspects, the sample is contacted with at least one oligonucleotide tag and at least two readout molecules, and the relative spatial order of the detection molecules hybridized to at least one oligonucleotide tag is detected (e.g., with a detector); where at least one oligonucleotide tag is hybridized to at least one target molecule, whereby the relative spatial order of the detection molecules allows identification of which oligonucleotide tag is hybridized to the target molecule at that position. In some embodiments of any of the above aspects, the detected data is output on a display.

[0034] As used herein, the term "oligonucleotide tag" refers to an oligonucleotide that includes a recognition domain and / or at least one street. In some embodiments of any of the above aspects, the oligonucleotide tag comprises or is composed of an oligonucleotide, such as, but not limited to, Oligopaint, multiplexed error-robust fluorescence in situ hybridization (MERFISH) oligo, seqFISH oligo, RNA sequential probing of targets (SPOTs) oligo, high-coverage microscopy-based technology (Hi-M) oligo, or optical reconstruction of chromatin architecture (ORCA) oligo, or any oligonucleotide used in FISH and / or any oligonucleotide having a sequence (e.g., a recognition domain) that is complementary to a target molecule (e.g., an oligonucleotide sequence, a portion of a DNA sequence, or a specific chromosome or a partial chromosomal region of a specific chromosome). For further details, see, e.g., Cardozo et al., Mol Cell. 2019 Apr 4;74(1):212-222; Mateo et al., Nature. 2019 Apr;568(7750):49-54; Wang et al., Scientific Reports volume 8, Article number: 4847 (2018); Shah et al., Neuron, Volume 92, Issue 2, 19 October 2016, Pages 342-357; Eng et al., Nat Methods. 2017 Dec;14(12):1153-1155; each of which is incorporated by reference in its entirety.

[0035] The term "street" as used herein refers to a portion of an oligonucleotide tag (e.g., an Oligopaint) that does not have identity to or hybridize to a target sequence. A "cassette" as used herein refers to a region of a street that includes a barcode region and at least one anchor region. A "barcode region" as used herein refers to a region of a cassette that includes at least one nucleotide. An "anchor region" as used herein refers to a region of a cassette that is unique and / or constant for a set of cassettes and / or is complementary to an anchor hybridizing region of at least one readout molecule. A "readout molecule" as used herein refers to a molecule that includes 1) a detection molecule or detection moiety and 2) an oligonucleotide sequence that is complementary to at least a portion of at least one cassette of at least one oligonucleotide tag (e.g., an Oligopaint) and / or specifically hybridizes to at least a portion of at least one cassette of at least one oligonucleotide tag (e.g., an Oligopaint).

[0036] In some embodiments of any of the above aspects, the oligonucleotide tag (e.g., Oligopaint) comprises at least one street, the street comprising at least one cassette. As used herein, "cassette" refers to a region of a street comprising a barcode region and at least one anchor region. As non-limiting examples, the street comprises 1 cassette, 2 cassettes, 3 cassettes, 4 cassettes, 5 cassettes, 6 cassettes, 7 cassettes, 8 cassettes, 9 cassettes, or at least 10 cassettes.

[0037] In some embodiments of any of the foregoing aspects, the oligonucleotide tag (e.g., Oligopaint) comprises at least one cassette; the population of oligonucleotide tags comprises subpopulations of oligonucleotide tags, each subpopulation defined by the type of cassette(s) present in the oligonucleotide tag. Each cassette type comprises at least one unique and / or identifiable anchor region (i.e., the anchor region can be specific to the cassette type), and each individual cassette comprises a unique and / or identifiable barcode region. As a non-limiting example, the population of oligonucleotide tags (e.g., Oligopaint) comprises type 1 cassettes and type 2 cassettes, where the set of type 1 cassettes share at least one anchor region that is different from the anchor region(s) shared by the set of type 2 cassettes. As a non-limiting example, the type 1 cassettes comprise anchor region A and anchor region B, and the type 2 cassettes comprise anchor region C and anchor region D. In some embodiments of any of the above aspects, the cassette subpopulations can share anchor regions, so long as at least one anchor region is unique and / or identifiable for each cassette subpopulation. As a non-limiting example, a type 1 cassette contains anchor region A and anchor region B, and a type 2 cassette contains anchor region A and anchor region C.

[0038] In some embodiments of any of the preceding aspects, the oligonucleotide tag (e.g., an Oligopaint) comprises multiple cassettes, each cassette being of a different type (e.g., oligonucleotide tag X comprises a type 1 cassette, a type 2 cassette, and a type 3 cassette). In some embodiments of any of the preceding aspects, the oligonucleotide tag (e.g., an Oligopaint) comprises multiple cassettes, at least two cassettes being of the same type (e.g., oligonucleotide tag Y comprises two type 1 cassettes and one type 2 cassette). The presence of multiple copies of the same cassette type in an oligonucleotide tag (e.g., an Oligopaint) can amplify the signal (e.g., recruit two readout molecules to the oligonucleotide tag). In some embodiments of any of the preceding aspects, the oligonucleotide tag (e.g., Oligopaint) or population of oligonucleotide tags comprises 1 type of cassette, 2 types of cassettes, 3 types of cassettes, 4 types of cassettes, 5 types of cassettes, 6 types of cassettes, 7 types of cassettes, 8 types of cassettes, 9 types of cassettes, or at least 10 types of cassettes. In some embodiments of any of the preceding aspects, each type of cassette can encode a specific identifier (e.g., chromosome number, chromosome arm, chromosome region, gene, etc.).

[0039] In some embodiments of any of the preceding aspects, each oligonucleotide tag (e.g., an Oligopaint) in a set of oligonucleotide tags has a unique arrangement of cassettes; for example, the sequence of cassettes may be unique, with each different oligonucleotide tag having cassettes arranged in a different spatial order within a street. Thus, in some embodiments of any of the preceding aspects, each oligonucleotide tag (e.g., an Oligopaint) in a set of oligonucleotide tags has a unique street, e.g., at least in that the spatial order of cassettes within a street of each oligonucleotide tag is different. In some embodiments of any of the preceding aspects, each oligonucleotide tag (e.g., an Oligopaint) in a set of oligonucleotide tags has a unique cassette or set of cassettes. In some embodiments of any of the preceding aspects, all cassettes within a street are identical to one another. In some embodiments of any of the preceding aspects, all cassettes within an oligonucleotide tag (e.g., an Oligopaint) are identical to one another. In some embodiments of any of the preceding aspects, at least one cassette is different from other cassettes within the same street or oligonucleotide tag (e.g., an Oligopaint). In some embodiments of any of the preceding aspects, any cassette is different from other cassettes within the same street or oligonucleotide tag (e.g., Oligopaint). In some embodiments of any of the preceding aspects, Main Street comprises a cassette or set of cassettes and Back Street comprises a different cassette or set of different cassettes. In some embodiments of any of the preceding aspects, Main Street and Back Street share the same cassette or set of cassettes. In some embodiments of any of the preceding aspects, each oligonucleotide tag within a set of oligonucleotide tags (e.g., Oligopaints) share the same cassette or set of cassettes; where the set of oligonucleotide tags comprise oligonucleotide tags that hybridize to different target molecules.

[0040] In some embodiments of any of the above aspects, the cassette comprises a barcode region. As used herein, "barcode region" refers to a region of the cassette that comprises at least one nucleotide. As non-limiting examples, the barcode region comprises 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, or 10 nucleotides. In some embodiments of any of the above aspects, at least one nucleotide of the barcode region comprises a modified nucleobase, as further described herein.

[0041] In some embodiments of any of the preceding aspects, the sequence of the barcode region is different from the barcode region of other oligonucleotide tags (e.g., Oligopaints). In some embodiments of any of the preceding aspects, every cassette has a different barcode region, e.g., has a barcode region that differs in nucleotide sequence. In some embodiments of any of the preceding aspects, every cassette in a street has a different barcode region, e.g., has a barcode region that differs in nucleotide sequence, from other cassettes in the street. In some embodiments of any of the preceding aspects, the sequence of the barcode region is the same as, and is shared with, at least one barcode region of other oligonucleotide tags (e.g., Oligopaints). In some embodiments of any of the preceding aspects, the barcode region is flanked on at least one side by an anchor region. In some embodiments of any of the preceding aspects, the barcode region is flanked on both sides by anchor regions.

[0042] In some embodiments of any of the above aspects, the cassette comprises at least one anchor region. As used herein, "anchor region" refers to a region of a cassette that is specific and / or constant for a set of cassettes and / or is complementary to the anchor hybridizing region of at least one readout molecule. In some embodiments of any of the above aspects, each cassette comprises at least one anchor region that is unique compared to at least one anchor region of all other cassettes. As a non-limiting example, the anchor region comprises at least one nucleotide. As a non-limiting example, the anchor region comprises 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, or 10 nucleotides. In some embodiments of any of the above aspects, the anchor region comprises at least 5 nucleotides.

[0043] In some embodiments of any of the above aspects, an anchor region comprising 5 nucleotides or less (e.g., 1, 2, 3, 4, or 5 nucleotides) allows for transient binding of the readout molecule to its oligonucleotide tag (e.g., Oligopaint). As used herein, the term "transient binding" refers to a weak, reversible, and / or temporary, specific interaction between molecules, i.e., the readout molecule has a binding affinity such that it can repeatedly bind and unbind. Such binding affinity can be measured using the dissociation constant Kd. In some embodiments of any of the above aspects, transient binding can be defined as having a Kd in the μM range. In some embodiments of any of the above aspects, anchor regions with low binding affinity for the target molecule may be used in methods where transient binding to the target molecule is desired (e.g., DNA-Exchange PAINT, see, e.g., US 2016 / 0161472 A1; Jungmann et al., Nano Lett. 2010, 10, 11, 4756-4761; each of which is incorporated herein by reference in its entirety).

[0044] Non-limiting examples of anchor regions include: GTCTC (SEQ ID NO: 1); CACTA (SEQ ID NO: 2); GCCCG (SEQ ID NO: 3); and TGTGC (SEQ ID NO: 4).

[0045] As non-limiting examples, the cassette comprises one anchor region or two anchor regions. In some embodiments of any of the above aspects, the anchor region is 5' to the barcode region and / or the anchor region is 3' to the barcode region. In some embodiments of any of the above aspects, the barcode region is flanked on both sides by anchor regions, e.g., one anchor region is 5' to the barcode region and one anchor region is 3' to the barcode region.

[0046] Each cassette can optionally contain additional sequences, such as linker or spacer sequences, to position it at a desired distance from other cassettes or elements of the oligonucleotide tag (eg, Oligopaint).

[0047] In some embodiments of any of the above aspects, the street of each oligonucleotide tag (e.g., an Oligopaint) is unique from the streets of other oligonucleotide tags by its cassette or set of cassettes. In some embodiments of any of the above aspects, the street of each oligonucleotide tag (e.g., an Oligopaint) is unique from the streets of other oligonucleotide tags at least in that the spatial order of the cassettes within the street is different. As a non-limiting example, a street containing three cassettes (e.g., cassettes "A", "B", and "C") in a spatial order of 5'-ABC-3' has a unique spatial order of the cassettes that is different from other streets containing cassettes in a spatial order selected from 5'-ACB-3', 5'-BAC-3', 5'-BCA-3', 5'-CAB-3', or 5'-CBA-3', each of the aforementioned streets being unique and different from one another in the spatial order of their cassettes.

[0048] In some embodiments of any of the foregoing aspects, the street of each oligonucleotide tag (e.g., Oligopaint) is unique relative to the streets of other oligonucleotide tags, at least in that the barcode regions within the street are distinct. As a non-limiting example, a street including a barcode including, for example, three nucleotides (e.g., nucleotides "X", "Y", and "Z") in the order 5'-XYZ-3' has a unique barcode that is distinct relative to other streets including a barcode region selected from 5'-XZY-3', 5'-YXZ-3', 5'-YZX-3', 5'-ZXY-3', or 5'-ZYX-3', each of the foregoing streets being unique and distinct from one another in their barcode regions.

[0049] In some embodiments of any of the above aspects, the street of each oligonucleotide tag (e.g., Oligopaint) is unique from the streets of other oligonucleotide tags at least in that the spatial order of the cassettes within the street differs, and the street of each oligonucleotide tag is unique from the streets of other oligonucleotide tags at least in that the barcode regions within the street differ.

[0050] In some embodiments of any of the above aspects, the methods described herein include contacting the sample with a readout molecule. As used herein, a "readout molecule" refers to a molecule that includes 1) a detection molecule or moiety and 2) an oligonucleotide sequence that is complementary to and / or specifically hybridizes to at least a portion of at least one cassette of at least one oligonucleotide tag (e.g., Oligopaint). As a non-limiting example, the readout molecule includes at least one region that is complementary to and specifically hybridizes to an anchor region of the cassette, e.g., an "anchor hybridizing region". As a non-limiting example, the readout molecule includes at least one region that is complementary to and specifically hybridizes to a barcode region of the cassette, e.g., a "barcode hybridizing region". As a non-limiting example, the readout molecule includes one anchor hybridizing region and / or two anchor hybridizing regions. In some embodiments of any of the preceding aspects, the anchor hybridizing region is 5' to the barcode hybridizing region and / or the anchor hybridizing region is 3' to the barcode hybridizing region. In some embodiments of any of the preceding aspects, the barcode hybridizing region is flanked on both sides by anchor hybridizing regions, e.g., one anchor hybridizing region is 5' to the barcode hybridizing region and one anchor hybridizing region is 3' to the barcode hybridizing region. In some embodiments of any of the preceding aspects, the anchor hybridizing region may be unique to the cassette type, as described above for anchor regions unique to the cassette type.

[0051] In some embodiments of any of the preceding aspects, the readout molecule comprises a detection molecule, e.g., an optically detectable detection molecule. In some embodiments of any of the preceding aspects, the detection molecule is a fluorophore and the detecting step is performed using a fluorescent microscope. In some embodiments of any of the preceding aspects, the detection molecule comprises a biotin, an amine, a metal, an anchoring molecule, or an acrydite. Non-limiting examples of detection molecules, fluorophores, and detection techniques are further described herein.

[0052] In some embodiments of any of the above aspects, the at least two readout molecules collectively constitute at least two distinguishable detection molecules. As non-limiting examples, two readout molecules collectively constitute two distinguishable detection molecules, three readout molecules collectively constitute three distinguishable detection molecules, four readout molecules collectively constitute four distinguishable detection molecules, or at least five readout molecules collectively constitute at least five distinguishable detection molecules. In some embodiments of any of the above aspects, the pool of readout molecules includes more readout molecules than distinguishable detection molecules, e.g., the same detection molecule may be present on multiple readout molecules.

[0053] In some embodiments of any of the preceding aspects, a detection molecule may be linked to the 5' end of the readout molecule, a detection molecule may be linked to the 3' end of the readout molecule, or a detection molecule may be linked to the 5' and 3' ends of the readout molecule. In some embodiments of any of the preceding aspects, the detection molecule linked to the 5' end of the readout molecule is the same type of detection molecule as the detection molecule linked to the 3' end of the readout molecule. In some embodiments of any of the preceding aspects, the detection molecule linked to the 5' end of the readout molecule is a different type of detection molecule than the detection molecule linked to the 3' end of the readout molecule.

[0054] In some embodiments of any of the above aspects, the sample is contacted with at least two readout molecules. In some embodiments of any of the above aspects, the sample is contacted with at least one readout molecule. As non-limiting examples, the sample is contacted with one readout molecule, two readout molecules, three readout molecules, four readout molecules, or at least five readout molecules.

[0055] In some embodiments of any of the above aspects, the method includes detecting a detection molecule, e.g., a detection molecule of a readout molecule hybridized to an oligonucleotide tag (e.g., an Oligopaint). In some embodiments of any of the above aspects, the method includes detecting a detection molecule, e.g., a detection molecule of a readout molecule hybridized to an oligonucleotide tag (e.g., an Oligopaint) and thus hybridized to one or more targets.

[0056] In some embodiments of any of the above aspects, the detecting step includes detecting a relative spatial order of the detection molecules hybridized to at least one oligonucleotide tag (e.g., an Oligopaint). The different labels (e.g., colors) of the readout molecules correlate with one or more cassettes, and the spatial order of the different readout molecules provides information about the order of the cassettes on a single oligonucleotide tag (e.g., an Oligopaint), such that multiple different oligonucleotide tags can be distinguished by the barcoding signal provided by a group of readout molecules hybridized to a single street. Thus, in some embodiments of any of the above aspects, the relative spatial order of the detection molecules allows identification of which oligonucleotide tag (e.g., an Oligopaint) is hybridized to the target molecule at that location.

[0057] In some embodiments of any of the above aspects, the detecting step comprises detecting a relative spatial order of the detection molecules of the readout molecule hybridized to at least one cassette, whereby the relative spatial order of the detection molecules allows for identifying which oligonucleotide tag (e.g., Oligopaint) is hybridized to the target molecule at that position.

[0058] In some embodiments of any of the above aspects, the detecting step comprises detecting a relative spatial order of readout molecules hybridized to at least one cassette, whereby the relative spatial order of the readout molecules allows for identifying which oligonucleotide tag (e.g., Oligopaint) is hybridized to the target molecule at that position.

[0059] In some embodiments of any of the above aspects, the sample is contacted with a pool of readout molecules, e.g., a "readout pool." In some embodiments of any of the above aspects, the readout pool comprises readout molecules for the same cassette type (e.g., readout molecules that share at least one unique anchor hybridizing region). In some embodiments of any of the above aspects, the readout pool comprises readout molecules for at least one, at least two, at least three, at least four, or at least five cassette types.

[0060] In some embodiments of any of the above aspects, each readout pool is directed to determining the identity of a nucleotide at a particular position of the barcode region of a set of oligonucleotide tags (e.g., an Oligopaint having a particular cassette type). As a non-limiting example, a sample is contacted with at least two readout pools sequentially or simultaneously to detect at least one nucleotide of the barcode region. As a non-limiting example, a sample is contacted with at least two readout pools sequentially to detect at a first and second cassette position of one or more streets. In some embodiments of any of the above aspects, a readout pool comprises two readout molecules, three readout molecules, four readout molecules, or at least five readout molecules. In some embodiments of any of the above aspects, a readout pool comprises two different detection molecules, three different detection molecules, four different detection molecules, or at least five different detection molecules.

[0061] In some embodiments of any of the above aspects, the readout pool comprises a set of readout molecules linked to the same type of detection molecule. As a non-limiting example, the set of readout molecules linked to the same type of detection molecule comprises the same nucleotide at one position of the barcode hybridizing region and at least one of: (1) comprises a different nucleotide at another position of the barcode hybridizing region, (2) the set is degenerate at other positions of the barcode hybridizing region, or (3) comprises a universal nucleotide at other positions of the barcode hybridizing region. A universal nucleotide comprises a universal base that can bind to any nucleotide. Non-limiting examples of universal bases include inosine, hypoxanthine, nitroazoles, isocarbostyril analogs, azolecarboxamides, or aromatic triazole analogs (see, e.g., Loakes et al., Nucleic Acids Res. 2001 Jun 15;29(12):2437-47; Berger et al., Nucleic Acids Res. 2000 Aug 1; 28(15): 2911-2914; Liang et al., RSC Advances 3(35); June 2013).

[0062] In some embodiments of any of the preceding aspects, the readout pool comprises at least two sets of readout molecules; where each set is linked to the same type of detection molecule that is different from the detection molecules linked to the readout molecules of the other set(s); and where each set detects the same nucleotide within the barcode region that is different from the nucleotide at the same position within the barcode region detected by the readout molecules of the other set(s). In some embodiments of any of the preceding aspects, the readout pool comprises at least 1 set, 2 sets, 3 sets, 4 sets, or at least 5 sets of readout molecules.

[0063] In some embodiments of any of the preceding aspects, the sample is contacted with a first readout pool that recognizes the first nucleotide of the barcode region of each cassette (or recognizes a different cassette at the first cassette position in a street). In some embodiments of any of the preceding aspects, the sample is contacted with a second readout pool that recognizes the second nucleotide of the barcode region of each cassette (or recognizes a different cassette at the second cassette position in a street). In some embodiments of any of the preceding aspects, the sample is contacted with a third readout pool that recognizes the third nucleotide of the barcode region of each cassette (or recognizes a different cassette at the third cassette position in a street). In some embodiments of any of the preceding aspects, the sample is contacted with a fourth readout pool that recognizes the fourth nucleotide of the barcode region of each cassette (or recognizes a different cassette at the fourth cassette position in a street). In some embodiments of any of the preceding aspects, the sample is contacted with a fifth readout pool that recognizes the fifth nucleotide of the barcode region of each cassette (or recognizes a different cassette at the fifth cassette position in a street). In some embodiments of any of the above aspects, the sample is contacted with an nth readout pool that recognizes the nth nucleotide of the barcode region of each cassette (or recognizes a different cassette at the nth cassette position within a street); where n corresponds to an integer from 1 to 10.

[0064] In some embodiments of any of the above aspects, the sample is contacted with each readout pool sequentially. In some embodiments of any of the above aspects, between contacting the sample with the nth readout pool and the (n+1)th readout pool, the readout pool is detected as described herein and the nth readout pool is washed away (e.g., with any buffer suitable for use in hybridization reactions, such as 60% formamide in 2XSSCT; where SSC refers to saline-sodium citrate buffer and T refers to TWEEN).

[0065] In some embodiments of any of the above aspects, sample is contacted with at least two readout pools simultaneously.As a non-limiting example, sample is contacted with at least two readout pools, at least three readout pools, at least four readout pools, or at least five readout pools simultaneously.Compared to contacting sample with one readout pool, contacting sample with at least two readout pools simultaneously can provide additional benefits, including but not limited to signal amplification, introduction of additional optically detectable markers (e.g., pseudocolor combinations of different fluorophores), and increasing the speed of the process.

[0066] In some embodiments of any of the preceding aspects, the method of analyzing at least one target molecule in a sample includes contacting the sample with at least one oligonucleotide tag (e.g., an Oligopaint), contacting the sample with at least two readout molecules, and detecting the relative spatial order of the readout molecules. In some embodiments of any of the preceding aspects, specific hybridization of the readout molecules to the cassette is determined by or dependent on the identity of the barcode region.

[0067] In some embodiments of any of the above aspects, the compositions and methods described herein include improvements on compositions and methods related to Oligopaint technology. As used herein, the term "Oligopaint" refers to a polynucleotide having a sequence complementary to a target molecule, e.g., an oligonucleotide sequence, a portion of a DNA sequence, or a specific chromosome or a partial chromosomal region of a specific chromosome.

[0068] Traditionally, probes for fluorescence in situ hybridization (FISH) are derived from cloned genomic regions or flow-sorted chromosomes that are either directly labeled by nick translation or PCR in the presence of fluorophore-conjugated nucleotides, or indirectly labeled with nucleotide-conjugated haptens such as biotin or digoxigenin, and then visualized with secondary detection reagents. Traditional FISH probes are limited by repetitive sequences and variability in efficacy. Furthermore, target regions are constrained by the availability of clones and the size of their genomic inserts. Although it is possible to target larger regions with traditional FISH probes, this approach is often cumbersome and expensive, as each clone must be individually prepared and optimized for hybridization.

[0069] Oligopaint is an improved FISH technology in which oligo libraries can be generated by massively parallel synthesis and used as a renewable source of probes. The oligo libraries can be amplified by PCR (optionally with fluorophore-conjugated primers). The amplification products can be enzymatically treated to obtain highly efficient single-stranded, strand-specific probes that can visualize regions ranging from tens of kilobases to megabases. Oligopaint can include synthetic probes and arrays, which are optionally computer-patterned and / or computer-designed.

[0070] For publications regarding Oligopaint and related techniques, see, e.g., Beliveau et al. OligoMiner provides a rapid, flexible environment for the design of genome-scale oligonucleotide in situ hybridization probes. Proc. Nat. Acad. Sci. USA 2018 115:E2183-E2192; Beliveau et al. In situ super-resolution imaging of genomic DNA with OligoSTORM and OligoDNA-PAINT. Methods Mol Biol 2017 1663:231-252; Wang et al. Spatial organization of chromatin domains and compartments in single chromosomes. Science 2016 353:598-602; Boettiger et al. Super-resolution imaging reveals distinct chromatin folding for different epigenetic states. Nature. 2016 529:418-22; Schmidt et al. Scalable amplification of strand subsets from chip-synthesized oligonucleotide libraries. Nat Commun 2015 Nov 16;6:8634; Murgha et al. Combined in vitro transcription and reverse transcription method to amplify and label complex synthetic oligonucleotide probe libraries. Biotechniques 2015 58:301-7; Beliveau et al.Single-molecule super-resolution imaging of chromosomes and in situ haplotype visualization using Oligopaint FISH probes. Nat Commun 2015 6:7147; Beliveau et al. Visualizing genomes with Oligopaint FISH probes. Curr Protocols Mol Biol 2014 14.23; Beliveau et al. A versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes. Proc. Nat. Acad. Sci. USA 2012 109:21301-6; US 2010 / 0304994 A1; US ​​2018 / 0223347 A1; WO 2018 / 045186 A1; US ​​2014 / 0364333 A1; US ​​2019 / 0032121 A1; US 2013 / 0143208 A1; US ​​10,119,160 B2; US 2018 / 0057867 A1; US ​​2019 / 0127786 A1; US ​​2018 / 0292318 A1; WO 2017 / 189525 A1; WO 2018 / 183851 A1; WO 2018 / 183860 A1; WO 2018 / 045181 A1; US ​​2016 / 0040235 A1; each of which is incorporated herein by reference in its entirety.

[0071] As used herein, the terms "Oligopainted" and "Oligopainted region" refer to a target nucleotide sequence (e.g., a chromosome) or a region of a target nucleotide sequence (e.g., a sub-chromosomal region), respectively, that has been hybridized to one or more Oligopaints. Oligopaints can be used to label target nucleotide sequences, such as chromosomes and sub-chromosomal regions of chromosomes, in various stages of the cell cycle, including, but not limited to, interphase, preprophase, prophase, prometaphase, metaphase, anaphase, telophase, and cytokinesis.

[0072] In some embodiments of any of the foregoing aspects, the FISH method can include: Oligopaint, MERFISH (multiplexed error-robust fluorescent in situ hybridization), seqFISH, SPOTs (RNA sequential probing of targets), Hi-M (high-coverage microscopy-based technique), or ORCA (optical reconstruction of chromatin structure), or any method that involves contacting a sample with an oligonucleotide having a sequence (e.g., a recognition domain) that is complementary to a target molecule (e.g., an oligonucleotide sequence, a portion of a DNA sequence, or a specific chromosome or a subchromosomal region of a specific chromosome). For further details, see, e.g., Cardozo et al., Mol Cell. 2019 Apr 4;74(1):212-222; Mateo et al., Nature. 2019 Apr;568(7750):49-54; Wang et al., Scientific Reports volume 8, Article number: 4847 (2018); Shah et al., Neuron, Volume 92, Issue 2, 19 October 2016, Pages 342-357; Eng et al., Nat Methods. 2017 Dec;14(12):1153-1155; each of which is incorporated by reference in its entirety.

[0073] In some embodiments of any of the above aspects, the method described herein includes analyzing at least one target molecule in the sample. In some embodiments of any of the above aspects, the target molecule includes a nucleic acid, a polypeptide, a cell surface molecule, and / or an inorganic material. In some embodiments of any of the above aspects, the target molecule includes DNA, including but not limited to, for example, genomic DNA, genomic DNA organized as chromosomes, or complementary DNA (cDNA). In some embodiments of any of the above aspects, the target molecule includes RNA, including but not limited to, for example, messenger RNA (mRNA) or ribosomal RNA (rRNA). In some embodiments of any of the above aspects, the target molecule includes a polypeptide, including but not limited to, for example, an intracellular protein, a transmembrane protein, or an extracellular protein. In some embodiments of any of the above aspects, the target molecule includes a cell surface molecule, including but not limited to, for example, a transmembrane protein, a membrane lipid, a membrane receptor, or a transmembrane receptor. In some embodiments of any of the above aspects, the target molecule includes an inorganic material, including any material derived from a non-biological source, including but not limited to, for example, glass, ceramic, metal, or any other solid substrate.

[0074] In some embodiments of any of the above aspects, one target molecule is analyzed.In some embodiments of any of the above aspects, at least two target molecules are analyzed simultaneously.As non-limiting examples, at least two target molecules, at least three target molecules, at least four target molecules, at least five target molecules, at least six target molecules, at least seven target molecules, at least eight target molecules, at least nine target molecules, at least ten target molecules, or at least twenty target molecules are analyzed simultaneously.

[0075] In some embodiments of any of the above aspects, multiple regions of a target molecule are analyzed simultaneously. As non-limiting examples, two regions, three regions, four regions, five regions, six regions, seven regions, eight regions, nine regions, ten regions, or more than ten regions of a target molecule or multiple target molecules are analyzed.

[0076] In some embodiments of any of the above aspects, the sample is a cell, cell culture, or tissue sample. As a non-limiting example, the cell, cell culture, or tissue sample is taken at a time or under conditions when individual chromosomes are discernible, e.g., during mitosis.

[0077] The term "sample" or "test sample" as used herein refers to a sample taken or separated from an organism, such as a blood or tissue sample from a subject. In some embodiments of any of the above aspects, the invention encompasses some examples of biological samples. In some embodiments of any of the above aspects, the biological sample is a cell, tissue, peripheral blood, or bodily fluid. Exemplary biological samples include, but are not limited to, biopsies, tumor samples, biological fluid samples; blood; serum; plasma; urine; sperm; mucus; tissue biopsies; organ biopsies; synovial fluid; bile; cerebrospinal fluid; mucosal secretions; exudates; sweat; saliva; and / or tissue samples. The term also includes mixtures of the above samples. The term "test sample" also includes untreated or pretreated (pre-treated) biological samples. In some embodiments of any of the above aspects, the test sample includes cells from a subject.

[0078] A test sample can be obtained by taking a sample from a subject, but can also be accomplished using a previously isolated sample (e.g., isolated at a previous time point and isolated by the same or another person).

[0079] In some embodiments of any of the above aspects, the test sample may be an unprocessed test sample. As used herein, the phrase "unprocessed test sample" refers to a test sample that has not undergone any prior pretreatment of the sample, except dilution and / or suspension in a solution. Exemplary methods for processing a test sample include, but are not limited to, centrifugation, filtration, sonication, homogenization, heating, freezing and thawing, and combinations thereof. In some embodiments of any of the above aspects, the test sample may be a frozen test sample, e.g., frozen tissue. The frozen sample may be thawed prior to utilizing the methods, assays, and systems described herein. After thawing, the frozen sample may be centrifuged and then subjected to the methods, assays, and systems described herein. In some embodiments of any of the above aspects, the test sample is a clarified test sample, e.g., obtained by centrifugation and recovery of the supernatant, which comprises the clarified test sample. In some embodiments of any of the above aspects, the test sample is a preprocessed test sample, e.g., a supernatant or filtrate obtained from a process selected from the group consisting of centrifugation, filtration, thawing, purification, and any combination thereof. In some embodiments of any of the above aspects, the test sample can be treated with chemical and / or biological reagents. Chemical and / or biological reagents can be utilized to protect and / or maintain the stability of the sample during processing, for example, the stability of the biological molecules (e.g., nucleic acids and proteins) contained therein. One exemplary reagent is a protease inhibitor, which is generally used to protect or maintain the stability of proteins during processing. Those skilled in the art are familiar with the methods and processes suitable for pre-processing biological samples required for measuring the level of expression products described herein.

[0080] In some embodiments of any of the preceding aspects, the methods, assays, and systems described herein can further include obtaining or obtaining a test sample from a subject. In some embodiments of any of the preceding aspects, the subject can be a human subject.

[0081] In some embodiments of any of the above aspects, the oligonucleotide tag (e.g., Oligopaint) comprises a recognition domain. As used herein, a "recognition domain" is a domain of an oligonucleotide tag (e.g., Oligopaint) that specifically binds to a target molecule and / or sequence to be analyzed. As a non-limiting example, the recognition domain can be a nucleic acid sequence that is complementary to a target molecule and / or sequence, e.g., a region of a chromosome. Thus, the sequence of the recognition domain will vary depending on the identity of the desired target. It is well within the skill of one of ordinary skill in the art to design a recognition domain that specifically hybridizes to a given target under certain conditions, for example, using software that is widely and freely available for this purpose (e.g., Primer3 or PrimerBank, both available on the World Wide Web). In some embodiments of any of the above aspects, the recognition domain can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a portion of the target molecule and / or to the target sequence. In some embodiments of any of the above aspects, the recognition domain comprises a domain that is "genome-homologous," i.e., a domain that specifically binds to a region of a genome. In some embodiments of any of the above aspects, multiple recognition domains found on the same or different oligonucleotide tags (e.g., Oligopaints) can specifically bind to a single target molecule and / or target sequence. As non-limiting examples, at least two recognition domains, at least three recognition domains, at least four recognition domains, at least five recognition domains, at least ten recognition domains, at least twenty recognition domains, at least thirty recognition domains, at least forty recognition domains, or at least fifty recognition domains can specifically bind to one target molecule and / or target sequence.

[0082] In some embodiments of any of the above aspects, the recognition domain comprises or consists of an oligonucleotide, including, but not limited to, an Oligopaint, a MERFISH (multiple error-robust fluorescent in situ hybridization) oligo, a seqFISH oligo, a SPOTs (RNA sequential probing of targets) oligo, a Hi-M (high coverage microscopy-based technique) oligo, or an ORCA (optical reconstitution of chromatin structure) oligo, or any oligonucleotide used in FISH methods and / or any oligonucleotide having a sequence (e.g., a recognition domain) complementary to a target molecule (e.g., an oligonucleotide sequence, a portion of a DNA sequence, or a specific chromosome or a subchromosomal region of a specific chromosome). For further details, see, e.g., Cardozo et al., Mol Cell. 2019 Apr 4;74(1):212-222; Mateo et al., Nature. 2019 Apr;568(7750):49-54; Wang et al., Scientific Reports volume 8, Article number: 4847 (2018); Shah et al., Neuron, Volume 92, Issue 2, 19 October 2016, Pages 342-357; Eng et al., Nat Methods. 2017 Dec;14(12):1153-1155; each of which is incorporated by reference in its entirety.

[0083] In some embodiments of any of the above aspects, the recognition domain comprises any nucleic acid binding composition, such as a non-nucleic acid, e.g., a DNA-binding polypeptide. As non-limiting examples, the recognition domain comprises a sequence-specific single-stranded DNA binding protein or factor, a sequence-specific double-stranded DNA binding protein or factor, a DNA-RNA binding protein or factor, or an RNA binding protein or factor. Non-limiting examples of such nucleic acid binding compositions include, but are not limited to, transcription factors, restriction enzymes, transcription activator-like effector nucleases (TALENs), CRISPR-Cas type factors, and the like. In some embodiments of any of the above aspects, the nucleic acid binding composition lacks nuclease activity.

[0084] In some embodiments of any of the above aspects, the target molecule comprises a non-nucleic acid, such as a polypeptide. Thus, the recognition domain comprises any composition that specifically binds to the target polypeptide. Non-limiting examples of such polypeptide-binding recognition domains include, but are not limited to, antibodies (e.g., nanobodies), aptamers, small molecules, ligands, known binding partners of a particular polypeptide, and the like.

[0085] In some embodiments of any of the above aspects, the oligonucleotide tag does not specifically recognize the target molecule in that the oligonucleotide tag is not linked to a recognition domain but is linked to an entity to detect the oligonucleotide tagged entity of interest. The oligonucleotide tag can be a nucleic acid that includes one anchor region and at least a barcode region, but lacks, for example, a recognition domain. As non-limiting examples, such oligonucleotide tagged entities can include small molecules (e.g., for drug screening purposes), polypeptides, cells, or non-biological materials (e.g., metals, chemicals, etc.). Methods for detecting such oligonucleotide tagged entities can be the same as those used to detect oligonucleotide tags (e.g., Oligopaints) as described herein (e.g., contacting with a pool of readout molecules that hybridize to a particular cassette type of oligonucleotide tag). In some embodiments of any of the above aspects, multiple types of target molecules and / or multiple types of tagged entities can be detected at once, for example, using at least one oligonucleotide tag (e.g., Oligopaints) that recognizes DNA, at least one oligonucleotide tag that recognizes a polypeptide, and / or at least one oligonucleotide tagged entity.

[0086] In some embodiments of any of the above aspects, the oligonucleotide tag (e.g., an Oligopaint) comprises at least one street. As used herein, the term "street" refers to a portion of an oligonucleotide tag (e.g., an Oligopaint) that does not have identity to or hybridize to a target sequence. A street includes a region for detection and / or a region for amplification. As a non-limiting example, the oligonucleotide tag (e.g., an Oligopaint) comprises two streets. In some embodiments of any of the above aspects, a street can be one or more of a "main street" and / or a "back street". As a non-limiting example, the main street is 5' to the recognition domain, the main street is 5' to the back street, and / or the main street is 5' to the recognition domain and the back street. As a non-limiting example, the back street is 3' to the recognition domain, the back street is 3' to the main street, and / or the back street is 3' to the recognition domain and the main street. As non-limiting examples, Main Street is 3' to the recognition domain, Main Street is 3' to Back Street, and / or Main Street is 3' to the recognition domain and Back Street. As non-limiting examples, Back Street is 5' to the recognition domain, Back Street is 5' to Main Street, and / or Back Street is 5' to the recognition domain and Main Street.

[0087] In some embodiments of any of the above aspects, a street (e.g., Main Street and / or Back Street) comprises at least one cassette and / or at least one universal priming region. As described herein, the cassette comprises at least one barcode region and at least one anchor region. As used herein, a "universal priming region" refers to a region that binds to a universal primer (e.g., a universal forward primer, a universal reverse primer). As used herein, a "universal primer" refers to a primer that is used for a plurality of individual oligonucleotide tags (e.g., Oligopaints) or a set of oligonucleotide tags. A universal primer can be used to amplify, e.g., an oligonucleotide tag (e.g., an Oligopaint), e.g., to create an oligonucleotide tag or a set of oligonucleotide tags, e.g., by PCR. In some embodiments of any of the above aspects, the universal priming region of each oligonucleotide tag (e.g., Oligopaint) is identical to the universal priming region of the remaining oligonucleotide tags (e.g., other oligonucleotide tags that are contacted with the sample).

[0088] In some embodiments of any of the preceding aspects, the street comprises at least one universal priming region and / or at least one cassette. As a non-limiting example, the universal priming region is 5' of at least one cassette. As a non-limiting example, the universal forward priming region that specifically binds to a universal forward primer is at the 5' end of an oligonucleotide tag (e.g., an Oligopaint). As a non-limiting example, the universal priming region is 3' of at least one cassette. As a non-limiting example, the universal reverse priming region that specifically binds to a universal reverse primer is at the 3' end of an oligonucleotide tag (e.g., an Oligopaint). In some embodiments of any of the preceding aspects, the universal priming region is adjacent (both 5' and 3') to any cassette present in the oligonucleotide tag (e.g., an Oligopaint). In some embodiments of any of the preceding aspects, the universal reverse priming region comprises a recognition site for a nicking endonuclease (NE), e.g., to cause the oligonucleotide tag (e.g., an Oligopaint) to become single stranded when exposed to the NE. In some embodiments of any of the above aspects, the oligonucleotide tags (e.g., Oligopaints) are not necessarily amplified (e.g., via PCR and / or universal priming regions). In some embodiments of any of the above aspects, the described oligonucleotide tags (e.g., Oligopaints) can be synthesized de novo and used "straight out of the tube."

[0089] In some embodiments of any of the above aspects, the detecting step is performed at least with single-cell resolution. As non-limiting examples, the detecting step can be performed at a resolution of at least 200 nm, at least 300 nm, at least 400 nm, at least 500 nm, at least 600 nm, at least 700 nm, at least 800 nm, at least 900 nm, at least 1 μm, at least 2 μm, at least 3 μm, at least 4 μm, at least 5 μm, at least 6 μm, at least 7 μm, at least 8 μm, at least 9 μm, or at least 10 μm. In some embodiments of any of the above aspects, the detecting step is performed at a resolution that allows individual target molecules, such as chromosomes, to be distinguished.

[0090] In some embodiments of any of the above aspects, the nucleic acid, e.g., an oligonucleotide tag (e.g., an Oligopaint), is chemically modified to enhance stability or other beneficial properties. The nucleic acids described herein can be synthesized and / or modified by methods well established in the art, such as, for example, those described in "Current protocols in nucleic acid chemistry," Beaucage, SL et al. (eds.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications include, for example: (a) terminal modifications, such as 5'-terminal modifications (phosphorylation, conjugation, reverse linkage, etc.), 3'-terminal modifications (conjugation, DNA nucleotides, reverse linkage, etc.), (b) base modifications, such as replacement with stabilizing bases, destabilizing bases, or bases that base-pair with an expanded repertoire of partners, removal of bases (nucleotides lacking a base), or conjugated bases, (c) sugar modifications (e.g., modifications at the 2' or 4' positions) or sugar replacement, and (d) backbone modifications, such as modification or replacement of phosphodiester bonds. Specific examples of nucleic acid compounds useful in the embodiments described herein include, but are not limited to, nucleic acids that contain modified backbones or do not contain natural internucleoside linkages. Nucleic acids with modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For purposes of this specification, and as sometimes referred to in the art, modified nucleic acids that do not have a phosphorus atom in the internucleoside backbone can also be considered to be oligonucleosides. In some embodiments of any of the above aspects, the modified nucleic acid has a phosphorus atom in its internucleoside backbone.

[0091] Modified nucleic acid backbones can include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates (including 3'-alkylene phosphonates) and chiral phosphonates, phosphinates, phosphoramidates (including 3'-amino phosphoramidates) and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates with normal 3'-5' linkages, 2'-5' linked analogs of these, and those with reverse polarity where adjacent pairs of nucleoside units are linked 3'-5' to 5'-3', or 2'-5' to 5'-2'. Also included are various salts, mixed salts, and free acid forms. Modified nucleic acid backbones that do not contain phosphorus atoms in the backbone have backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages. These include those with: morpholino linkages (formed in part from the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; alkene-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; N , others containing a mixture of O, S, and CH2 moieties, as well as oligonucleosides having heteroatom backbones, particularly oligonucleosides having -CH2-NH-CH2-, -CH2-N(CH3)-O-CH2- [known as a methylene (methylimino) or MMI backbone], -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, and -N(CH3)-CH2-CH2- [the naturally occurring phosphodiester backbone is represented as -OPO-CH2-].

[0092] Modified nucleic acids can also include one or more substituted sugar moieties. The nucleic acids described herein can include one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl; where the alkyl, alkenyl, and alkynyl can be substituted or unsubstituted C1-C10 alkyl or C2-C10 alkenyl and alkynyl. Exemplary suitable modifications include O[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. In some embodiments of any of the foregoing aspects, the dsRNA comprises one of the following at the 2' position: C1-C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group that improves the pharmacokinetic properties of a nucleic acid, or a group that improves the pharmacodynamic properties of a nucleic acid, and other substituents with similar properties. In some embodiments of any of the above aspects, the modification comprises 2'-methoxyethoxy (2'-O-CH2CHOCH3; also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Another exemplary modification is the O(CH2)2ON(CH3)2 group, also known as 2'-dimethylaminooxyethoxy, i.e., 2'-DMAOE (described in the Examples herein below), and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH2)2 (also described in the Examples herein below).

[0093] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions in nucleic acids, particularly the 3' position of the sugar on the 3' terminal nucleotide or the 5' position of the 5' terminal nucleotide in 2'-5' linked dsRNA. Nucleic acids may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

[0094] Nucleic acids can also include modifications or substitutions of nucleobases (often referred to in the art simply as "bases"). As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases can include synthetic and natural nucleobases, including, but not limited to, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, 6-amino ... , cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine. Some of these nucleobases are particularly useful for increasing the binding affinity of the inhibitory nucleic acids featured in the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6 and 0-6 substituted purines, such as 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are an exemplary base substitution, especially when combined with 2'-O-methoxyethyl sugar modifications.In some embodiments of any of the above aspects, the modified nucleobases can include d5SICS and dNAM, which are non-limiting examples of unnatural nucleobases that can be used separately or together as base pairs (see, e.g., Leconte et. al. J. Am. Chem. Soc.2008, 130, 7, 2336-2343; Malyshev et. al. PNAS. 2012. 109 (30) 12005-12010). In some embodiments of any of the above aspects, the oligonucleotide tag (e.g., Oligopaint) includes any modified nucleobase known in the art, i.e., any nucleobase modified from an unmodified and / or natural nucleobase.

[0095] The preparation of the above modified nucleic acids, backbones, and nucleobases is well known in the art.

[0096] Another modification of the nucleic acids featured in the invention involves chemically linking to the nucleic acid one or more ligands, moieties or conjugates that enhance the activity, cellular distribution, pharmacokinetic properties, or cellular uptake of the nucleic acid. Such moieties include, but are not limited to, lipid moieties, such as cholesterol moieties (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), thioethers, such as beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterols (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 75:49-54). 18:3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0097] In some embodiments of any of the above aspects, measuring and / or detecting a target molecule, e.g., a DNA target molecule, an RNA target molecule, or a polypeptide target molecule, comprises contacting a sample obtained from a subject with a reagent or reagents described herein. In some embodiments of any of the above aspects, the reagent is detectably labeled. In some embodiments of any of the above aspects, the reagent is capable of generating a detectable signal. In some embodiments of any of the above aspects, the reagent generates a detectable signal when the target molecule is present.

[0098] In some embodiments of any of the above aspects, one or more of the reagents described herein comprise a detectable label and / or are capable of generating a detectable signal (e.g., by catalyzing a reaction that converts a compound into a detectable product). Detectable labels can comprise, for example, light-absorbing dyes, fluorescent dyes, or radioactive labels. Detectable labels, methods for detecting them, and methods for incorporating them into the reagents described herein are well known in the art.

[0099] In some embodiments of any of the above aspects, detectable labels, molecules, and / or moieties may include those that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as fluorescence, chemiluminescence, or chemiluminescence, or any other suitable means. Detectable labels used in the methods described herein may be primary labels (where the label contains a moiety that is directly detectable or that produces a directly detectable moiety) or secondary labels (where the detectable label binds to another moiety to produce a detectable signal, as is common in immunological labeling using secondary and tertiary antibodies). Detectable labels may be linked to the reagent by covalent or non-covalent means. Alternatively, detectable labels may be linked by directly labeling a molecule that achieves binding to the reagent, for example, through a ligand-receptor binding pair arrangement or other such specific recognition molecule. Detectable labels include, but are not limited to, radioisotopes, bioluminescent compounds, chromophores, antibodies, chemiluminescent compounds, fluorescent compounds, metal chelates, and enzymes.

[0100] In other embodiments, the detection reagent is labeled with a fluorescent compound. When a fluorescently labeled reagent is exposed to light of the appropriate wavelength, its presence can be detected by fluorescence. In some embodiments of any of the above aspects, the detectable label can be a fluorescent dye molecule, or fluorophore, including, but not limited to, fluorescein, phycoerythrin, phycocyanin, o-phthalaldehyde, fluorescamine, Cy3™, Cy5™, allophycocyanin, Texas Red, peridinin chlorophyll, cyanin, tandem conjugates such as phycoerythrin-Cy5™, green fluorescent protein, rhodamine, fluorescein isothiocyanate (FITC) and Oregon Green™, rhodamine and derivatives (e.g., Texas Red and tetrarhodamine isothiocyanate (TRITC)), biotin, phycoerythrin, AMCA, CyDyes™, 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; 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; BODIPY dyes and quinoline dyes. In some embodiments of any of the above aspects, the detectable label is 3 H, 125 I, 35 S, 14 C. 32 P, and 33In some embodiments of any of the above aspects, the detectable label may be an enzyme, including, but not limited to, horseradish peroxidase and alkaline phosphatase. Enzyme labels can generate, for example, a chemiluminescent, color, or fluorescent signal. Enzymes contemplated for use in detectably labeling antibody reagents include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase. In some embodiments of any of the preceding aspects, the detectable label is a chemiluminescent label, including but not limited to, lucigenin, luminol, luciferin, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts, and oxalate esters. In some embodiments of any of the preceding aspects, the detectable label can be a spectral colorimetric label, including but not limited to, colloidal gold, or colored glass or plastic (e.g., polystyrene, polypropylene, and latex) beads.

[0101] In some embodiments of any of the above aspects, the detection reagent can be labeled with a detectable tag, such as c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS, or biotin. Other detection systems can also be used, such as the biotin-streptavidin system, in which an antibody immunoreactive with (i.e., specific for) the biomarker of interest is biotinylated. The amount of biotinylated antibody bound to the biomarker is measured using a streptavidin-peroxidase conjugate and a chromogenic substrate. Such streptavidin-peroxidase detection kits are commercially available, for example, from DAKO (Carpinteria, Calif.). The reagent can also be 152 They can also be detectably labeled with fluorescent metals such as Eu or other metals of the lanthanide series, which can be attached to the reagent using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0102] The detection method(s) used will depend on the particular detectable label used in the readout molecule. In an exemplary embodiment, chromosomes and / or chromosomal regions having one or more oligonucleotide tags (e.g., Oligopaints) and / or readout molecules bound thereto can be selected and / or screened using a microscope, a spectrophotometer, a tube or plate luminometer, X-ray film, a scintillator, a fluorescence activated cell sorting (FACS) machine, a microfluidics machine, or the like.

[0103] In some embodiments of any of the preceding aspects, the detection molecule comprises a fluorophore or fluorescent compound. Systems and devices for measuring fluorescence are well known in the art. Fluorescence measurements require a light source that emits light comprising an appropriate absorption or excitation wavelength. The absorption or excitation wavelength of the compounds described herein is about 300-800 nm. In some embodiments of any of the preceding aspects, the light source emits light comprising, consisting essentially of, or consisting of wavelengths between 300 and 870 nm. When the light contacts the sample, it excites electrons of certain substances in the sample, also known as fluorophores, causing the substances to emit light in the form of fluorescence (emission).

[0104] The emitted light is then detected by a system or device that measures fluorescence. In some embodiments, the system or device can include a filter or monochromator so that only light of the desired wavelength reaches the detector of the system or device. In some embodiments of any of the above aspects, the system or device is configured to detect light comprising, consisting essentially of, or consisting of wavelengths between 300 and 800 nm. In some embodiments of any of the above aspects, the system or device is configured to detect light comprising, consisting essentially of, or consisting of wavelengths between 300 and 800 nm. Suitable systems and equipment are commercially available and may include, for example, the 20 / 30 PV™ Microspectrometer or 508 PV™ Microscope Spectrometer manufactured by CRAIC (San Dimas, CA), the Duetta™, FluoroMax™, Fluorolog™, QuantaMaster 8000™, DeltaFlex™, DeltaPro, or Nanolog™ manufactured by Horiba (Irvine, CA), or the SP8 Lightning™, SP8 Falcon™, SP8 Dive™, TCS SPE™, HCS A™, or TCS SP8 X™ manufactured by Leica (Buffalo Grove, IL).

[0105] In some embodiments of any of the above aspects, fluorescence microscopy can be used to detect and record the results of in situ hybridization using routine methods known in the art. Alternatively, digital (computer-implemented) fluorescence microscopy with image processing can be used. Two well-known systems for imaging FISH of chromosomes with multiple color labels include multiplex-FISH (M-FISH) and spectral karyotyping (SKY). For reviews of methods for painting chromosomes and detecting painted chromosomes, see Schrock et al. (1996) Science 273:494; Roberts et al. (1999) Genes Chrom. Cancer 25:241; Fransz et al. (2002) Proc. Natl. Acad. Sci. USA 99:14584; Bayani et al. (2004) Curr. Protocol. Cell Biol. 22.5.1-22.5.25; Danilova et al. (2008) Chromosoma 117:345; U.S. Patent No. 6,066,459; and the FISH TAG™ DNA Multicolor Kit instructions (Molecular probes, Inc.).

[0106] In an exemplary embodiment, images of the fluorescently labeled chromosomes are detected and recorded using a computerized imaging system, such as the Applied Imaging CytoVision™ system (Applied Imaging Corporation, Santa Clara, Calif.) with modifications (e.g., software, Chroma 84000 filter set, and intensifying filter wheel). Other suitable systems include computerized imaging systems using a cooled CCD camera (Photometrics, NU200 series with Kodak KAF 1400 CCD) connected to a Zeiss Axiophot microscope, and images are processed as described in Ried et al. (1992) Proc. Natl. Acad. Sci. USA 89:1388. Other suitable imaging and analysis systems are described in Schrock et al. supra; and Speicher et al. (1996) Nature Genet. 12:368. In some embodiments of any of the above aspects, the oligonucleotide tags (e.g., Oligopaints) are visualized by super-resolution microscopy (e.g., Stochastic Optical Reconstruction Microscopy (STORM) imaging).

[0107] The in situ hybridization methods described herein can be performed on a variety of biological or clinical samples, on cells in any (or all) stages of the cell cycle (e.g., mitosis, meiosis, interphase, G0, G1, S and / or G2). Examples include any kind of cell culture, animal or plant tissue, peripheral blood lymphocytes, buccal smears, touch preparations made from uncultured primary tumors, cancer cells, bone marrow, cells obtained from biopsies or cells in body fluids (e.g., blood, urine, sputum, etc.), cells from amniotic fluid, cells from maternal blood (e.g., fetal cells), cells from testes and ovaries, etc. Samples are prepared for the assays of the present invention using conventional techniques, which usually depend on the source from which the sample or specimen is taken. These examples should not be construed as limiting the types of samples that can be applied to the methods and / or compositions described herein.

[0108] Hybridization of the oligonucleotide tags of the invention (e.g., Oligopaints) to target chromosomal sequences can be accomplished by standard in situ hybridization (ISH) techniques (see, e.g., Gall and Pardue (1981) Meth. Enzymol. 21:470; Henderson (1982) Int. Review of Cytology 76:1). In general, ISH involves the following major steps: (1) fixation of the biological structure to be analyzed (e.g., chromosome spread); (2) prehybridization treatment of the biological structure to increase the accessibility of the target DNA (e.g., denaturation by heat or alkali); (3) optional prehybridization treatment to reduce nonspecific binding (e.g., by blocking the hybridization ability of repetitive sequences); (4) hybridization of a mixture of nucleic acids to the nucleic acids in the biological structure or tissue; (5) posthybridization washes to remove nucleic acid fragments not bound by hybridization; and (6) detection of hybridized labeled oligonucleotides (e.g., hybridized oligonucleotide tags (e.g., Oligopaint)). The reagents used in each of these steps and the conditions of their use vary depending on the particular situation. For example, the recognition domains described herein can be designed to avoid repetitive sequences, so that step 3 is not necessarily required. Hybridization conditions are also described in U.S. Pat. No. 5,447,841. It will be appreciated that numerous variations of in situ hybridization protocols and conditions are known and can be used with the present invention by one of skill in the art following the guidance provided herein.

[0109] The term "hybridization" as used herein refers to the process by which two single-stranded polynucleotides bind non-covalently to form a stable double-stranded polynucleotide. The term "hybridization" may also refer to triplex hybridization. The resulting (usually) double-stranded polynucleotide is a "hybrid" or "duplex." "Hybridization conditions" will typically include a salt concentration of less than about 1M, more typically less than about 500 mM, and even more typically less than about 200 mM. Hybridization temperatures can be as low as 5°C, but are typically greater than 22°C, more typically greater than about 30°C, and often greater than about 37°C. Hybridization is usually performed under stringent conditions, i.e., conditions under which the probe hybridizes to its target subsequence. Stringent conditions are sequence-dependent and will vary from situation to situation. Longer fragments may require higher hybridization temperatures for specific hybridization. Hybridization stringency can be influenced by other factors, such as the base composition and length of the complementary strand, the presence of organic solvents, and the degree of base mismatching, so the combination of parameters is more important than any one absolute measure. Typically, stringent conditions are selected to be about 5°C lower than the Tm of a particular sequence at a defined ionic strength and pH. Exemplary stringent conditions include a salt concentration of at least 0.01M to 1M Na ion (or other salts) at pH 7.0-8.3 and a temperature of at least 25°C. For example, conditions of 5xSSPE (750mM NaCl, 50mM Na phosphate, 5mM EDTA, pH 7.4) and a temperature of 25-30°C are suitable for allele-specific probe hybridization.For stringent conditions, see, e.g., Sambrook, Fritsche and Maniatis, Molecular Cloning A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press (1989) and Anderson Nucleic Acid Hybridization, 1st Ed., BIOS Scientific Publishers Limited (1999). "Specifically hybridizes to" or "specifically hybridizes to" or similar phrases mean that a molecule will bind, duplex, or hybridize substantially to, or only to, a particular nucleotide sequence when present in a complex mixture (e.g., total DNA or RNA in a cell) under stringent conditions.

[0110] As used herein, the term "specific binding" refers to a chemical interaction between two molecules, compounds, cells and / or particles, where a first entity binds to a second target entity with greater specificity and affinity than it binds to a third, non-target entity. In some embodiments, specific binding can refer to an affinity of the first entity for the second target entity that is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times, or more, greater than its affinity for the third, non-target entity. A reagent specific for a given target is one that exhibits specific binding to that target under the conditions of the assay employed.

[0111] The term "oligonucleotide" as used herein is intended to include, but is not limited to, single-stranded DNA or RNA molecules, typically prepared by synthetic means. The nucleotides of the present invention are typically naturally occurring nucleotides, such as those derived from adenosine, guanosine, uridine, cytidine, and thymidine. When an oligonucleotide is referred to as "double-stranded", as will be understood by those skilled in the art, a pair of oligonucleotides are present in a hydrogen-bonded helical arrangement, which is generally associated with, for example, DNA. In addition to the 100% complementary form of double-stranded oligonucleotide, the term "double-stranded" as used herein is also meant to include forms that include structural features such as bulges and loops (see Stryer, Biochemistry, Third Ed. (1988); incorporated herein by reference in its entirety for all purposes). The term "polynucleotide" as used herein is intended to include, but is not limited to, two or more oligonucleotides that are joined together (e.g., by hybridization, ligation, polymerization, etc.).

[0112] Nucleic acid and ribonucleic acid (RNA) molecules can be separated from a particular biological sample using any of many techniques known in the art, and the particular separation technique selected is appropriate for the particular biological sample.For example, freeze-thaw and alkaline lysis techniques can be useful for obtaining nucleic acid molecules from solid materials; heating and alkaline lysis techniques can be useful for obtaining nucleic acid molecules from urine; proteinase K extraction can be used to obtain nucleic acid from blood (Roiff, A et al. PCR: Clinical Diagnostics and Research, Springer (1994)).

[0113] In certain exemplary embodiments, universal primers can be used to amplify nucleic acid sequences, such as oligonucleotide tags (e.g., Oligopaints). The term "universal primers" refers to a set of primers (e.g., forward and reverse primers) that can be used for chain extension / amplification of multiple polynucleotides, e.g., the primers hybridize to a site common to multiple polynucleotides. For example, a universal primer can be used for amplification of all or essentially all polynucleotides contained in a single pool. In some embodiments of any of the above aspects, the forward primer and the reverse primer have the same sequence. In some embodiments of any of the above aspects, the sequence of the forward primer is different from the sequence of the reverse primer. In still other aspects, multiple universal primers are provided, e.g., tens, hundreds, thousands, or more.

[0114] In some embodiments of any of the above aspects, the universal primer can be a temporary primer that can be removed after amplification by enzymatic or chemical cleavage. In some embodiments of any of the above aspects, the universal primer can be a temporary primer that can be removed after amplification by enzymatic or chemical cleavage. In other embodiments, the universal primer can include a modification that becomes incorporated into the polynucleotide molecule during chain extension. Exemplary modifications include, for example, 3' or 5' end caps, labels (e.g., fluorescein), or tags (e.g., tags that facilitate immobilization or isolation of polynucleotides, such as biotin).

[0115] In some embodiments of any of the above aspects, the methods disclosed herein include amplification of an oligonucleotide sequence, such as an oligonucleotide tag (e.g., an Oligopaint), etc. Amplification methods can include contacting a nucleic acid with one or more primers that specifically hybridize to the nucleic acid (e.g., universal primers) under conditions that promote hybridization and chain extension. Exemplary methods for amplifying nucleic acids include: polymerase chain reaction (PCR) (see, e.g., Mullis et al. (1986) Cold Spring Harb. Symp. Quant. Biol. 51 Pt 1:263 and Cleary et al. (2004) Nature Methods 1:241; and U.S. Pat. Nos. 4,683,195 and 4,683,202), anchored PCR, RACE PCR, ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc. Natl. Acad. Sci. USA 91:360-364), self sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 91:360-364), and the like. 87:1874), transcription amplification systems (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173), Q-beta replicase (Lizardi et al. (1988) BioTechnology 6:1197), recursive PCR (Jaffe et al. (2000) J. Biol. Chem. 275:2619; and Williams et al. (2002) J. Biol. Chem. 277:7790), the amplification methods described in U.S. Patent Nos. 6,391,544, 6,365,375, 6,294,323, 6,261,797, 6,124,090, and 5,612,199, or other nucleic acid amplification methods using techniques well known to those of skill in the art.In an exemplary embodiment, the methods disclosed herein utilize PCR amplification.

[0116] Generally, PCR refers to a method of gene amplification, which includes (i) sequence-specific hybridization of primers to specific genes or sequences in a nucleic acid sample or library; (ii) subsequent amplification with multiple rounds of annealing, extension, and denaturation using a thermostable DNA polymerase; and (iii) screening of PCR products for bands of the correct size. The primers used are oligonucleotides of sufficient length and appropriate sequence to cause the initiation of polymerization, i.e., each primer is specifically designed to be complementary to a strand of the genomic locus to be amplified. In another embodiment, the mRNA level of the gene expression products described herein can be measured by reverse transcription (RT) PCR and by quantitative RT-PCR (QRT-PCR) or real-time PCR. The methods of RT-PCR and QRT-PCR are well known in the art.

[0117] In some embodiments of any of the above aspects, the oligonucleotide tags (e.g., Oligopaints) are not necessarily amplified (e.g., via PCR and / or universal priming regions). In some embodiments of any of the above aspects, the described oligonucleotide tags (e.g., Oligopaints) can be synthesized de novo and used "direct from the tube". Methods of de novo synthesis of oligonucleotides are well known to those of skill in the art. As used herein, "oligonucleotide synthesis" refers to the chemical synthesis of relatively short fragments of nucleic acid with defined chemical structures. As non-limiting examples, methods of oligonucleotide synthesis include phosphoramidite solid phase synthesis, phosphoramidite synthesis, phosphodiester synthesis, phosphotriester synthesis, or phosphite triester synthesis. See, e.g., Beaucage et al. Tetrahedron Volume 48, Issue 12, 20 March 1992, Pages 2223-2311; Caruthers, J Biol Chem. 2013 Jan 11, 288(2):1420-7. In some embodiments, each oligonucleotide is synthesized separately. In some embodiments, the entire oligonucleotide set is synthesized in one reaction. In some embodiments, a subset of the entire oligonucleotide set is synthesized in one reaction. In some embodiments, the entire oligonucleotide set is synthesized in multiple separate reactions. In some embodiments, the reaction products are isolated, for example, by high performance liquid chromatography (HPLC), to obtain the desired oligonucleotides in high purity.

[0118] In certain exemplary embodiments, kits are provided. As used herein, the term "kit" refers to any delivery system for providing oligonucleotide tags (e.g., Oligopaints), readout molecules, and / or reagents for carrying out the methods described herein. In the context of an assay, such a kit includes a system for storing, transporting, or providing reaction reagents (e.g., enclosures providing one or more of oligonucleotide tags, readout molecules, primers (e.g., primers specific for all oligonucleotide tags present and / or one or more subsets of primers specific for one or more subsets of oligonucleotide tag sequences), primers with one or more detectable and / or retrievable labels attached), supports to which oligonucleotides are attached (e.g., microarrays, pallets, etc.), and / or auxiliary materials (e.g., enclosures providing buffers, instructions for carrying out the assays described herein, etc.) from one location to another. For example, the kit includes one or more enclosures (e.g., boxes) that include reaction reagents and / or auxiliary materials associated with the assays described herein. In one aspect, the kit of the invention includes oligonucleotide tags (e.g., Oligopaints) specific to one or more target nucleotide sequences (e.g., chromosomes) or one or more regions (e.g., sub-chromosomal regions) of one or more target nucleotide sequences. In one aspect, the kit of the invention includes readout molecules specific to one or more oligonucleotide tags (e.g., Oligopaints). In another aspect, the kit includes one or more primer sequences, one or more supports to which a plurality of synthetic oligonucleotide sequences are attached, and one or more detectable and / or retrievable labels. Such contents can be delivered to an intended recipient together or separately. For example, a first container includes primer sequences for use in an assay and a second container includes a support to which a plurality of synthetic oligonucleotide sequences are attached.

[0119] In some embodiments of any of the foregoing aspects, the kit provides one or more arrays and / or palettes to which a plurality of specific oligonucleotide sequences (e.g., oligonucleotide tags (e.g., Oligopaints) and / or readout molecules) are attached. In some embodiments of any of the foregoing aspects, the arrays and / or palettes provide a plurality of oligonucleotide tag sequences (e.g., Oligopaints) specific to a set of binding patterns on a genome (e.g., the human genome). In some embodiments of any of the foregoing aspects, the arrays or palettes are specific to a set of chromosomal abnormalities (e.g., one or more of translocations, insertions, inversions, deletions, duplications, rearrangements, aneuploidies, polyploidies, complex rearrangements, and telomere losses) associated with one or more diseases described herein. In some embodiments of any of the foregoing aspects, the kits described herein are particularly suitable for diagnostic and / or prognostic uses (e.g., for patient diagnosis and / or prognosis, prenatal diagnosis and / or prognosis, etc.) for detecting one or more diseases described herein in a clinical setting (e.g., a hospital, clinic, doctor's office, clinical laboratory, research laboratory, etc.).

[0120] In some embodiments of any of the preceding aspects, the kit provides instructions for amplifying a plurality of specific oligonucleotide tag sequences (e.g., Oligopaints) provided in the kit. In some embodiments of any of the preceding aspects, the kit provides instructions for detectably and / or retrievably labeling one or more target nucleic acid sequences (e.g., one or more chromosomes or sub-chromosomal regions) with the amplified oligonucleotide tags (e.g., Oligopaints). In some embodiments of any of the preceding aspects, the kit provides instructions for detectably and / or retrievably labeling one or more target nucleic acid sequences (e.g., one or more chromosomes or sub-chromosomal regions) with the oligonucleotide tags (e.g., Oligopaints) and readout molecules. In some embodiments of any of the preceding aspects, the kit provides instructions for effectively removing one or more of the plurality of specific oligonucleotide tag sequences (e.g., Oligopaints) during the amplification step by including one or more unlabeled amplification primers that hybridize to one or more oligonucleotide sequences one wishes to remove, such that the one or more target nucleic acid sequences are no longer detectably and / or retrievably labeled.

[0121] In some embodiments of any of the above aspects, the system and method described herein can be implemented in any type of hardware and / or software, which may be a pre-programmed general-purpose computer device.For example, the system can be implemented using a server, a personal computer, a portable computer, a thin client, or any suitable device.The present disclosure and / or its components may be a single device in a single location, or multiple devices in a single or multiple locations, and the multiple devices are connected together using any communication medium, such as electrical cable, fiber optic cable, or wirelessly, using a suitable communication protocol.

[0122] It should also be noted that the present disclosure is illustrated and described herein as having a number of modules performing specific functions. It should be understood that these modules are merely illustrated generally based on their functionality for clarity purposes only, and do not necessarily represent specific hardware or software. In this regard, these modules may be hardware and / or software implemented to substantially perform the specific functions described. Furthermore, these modules may be combined together or divided into additional modules within the present disclosure based on the specific functions desired. Thus, the present disclosure should not be construed as limiting the present invention, but should be understood as merely illustrating one example of its implementation.

[0123] A computing system may include clients and servers. Clients and servers are usually remote from each other and typically interact through a communications network. The relationship of client and server arises by virtue of computer programs running on the respective computers and having a client-server relationship to each other. In some implementations, a server sends data (e.g., HTML pages) to a client device (e.g., to display the data to a user interacting with the client device and receive user input from the user). Data created at the client device (e.g., a result of a user interaction) can be received at the server from the client device.

[0124] Implementations of the subject matter described herein may be performed on a computing system that includes a back-end component, e.g., as a data server, includes a middleware component, e.g., an application server, includes a front-end component, e.g., a client computer having a graphical user interface or web browser through which a user can interact with implementations of the subject matter described herein, or includes any combination of one or more such back-end, middleware, or front-end components. The components of the system may be interconnected by any form or medium of digital data communication, e.g., a communications network. Examples of communications networks include local area networks ("LANs") and wide area networks ("WANs"), internetworks (e.g., the Internet), and peer-to-peer networks (e.g., ad-hoc peer-to-peer networks).

[0125] Implementation of the subject matter and operations described herein can be performed in digital electronic circuitry, or in computer software, firmware, or hardware, including the structures disclosed herein and their structural equivalents, or any combination of one or more of them. Implementation of the subject matter described herein can be performed as one or more computer programs, i.e., as one or more modules of computer program instructions encoded on a computer storage medium for execution by or to control the operation of a data processing device. Alternatively or additionally, the program instructions can be encoded into an artificially generated propagated signal, such as a machine-generated electrical, optical, or electromagnetic signal, which is generated to encode information for transmission to a receiving device suitable for execution by a data processing device. The computer storage medium can be or be included in a computer readable storage device, a computer readable storage substrate, a random access or serial access memory array or device, or any combination of one or more of them. Furthermore, although the computer storage medium is not a propagating signal, the computer storage medium can be the source or destination of computer program instructions encoded into an artificially generated propagated signal. The computer storage medium may also be, or be contained in, one or more separate physical components or media (eg, multiple CDs, disks, or other storage devices).

[0126] The operations described herein may be performed as operations performed by a "data processing apparatus" on data stored in one or more computer-readable storage devices or received from other sources.

[0127] The term "data processing apparatus" encompasses all kinds of apparatus, devices, and machines for processing data, including, for example, programmable processors, computers, systems on chips, or composites or combinations of the above. The apparatus may include special purpose logic circuitry, such as a field programmable gate array (FPGA) or an application specific integrated circuit (ASIC). In addition to hardware, the apparatus may also include code that creates an execution environment for the computer program in question, such as code that constitutes processor firmware, protocol stacks, database management systems, operating systems, cross-platform runtime environments, virtual machines, or any combination thereof. The apparatus and execution environments may implement infrastructures for a variety of different computing models, such as infrastructures for web services, distributed computing, and grid computing.

[0128] A computer program (also known as a program, software, software application, script, or code) can be written in any form of programming language, such as compiled or interpreted languages, declarative or procedural languages, and can be deployed in any form, for example as a stand-alone program or as a module, component, subroutine, object, or other unit suitable for use in a computing environment. A computer program may correspond to a file in a file system, but this need not be the case. A program can be stored as part of a file that holds other programs or data (e.g., one or more scripts stored in a markup language document), in a single file dedicated to the program in question, or in several coordinating files (e.g., files that store one or more modules, subprograms, or portions of code). A computer program can be deployed to be executed on one computer or on several computers located at one site or distributed across several sites and interconnected by a communication network.

[0129] The processes and logic flows described herein may be performed by one or more programmable processors executing one or more computer programs to take action by operating on input data to generate output. The processes and logic flows may also be performed by, and devices may be implemented as, special purpose logic circuitry, such as an FPGA (field programmable gate array) or an ASIC (application specific integrated circuit).

[0130] Processors suitable for executing computer programs include, by way of example, both general purpose and special purpose microprocessors, as well as any one or more processors of any kind of digital computer. Generally, a processor receives instructions and data from a read-only memory or a random access memory, or both. The essential elements of a computer are a processor for performing operations according to the instructions, and one or more storage devices for storing instructions and data. Generally, a computer will include or be operatively coupled to one or more mass storage devices for storing data, such as magnetic disks, magneto-optical disks, or optical disks, for receiving data, transferring data, or both. However, a computer need not have such devices. Furthermore, a computer can be incorporated into another device, such as a mobile phone, a personal digital assistant (PDA), a portable audio or video player, a home game console, a Global Positioning System (GPS) receiver, or a portable storage device (e.g., a Universal Serial Bus (USB) flash drive), to name just a few. Devices suitable for storing computer program instructions and data include all forms of non-volatile memory, media and memory devices, including, by way of example, semiconductor memory devices, such as EPROM, EEPROM, and flash memory devices; magnetic disks, such as internal hard disks or removable disks; magneto-optical disks; and CD ROM and DVD-ROM disks. The processor and the memory can be supplemented by, or incorporated in, special purpose logic circuitry.

[0131] For convenience, the meanings of some terms and phrases used in the specification, examples and the appended claims are provided below. Unless otherwise specified or implied from the context, the following terms and phrases shall have the meanings given below. The definitions are provided to assist in the description of certain embodiments and are not intended to limit the claimed invention; since the scope of the present invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. In the event of an apparent discrepancy between the use of a term in the art and its definition provided herein, the definition provided herein shall prevail.

[0132] For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

[0133] The term "chromosome" as used herein refers to the genetic structure that carries heredity in living cells, including DNA, proteins, RNA, and other associated factors. The conventional international system for identifying and numbering the chromosomes of the human genome is used herein. The size of individual chromosomes can vary both within a multi-chromosomal genome and from genome to genome. Chromosomes can be obtained from any species. Chromosomes can be obtained from adult subjects, juvenile subjects, infant subjects, pre-born subjects (e.g., from a fetus, e.g., via prenatal testing such as amniocentesis, chorionic villus sampling, or directly from a fetus, e.g., during fetal surgery), from biological samples (e.g., biological tissues, fluids, or cells (e.g., sputum, blood, blood cells, tissue or fine needle biopsy samples, urine, cerebrospinal fluid, peritoneal fluid, and pleural fluid, or cells obtained therefrom)), or from cell culture samples (e.g., primary cells, immortalized cells, partially immortalized cells, etc.). In certain exemplary embodiments, the one or more chromosomes are from the genera Homo, Drosophila, Caenorhabiditis, Danio, Cyprinus, Equus, Canis, Ovis, Ocorynchus, Salmo, Bos, Sus scrofa, and the like. The species may be obtained from one or more genera, including, but not limited to, Brassica napus, Brassica napus, Brassica rapa ...

[0134] As used herein, the term "chromosome banding" refers to differential staining of chromosomes resulting in a pattern of transverse bands of distinguishable (e.g., differently or alternatingly colored) regions characteristic of individual chromosomes or chromosomal regions (i.e., a "banding pattern"). Classical banding techniques include G banding (Giemsa staining), Q banding (quinacrine mustard staining), R banding (reverse Giemsa), and C banding (centromere banding).

[0135] The term "karyotype" as used herein refers to the chromosomal characteristics of an individual cell, cell line, or genome of a given species of organism, defined by both the number and morphology of chromosomes. Karyotype can refer to various chromosomal rearrangements, including but not limited to translocations, insertion translocations, inversions, deletions, duplications, translocations, aneuploidies, complex rearrangements, telomere losses, and the like. Typically, karyotypes are presented as systematic arrangements of chromosomes in prophase or metaphase (or other condensed states) from photomicrographs or computer-generated images. Interphase chromosomes may also be examined.

[0136] The term "chromosomal aberration" or "chromosome abnormality" as used herein refers to a deviation between the structure of a chromosome or karyotype of interest and a normal (i.e., aberrant) homologous chromosome or karyotype. The deviation may be one base pair or many base pairs. The term "normal" or "abnormal" when referring to a chromosome or karyotype refers to the karyotype or banding pattern found in healthy individuals of a particular species and sex. Chromosomal abnormalities are numerical or structural in nature and include, but are not limited to, aneuploidy, polyploidy, inversion, translocation, deletion, duplication, and the like. Chromosomal abnormalities may be correlated with the presence of a pathological condition or a predisposition to develop a pathological condition. Chromosomal abnormalities may also refer to changes that are not associated with a disease, disorder, and / or phenotypic change. Such abnormalities may be rare or present at a low frequency (e.g., a few percent of the population (e.g., polymorphisms)).

[0137] Disorders associated with one or more chromosomal abnormalities include, but are not limited to, the following: autosomal abnormalities (e.g., trisomy (Down syndrome (chromosome 21)), Edwards syndrome (chromosome 18), Patau syndrome (chromosome 13), trisomy 9, Walkanyi syndrome (chromosome 8), trisomy 22 / cat eye syndrome, trisomy 16); monosomies and / or deletions (Wolf-Hirschhorn syndrome (chromosome 4), Cri du chat / chromosome 5q deletion syndrome (chromosome 5), Williams syndrome (chromosome 7), Jacobsen syndrome (chromosome 11), Miller-Dicker syndrome / Smith-Magenis syndrome (chromosome 17), Di George syndrome (chromosome 22), genomic imprinting (Angelman syndrome / Prader-Willi syndrome (chromosome 15))); X / Y-linked abnormalities (e.g., monosomy (Turner syndrome (XO), trisomy or tetrasomy MIS and / or other karyotypes or mosaicism (Klinefelter syndrome (47(XXY)), 48(XXYY), 48(XXXY), 49(XXXYY), 49(XXXXY), triple X syndrome (47(XXX)), 48(XXXX), 49(XXXXX), 47(XYY), 48(XYYY, 49(XYYYY), 46(XX / XY)); translocations (e.g., leukemia or lymphoma (e.g., lymphoid (e.g., Burkitt lymphoma t(8 MYC;14 IGH), follicular lymphoma t(14 IGH;18 BCL2), mantle cell lymphoma / multiple myeloma t(11 CCND1;14 IGH), anaplastic large cell lymphoma t(2 ALK;5 NPM1), acute lymphoblastic leukemia) or myeloid (e.g. Philadelphia chromosome t(9 ABL;22 BCR), acute myeloblastic leukemia with maturation t(8 RUNX1T1;21 RUNX1), acute promyelocytic leukemia t(15 PML,17 RARA), acute megakaryoblastic leukemia t(1 RBM15;22 MKL1))) or other (e.g. Ewing sarcoma t(11 FiI1;22 EWS), synovial sarcoma t(x SYT;18 SSX), dermatofibrosarcoma protuberans t(17 COL1A1;22 PDGFB), myxoid liposarcoma t (12 DDIT3; 16 FUS), desmoplastic small round cell tumor t (11 WT1;22 EWS), alveolar rhabdomyosarcoma t(2 PAX3;13 FOXO1)t(1 PAX7;13 FOXO1)); gonadal dysgenesis (e.g., mixed gonadal dysgenesis, XX gonadal dysgenesis); and other abnormalities (e.g., fragile X syndrome, uniparental disomy). Disorders associated with one or more chromosomal abnormalities also include, but are not limited to: Beckwith-Wiedemann syndrome, branchio-oto-renal syndrome, cri-cry syndrome, de Lange syndrome, holoprosencephaly, Rubinstein-Taybi syndrome, and WAGR syndrome.;

[0138] Diseases associated with one or more chromosomal abnormalities also include cell proliferative disorders (e.g., cancer). The term "cell proliferative disorders" as used herein includes disorders characterized by unwanted or inappropriate proliferation of one or more cell subsets in a multicellular organism. The term "cancer" refers to various types of malignant neoplasms, many of which can invade surrounding tissues and metastasize to different sites (see, e.g., PDR Medical Dictionary, 1st ed., 1995). The terms "neoplasm" and "tumor" refer to abnormal tissues that grow by cell proliferation more rapidly than normal and continue to grow after the stimuli that initiated the proliferation are removed (see, e.g., PDR Medical Dictionary, 1st ed., 1995). Such abnormal tissues show partial or complete lack of structural organization and functional coordination with normal tissues and can be either benign (i.e., benign tumors) or malignant (i.e., malignant tumors).

[0139] Diseases associated with one or more chromosomal abnormalities also include brain diseases, including, but not limited to, acoustic neuroma, acquired brain injury, Alzheimer's disease, amyotrophic lateral sclerosis, aneurysm, aphasia, arteriovenous malformation, attention deficit hyperactivity disorder, autism, Batten disease, Behcet's disease, blepharospasm, brain tumors, cerebral palsy, Charcot-Marie-Tooth disease, Chiari malformation, CIDP, non-Alzheimer's dementia, autonomic neuropathy, dyslexia, movement disorder, dystonia, epilepsy, essential tremor, and Friedreich's ataxia. encephalopathy, Gaucher's disease, Guillain-Barre syndrome, headache, migraine, Huntington's disease, hydrocephalus, Meniere's disease, motor neuron disease, multiple sclerosis, muscular dystrophy, myasthenia gravis, narcolepsy, Parkinson's disease, peripheral neuropathy, progressive supranuclear palsy, restless legs syndrome, Rett syndrome, schizophrenia, Shy-Drager syndrome, stroke, subarachnoid hemorrhage, Sydenham syndrome, Tay-Sachs disease, Tourette's syndrome, transient ischemic attack, transverse myelitis, trigeminal neuralgia, tuberous sclerosis, and von Hippel-Lindau syndrome.

[0140] The terms "reduction", "reduction", "decreasing" or "suppression" as used herein all refer to a decrease by a statistically significant amount. In some embodiments, "reduction", "reduction" or "decreasing" or "suppression" typically refers to a decrease of at least 10% compared to a reference level (e.g., in the absence of a given treatment or drug), and can include, for example, a decrease of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. As used herein, "reduction" or "suppression" does not encompass a complete suppression or reduction when compared to a reference level. "Complete suppression" refers to a 100% suppression compared to a reference level. The decrease can preferably be to a level accepted as being within the normal range for a given disease-free individual.

[0141] As used herein, the terms "increase," "increase," "enhance," or "activate" all mean an increase by a statistically significant amount. In some embodiments, the terms "increase," "increase," "enhance," or "activate" can mean an increase of at least 10% compared to a base level, e.g., an increase of at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or up to and including 100% compared to a base level, or any increase between 10-100%, or an increase of at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold compared to a base level, or any increase between 2-fold and 10-fold, or more. In the context of a marker or condition, an "increase" is a statistically significant increase in such level.

[0142] In some embodiments of any of the above aspects, the reference sample or level is the level of the sample or the sample itself before contacting with the composition described herein. In some embodiments of any of the above aspects, the reference sample or level is the sample or level of the composition described in the present invention before contacting with the sample. In some embodiments of any of the above aspects, the reference can be a sample contacted with a composition that does not include a detection molecule. In some embodiments of any of the above aspects, the reference can be a sample contacted with a composition that includes a recognition domain that is not specific to the sample. In some embodiments of any of the above aspects, the reference can also be a level obtained from a control sample that is a pooled sample of control individuals, or a value or range of values ​​based thereon.

[0143] As used herein, "subject" refers to a human or an animal. Typically, an animal is a vertebrate, such as a primate, a rodent, a livestock animal, or a game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Domestic and game animals include cattle, horses, pigs, deer, bison, buffalo, felines, such as house cats, canines, such as dogs, foxes, wolves, birds, such as chickens, emus, ostriches, and fish, such as trout, catfish, and salmon. In some embodiments, the subject is a mammal, such as a primate, such as a human. As used herein, the terms "individual," "patient," and "subject" are used interchangeably.

[0144] Preferably, the subject is a mammal, which may be, but is not limited to, a human, non-human primate, mouse, rat, dog, cat, horse, or cow.

[0145] As used herein, the terms "protein" and "polypeptide" are used interchangeably to refer to a series of amino acid residues linked together by peptide bonds between the α-amino and carboxy groups of adjacent residues. The terms "protein" and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylation, glycation, glycosylation, etc.) and amino acid analogs, regardless of their size or function. Although "protein" and "polypeptide" are often used in reference to relatively large polypeptides, while the term "peptide" is often used in reference to small polypeptides, the use of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to gene products and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments, and other equivalents, variants, fragments, and analogs of the foregoing.

[0146] In various aspects described herein, it is further intended to encompass any variant (naturally occurring or not), allele, homolog, conservatively modified variant, and / or conservatively substituted variant of the specific polypeptide described.With respect to amino acid sequences, as will be recognized by those skilled in the art, individual substitutions, deletions, or additions to nucleic acid, peptide, polypeptide, or protein sequences that modify a single amino acid or a small percentage of amino acids in the encoded sequence are "conservatively modified variants" where the modification results in the replacement of an amino acid with a chemically similar amino acid and retains the desired activity of the polypeptide.Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles consistent with the present disclosure.

[0147] A given amino acid can be replaced with a residue having similar physicochemical properties, for example, one aliphatic residue can be replaced with another (e.g., Ile, Val, Leu, or Ala for each other), or one polar residue can be replaced with another (e.g., between Lys and Arg, between Glu and Asp, or between Gln and Asn). Other such conservative substitutions, such as the substitution of entire regions with similar hydrophobic properties, are well known. Polypeptides containing conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that the desired activity, e.g., the activity and specificity of the native or reference polypeptide, is retained.

[0148] Amino acids can be grouped according to the similarity of their side chain properties (AL Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)):(1) nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Nonconservative substitutions entail exchanging a member of one of these classes for another class. Particular conservative substitutions include, for example, replacement of Ala with Gly or Ser; Arg with Lys; Asn with Gln or His; Asp with Glu; Cys with Ser; Gln with Asn; Glu with Asp; Gly with Ala or Pro; His with Asn or Gln; Ile with Leu or Val; Leu with Ile or Val; Lys with Arg, Gln or Glu; Met with Leu, Tyr or Ile; Phe with Met, Leu or Tyr; Ser with Thr; Thr with Ser; Trp with Tyr; Tyr with Trp; and / or Phe with Val, Ile or Leu.

[0149] In some embodiments, the polypeptides described herein (or nucleic acids encoding such polypeptides) can be functional fragments of one of the amino acid sequences described herein. As used herein, a "functional fragment" refers to a fragment or segment of a peptide that retains at least 50% of the activity of a wild-type reference polypeptide according to the assays described hereinafter. Functional fragments can include conservative substitutions of the sequences disclosed herein.

[0150] In some embodiments, the polypeptides described herein may be variants of the sequences described herein. In some embodiments, the variants are conservatively modified variants. Conservative substitution variants can be obtained, for example, by mutation of a natural nucleotide sequence. As used herein, a "variant" refers to a polypeptide that is substantially homologous to a natural or reference polypeptide, but has an amino acid sequence that differs from the amino acid sequence of the natural or reference polypeptide due to one or more deletions, insertions, or substitutions. A DNA sequence encoding a variant polypeptide includes sequences that encode a variant protein or fragment thereof that contains one or more nucleotide additions, deletions, or substitutions compared to the natural or reference DNA sequence, but retains activity. A wide variety of PCR-based site-directed mutagenesis approaches are known in the art and can be applied by those skilled in the art.

[0151] The amino acid or DNA sequence of the variant may be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to the native or reference sequence. The degree of homology (percent identity) between a native sequence and a mutant sequence can be determined by comparing the two sequences, for example, using freely available computer programs commonly used for this purpose on the World Wide Web (e.g., BLASTp or BLASTn with default settings).

[0152] Modification of the native amino acid sequence can be accomplished by any of a number of techniques known to those skilled in the art. Mutations can be introduced at specific loci, for example, by synthesizing oligonucleotides containing the mutated sequence (flanked on either side by restriction sites that allow ligation to fragments of the native sequence). After ligation, the resulting reconstituted sequence encodes an analog with the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis can be used to provide modified nucleotide sequences with specific codons modified by the required substitution, deletion, or insertion. Techniques for making such modifications are well established and include, for example, those disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and U.S. Patent Nos. 4,518,584 and 4,737,462, which are incorporated herein by reference in their entirety. Cysteine ​​residues that are not involved in maintaining the proper conformation of the polypeptide can also be substituted, typically with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine ​​bond(s) can be added to a polypeptide to improve the stability of the polypeptide or promote oligomerization.

[0153] The term "nucleic acid" or "nucleic acid sequence" as used herein refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or analogs thereof. A nucleic acid can be either single-stranded or double-stranded. A single-stranded nucleic acid can be one nucleic acid strand of a denatured double-stranded DNA. Alternatively, it can be a single-stranded nucleic acid that is not derived from double-stranded DNA. In some embodiments of any of the above aspects, the single-stranded nucleic acid is generated by in vitro transcription followed by reverse transcription. In some embodiments of any of the above aspects, the single-stranded nucleic acid is generated by exposure to a nicking endonuclease. In some embodiments of any of the above aspects, the single-stranded nucleic acid is synthesized de novo. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable DNA includes, for example, genomic DNA or cDNA. Suitable RNA includes, for example, mRNA.

[0154] The term "expression" refers to the cellular processes involved in the production of RNA and protein, and optionally secretion of protein, including, but not limited to, transcription, processing of the transcript, translation, and protein folding, modification and processing, as applicable. Expression can refer to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment(s) of the invention, and / or the translation of mRNA into a polypeptide.

[0155] In some embodiments, expression of the biomarker(s), target(s), or genes / polypeptides described herein is tissue specific. In some embodiments, expression of the biomarker(s), target(s), or genes / polypeptides described herein is global. In some embodiments, expression of the biomarker(s), target(s), or genes / polypeptides described herein is systemic.

[0156] "Expression product" includes RNA transcribed from a gene and polypeptides resulting from translation of mRNA transcribed from a gene. "Gene" refers to a nucleic acid sequence (DNA) that is transcribed into RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. A gene may or may not include regions preceding and following the coding region, such as 5' untranslated (5'UTR) or "leader" sequences and 3'UTR or "trailer" sequences, and intervening sequences (introns) between individual coding segments (exons).

[0157] In some embodiments, the methods described herein relate to measuring, detecting, or determining the level of at least one target molecule. The term "detection" or "measurement" as used herein means observing a signal, for example, from a probe, a label, or a target molecule, to indicate the presence of an analyte in a sample. Any method known in the art for detecting a particular label moiety can be used for detection. Exemplary detection methods include, but are not limited to, spectroscopic, fluorescent, photochemical, biochemical, immunochemical, electrical, optical, or chemical methods. In some embodiments of any of the above aspects, the measurement can be a quantitative observation.

[0158] In some embodiments of any of the above aspects, the polypeptide, nucleic acid, or cell described herein may be artificially modified. As used herein, "artificially modified" refers to an aspect that has been manipulated by the hand of man. For example, a polypeptide is considered to be "artificially modified" if at least one aspect of the polypeptide, such as its sequence, has been manipulated by the hand of man to be different from that aspect that exists in nature. As is common practice and will be understood by those skilled in the art, the progeny of an artificially modified cell is generally still considered to be "artificially modified", even if actual manipulation has been performed on the previous entity.

[0159] In some embodiments of any of the above aspects, the nucleic acid described herein (e.g., an oligonucleotide tag (e.g., an Oligopaint)) is exogenous. In some embodiments of any of the above aspects, the nucleic acid described herein (e.g., an oligonucleotide tag (e.g., an Oligopaint)) is ectopic. In some embodiments of any of the above aspects, the nucleic acid described herein (e.g., an oligonucleotide tag (e.g., an Oligopaint)) is not endogenous.

[0160] The term "exogenous" refers to a substance present in a cell other than its natural source. As used herein, the term "exogenous" can refer to a nucleic acid (e.g., a nucleic acid encoding a polypeptide) or a polypeptide that has been introduced by a process requiring human intervention into a biological system, such as a cell or organism in which the nucleic acid or polypeptide is not normally found (one wishes to introduce the nucleic acid or polypeptide into such a cell or organism). Alternatively, "exogenous" can refer to a nucleic acid or polypeptide that has been introduced by a process requiring human intervention into a biological system, such as a cell or organism in which the nucleic acid or polypeptide is only found in relatively small amounts (one wishes to increase the amount of the nucleic acid or polypeptide in the cell or organism, e.g., to produce ectopic expression or levels). In contrast, the term "endogenous" refers to a substance that is naturally found in a biological system or cell. As used herein, "ectopic" refers to a substance that is present in a different location and / or amount than normal. An ectopic substance can be a substance that is normally found in a given cell, but in much smaller amounts and / or at a different time. Ectopic also includes materials, such as polypeptides or nucleic acids, that are not naturally found or expressed in a given cell under its natural environment.

[0161] As used herein, "contact" refers to any suitable means for delivering or exposing an agent to at least one cell.Exemplary delivery methods include, but are not limited to, direct delivery into cell culture medium, perfusion, injection, or other delivery methods well known to those skilled in the art.In some embodiments, contact includes physical human activities, such as injection; dispensing, mixing, and / or decanting; and / or operating a delivery device or machine.

[0162] The terms "statistically significant" or "significantly" refer to statistical significance, generally meaning a difference of 2 standard deviations (2 SD) or greater.

[0163] Except in the examples or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as being modified in all instances by the term "about." The term "about," when used in connection with percentages, can mean ±1%.

[0164] As used herein, the term "comprising" means that other elements may be present in addition to the particular elements listed. The use of "comprising" indicates inclusion rather than limitation.

[0165] The term "consisting of" refers to compositions, methods, and each component thereof described herein, excluding any element not recited in the description of that embodiment.

[0166] As used herein, the term "consisting essentially of" refers to elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of the embodiment of the invention.

[0167] As used herein, the term "corresponding to" refers to an amino acid or nucleotide at a recited position in a first polypeptide or nucleic acid, or an amino acid or nucleotide that is equivalent to the recited amino acid or nucleotide in a second polypeptide or nucleic acid. The equivalent recited amino acid or nucleotide can be determined by alignment of candidate sequences using homology programs known in the art, such as BLAST.

[0168] The singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The abbreviation "eg" is derived from the Latin exempli gratia and is used herein to denote a non-limiting example. Thus, the abbreviation "eg" is synonymous with the term "for example."

[0169] Groupings of alternative elements or aspects of the invention disclosed herein should not be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group may be included in or deleted from a group for reasons of convenience and / or patentability. When such inclusion or deletion occurs, the specification is deemed to include the group so modified that satisfies the description of all Markush groups used in the appended claims.

[0170] Unless otherwise defined herein, technical and scientific terms used in connection with this application shall have the meaning commonly understood by those skilled in the art to which this disclosure belongs. It is understood that the present invention is not limited to the particular methodology, protocols, reagents, etc. described herein, which may therefore vary. The terms used herein are for the purpose of describing particular embodiments, and are not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in: The Merck Manual of Diagnosis and Therapy, 20th edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), WWNorton & Company, 2016 (ISBN 0815345054, 978-0815345053); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe (eds.), John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737); the contents of all of which are incorporated herein by reference in their entireties.

[0171] Other terms are defined herein within the description of various aspects of the invention.

[0172] All patents and other publications cited throughout this application (including literature references, issued patents, published patent applications, and co-pending patent applications) are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that may be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of this application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by reason of prior invention or for any other reason. All statements as to the date or contents of these documents are based on the information available to the applicant and do not constitute any admission as to the accuracy of the dates or contents of these documents.

[0173] The description of the aspects of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. Although specific aspects and examples of the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as will be understood by those skilled in the relevant art. For example, while method steps or functions are presented in a given order, in alternative embodiments, the functions can be performed in a different order or substantially simultaneously. The teachings of the disclosure provided herein can be applied to other procedures or methods, as appropriate. The various aspects described herein can be combined to provide further aspects. Aspects of the disclosure can be modified, as appropriate, to employ the structure, functions, and concepts of the above documents and applications to provide further aspects of the disclosure. These and other modifications can be made to the disclosure in light of the detailed description. All such modifications are intended to be within the scope of the appended claims.

[0174] Particular elements of any of the foregoing embodiments may be combined with or substituted for elements of other embodiments. Additionally, although advantages associated with particular embodiments of the present disclosure have been described in connection with those embodiments, other embodiments may also exhibit such advantages, and not all embodiments necessarily need to exhibit such advantages in order to fall within the scope of the present disclosure.

[0175] The technology described herein is further illustrated by the following examples, which should in no way be construed as further limiting.

[0176] Some aspects of the technology described herein are defined according to any of the following numbered paragraphs: 1. A method for analyzing at least one target molecule in a sample, comprising the steps of: a. contacting the sample with at least one oligonucleotide tag, wherein each oligonucleotide tag comprises: i. a recognition domain that specifically binds to the target molecule to be analyzed; and ii. At least one street containing at least one cassette each cassette comprising: 1. A barcode region containing at least one nucleotide, flanked on at least one side by an anchor region. Including, wherein each street of oligonucleotide tags is unique relative to the streets of other oligonucleotide tags of step (a) in at least the following manner: A. the spatial order of the cassettes within the street is different; or B. the sequence of the barcode region is different from the barcode regions of the other oligonucleotide tags of step (a); b. contacting the sample with at least two readout molecules, each readout molecule comprising: i. an oligonucleotide that specifically hybridizes to at least one oligonucleotide tag cassette used in step (a); and ii. Detection molecules Including, wherein the at least two readout molecules collectively constitute at least two distinguishable detection molecules; and c. detecting the relative spatial order of the detection molecules hybridized to at least one oligonucleotide tag, where at least one oligonucleotide tag is hybridized to at least one target molecule, whereby the relative spatial order of the detection molecules allows identification of which oligonucleotide tag is hybridized to the target molecule at that position. A method comprising: 2. The method of any one of the preceding items, wherein the barcode region comprises 1 to 10 nucleotides. 3. The method of any one of the preceding claims, wherein the street comprises at least three cassettes. 4. The method of any one of the preceding items, wherein the barcode region is flanked on both sides by anchor regions. 5. The method of any one of the preceding items, wherein the anchor regions of all oligonucleotide tags are constant. 6. The method of any one of the preceding items, wherein specific hybridization of the readout molecule to the cassette is determined by the identity of the barcode region. 7. The method of any one of the preceding items, wherein the detection molecule is a fluorophore. 8. The method of any one of the preceding claims, wherein the detecting step is carried out by fluorescence microscopy. 9. The method of any one of the preceding items, wherein the detection molecule comprises biotin, an amine, a metal, an anchoring molecule, or an acrydite. 10. The method according to any one of the preceding items, wherein the detecting step is carried out at least by a single-cell resolution method. 11. The method of any one of the preceding claims, wherein step (b) comprises contacting the sample with at least four readout molecules. 12. The method of any one of the preceding items, wherein step (b) comprises contacting the sample with a group of readout molecules collectively constituting at least three distinguishable detection molecules. 13. The method of any one of the preceding items, wherein step (b) comprises contacting the sample with a group of readout molecules collectively constituting at least four distinguishable detection molecules. 14. The method of any one of the preceding items, wherein at least two target molecules are analyzed simultaneously. 15. The method of any one of the preceding items, wherein at least three target molecules are analyzed simultaneously. 16. The method of any one of the preceding items, wherein at least 10 target molecules are analyzed simultaneously. 17. The method of any one of the preceding items, wherein at least 20 target molecules are analyzed simultaneously. 18. The method of any one of the preceding items, wherein the target molecule is a nucleic acid, a polypeptide, a cell surface molecule, or an inorganic substance. 19. The method of any one of the preceding items, wherein the target molecule is DNA or mRNA. 20. The method of any one of the preceding items, wherein the sample is a cell, a cell culture, or a tissue sample. 21. A system for analyzing at least one target molecule in a sample, comprising: a. a detector capable of detecting at least two detectable molecules; b. at least one oligonucleotide tag, each oligonucleotide tag comprising: i. a recognition domain that specifically binds to the target molecule to be analyzed; and ii. A street containing at least one cassette each cassette comprising: 1. A barcode region containing at least one nucleotide, flanked on at least one side by an anchor region. Including, wherein each street of oligonucleotide tags is unique relative to the streets of other oligonucleotide tags of (b) in at least: A. the spatial order of the cassettes within the street is different; or B. the sequence of the barcode region is different from the barcode regions of the other oligonucleotide tags of (b); and c. at least two readout molecules, each readout molecule comprising: i. an oligonucleotide that specifically hybridizes to at least one of the oligonucleotide tag cassettes used in (b); and ii. Detection molecules wherein the at least two readout molecules collectively constitute at least two distinguishable detection molecules. Including, wherein a sample is contacted with said at least one oligonucleotide tag and said at least two readout molecules, and the relative spatial order of detection molecules hybridized to at least one oligonucleotide tag is detected; and wherein said at least one oligonucleotide tag is hybridized to said at least one target molecule, whereby the relative spatial order of the detection molecules enables identification of which oligonucleotide tag is hybridized to the target molecule at that position. EXAMPLES

[0177] Example 1 Compared to existing technologies, the methods and compositions described herein utilize a multiplexing technology, OligoCASSEQ, to enable the analysis of DNA, mRNA, proteins, etc., with microscopy at the single-cell level by visualizing and identifying thousands of targets within the same cell, providing a more global picture of cellular processes. Unlike current microscopy technologies that are limited to analyzing 4-5 targets at a time, OligoCASSEQ achieves a high level of multiplexing in an efficient and cost-effective manner. First, OligoCASSEQ does not use enzymes, which can be costly and inefficient. Second, OligoCASSEQ does not require lengthy oligos, which increase costs while decreasing accuracy.

[0178] The technology described herein, sometimes referred to as "OligoCASSEQ," is a technology that allows for highly multiplexed target discrimination at the single cell level. In this exemplary embodiment, DNA is targeted by an Oligopaint with "streets" that contain "cassettes" (see, e.g., FIG. 1A). The cassettes consist of variable barcode regions flanked on either side by constant anchor regions (see, e.g., FIG. 1B). The barcodes are sequenced using fluorescently labeled oligos (e.g., readouts; also called readout molecules) that are specific for the nucleotide at each barcode position (see, e.g., FIG. 1C). The readouts recognize the cassette via the anchor regions and competitively bind to the cassette. Complementary binding is directed by the readout recognition of a specific barcode sequence (see, e.g., FIG. 1D). This design allows for compact cassettes and reduced amounts of readouts required. A 5-nucleotide barcode can be used to identify 1024 targets (4 5 ) can be identified.

[0179] OligoCASSEQ is complementary to the Oligopaint technology; that is, it can function to multiplex / decode anything that is oligonucleotide tagged, including but not limited to oligonucleotide tagged oligos (e.g., Oligopaints or other DNA-binding entities), oligonucleotide tagged antibodies (e.g., nanobodies), oligonucleotide tagged small molecules (e.g., for drug screening), oligonucleotide tagged cells, and oligonucleotide tagged non-biological materials (metals, chemicals, etc.). The DNA target hybridizes with the Oligopaint. The crucial difference in the case of OligoCASSEQ is that the nucleotide cassette sequences are encoded into the non-genomic targeting "streets" of the Oligopaint. These cassettes allow for barcoding of the Oligopaints, and therefore high levels of multiplexing.

[0180] As described herein, the target can be DNA. OligoCASSEQ is also suitable for other targets (e.g., RNA, protein, etc.). OligoCASSEQ allows for identification of a vast number of targets using microscopy. These targets include, but are not limited to, DNA, RNA, protein, cells, and inorganic matter.

[0181] Herein, the use of OligoCASSEQ to trace chromosomes in situ is demonstrated. As a non-limiting example, OligoCASSEQ can be used to determine the localization of five loci along human chromosome 2 (Chr.2) (see, e.g., Figure 1E-H). A two-nucleotide, four-color barcode was used (see, e.g., Figure 1E). Images of micrographs of PGP1-F cells show OligoCASSEQ interrogation of two barcode locations (see, e.g., top and middle rows) followed by re-interrogation of the first location in the same nucleus (see, e.g., bottom row). Schematics above each micrograph show the color codes of specific loci at different barcode locations (see, e.g., Figure 1F). Readouts on the barcodes were identified and then decoded (see, e.g., Figure 1G). The spatial location of each chromosome was then traced (see, e.g., Figure 1H).

[0182] OligoCASSEQ addresses the need to target and identify multiple (>5) targets at once. Currently, microscopes can only interrogate 4-5 targets at a time because they can only distinguish 4-5 colors at a time. OligoCASSEQ solves this problem through barcoding and multiplexing. Instead of one target being represented by one color, targets are represented by a sequence of 4 colors, which exponentially increases the number of targets that can be interrogated.

[0183] OligoCASSEQ is superior to other options because: 1) it does not require enzymes; 2) it allows for shorter oligo lengths; and 3) it reduces the complexity and number of readout oligos required.

Claims

1. 1. A method for analyzing at least one target molecule in a sample, comprising the steps of: a. contacting the sample with oligonucleotide tags, wherein each oligonucleotide tag comprises: i. a recognition domain that specifically binds to the target molecule to be analyzed; and ii. At least one street containing a cassette each cassette comprising:

1. a barcode region comprising at least one nucleotide flanked on each side by anchor regions; wherein the anchor regions of the oligonucleotide tag are constant for a set of cassettes and are complementary to the anchor hybridization region of at least one readout molecule; Including, wherein each street of oligonucleotide tags is unique relative to the streets of other oligonucleotide tags of step (a) in at least: A. the spatial order of the cassettes within the street is different; or B. the sequence of the barcode region is different from the barcode regions of the other oligonucleotide tags of step (a); b. contacting the sample with at least two readout molecules, each readout molecule comprising: i. an oligonucleotide that specifically hybridizes to at least one oligonucleotide tag cassette used in step (a); wherein the oligonucleotide comprises an anchor hybridization region adjacent to a barcode hybridization region; and ii. Detection molecules Including, wherein the at least two readout molecules collectively constitute at least two distinguishable detection molecules; and c. detecting the relative spatial order of the detection molecules hybridized to the oligonucleotide tags, where the oligonucleotide tags are hybridized to at least one target molecule, whereby the relative spatial order of the detection molecules allows identification of which oligonucleotide tag is hybridized to the target molecule at that position. A method comprising:

2. 2. The method of claim 1, wherein the barcode region comprises 1 to 10 nucleotides.

3. The method of claim 1 or 2, wherein the street comprises at least three cassettes.

4. The method according to any one of claims 1 to 3, wherein the detection molecule is a fluorophore.

5. The method according to any one of claims 1 to 4, wherein the detecting step is carried out by fluorescence microscopy.

6. The method of any one of claims 1 to 5, wherein the detection molecule comprises biotin, an amine, a metal, an anchoring molecule, or an acrydite.

7. The method according to any one of claims 1 to 6, wherein the detecting step is carried out with at least single-cell resolution.

8. The method of any one of claims 1 to 7, wherein step (b) comprises contacting the sample with at least four readout molecules.

9. The method of any one of claims 1 to 8, wherein step (b) comprises contacting the sample with a group of readout molecules collectively constituting at least three distinguishable detection molecules.

10. 10. The method of any one of claims 1 to 9, wherein step (b) comprises contacting the sample with a group of readout molecules collectively constituting at least four distinguishable detection molecules.

11. The method according to any one of claims 1 to 10, wherein at least two target molecules are analysed simultaneously.

12. The method according to any one of claims 1 to 11, wherein at least three target molecules are analysed simultaneously.

13. The method according to any one of claims 1 to 12, wherein at least 10 target molecules are analysed simultaneously.

14. The method according to any one of claims 1 to 13, wherein at least 20 target molecules are analysed simultaneously.

15. The method according to any one of claims 1 to 14, wherein the target molecule is a nucleic acid.

16. The method according to any one of claims 1 to 15, wherein the target molecule is DNA or mRNA.

17. The method of any one of claims 1 to 16, wherein the sample is a cell, a cell culture, or a tissue sample.

18. 1. A system for analyzing at least one target molecule in a sample, comprising: a. a detector capable of detecting at least two detectable molecules; b. oligonucleotide tags, each oligonucleotide tag comprising: i. a recognition domain that specifically binds to the target molecule to be analyzed; and ii. Street, including cassettes each cassette comprising:

1. a barcode region comprising at least one nucleotide flanked on each side by anchor regions; wherein the anchor regions of the oligonucleotide tag are constant and complementary to the anchor hybridization region of a readout molecule; Including, wherein each oligonucleotide tag street is unique relative to the other oligonucleotide tags streets of (b) in at least: A. the spatial order of the cassettes within the street is different; or B. the sequence of the barcode region is different from the barcode regions of the other oligonucleotide tags of (b); and c. at least two readout molecules, each readout molecule comprising: i. an oligonucleotide that specifically hybridizes to at least one of the oligonucleotide tag cassettes used in (b); wherein the oligonucleotide comprises an anchor hybridization region adjacent to a barcode hybridization region; and ii. Detection molecules wherein the at least two readout molecules collectively constitute at least two distinguishable detection molecules. Including, wherein a sample is contacted with the oligonucleotide tags and the at least two readout molecules, and the relative spatial order of detection molecules hybridized to the oligonucleotide tags is detected; and wherein the oligonucleotide tags are hybridized to the at least one target molecule, whereby the relative spatial order of the detection molecules enables identification of which oligonucleotide tag is hybridized to the target molecule at that position.

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