Multiplex fluorescent cell and tissue imaging with DNA-encoded thermal channels and uses thereof

Abstract

Provided herein are probes, methods, and kits for detecting target molecules at single cell or tissue levels, for example in multiplex ways.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 319,021, filed on March 11, 2022, under §119(e), the contents of which are incorporated herein by reference in their entirety. Technical Field

[0002] The field of the invention relates to the detection of target molecules, for example in a multiplex format. Government support

[0003] This invention was made with government support under Grants GM133052 and GM124401 awarded by the National Institutes of Health and Grant N00014-18-1-2549 awarded by the Office of Naval Research. The U.S. Government has certain rights in this invention. Background Art

[0004] Spatial biology enables researchers to map spatial structures within individual cells and determine how they interact with probes in the tissue environment. The spatial and quantitative information revealed at the single-cell level greatly expands the understanding of heterogeneity in human cell types and disease lesion microenvironments. Sequencing and imaging are two major platforms for in situ spatial biology research and can be performed without dissecting cells. Compared with sequencing, imaging-based methods can provide higher spatial resolution in intact biological samples, but are often subject to the low multiplicity of the biological targets analyzed. The ability to visualize multiple different types of biomolecules in single cells and tissues has become an increasingly important tool to help understand complex biological systems, such as cellular heterogeneity and signal regulation pathways in cancer tumor environments (Lewis, SM et al., Spatialomics and multiplexed imaging to explore cancer biology. Nature methods, 1-16 (2021)).

[0005] However, due to spectral overlap, the multiplexity of fluorescence microscopy is usually limited by the color palette (usually 3-4 colors). To overcome this bottleneck, geometric encoding of fluorophores and iterative combinations of imaging agents on larger entities have been developed. This iterative approach requires multiple rounds of washing to remove the signal of the previous round and accumulate the signal of the current round, which is achieved by automated microfluidic systems. The experimental setup is usually complex and the washing process is time-consuming. Geometric encoding is implemented on giant entities, which are usually too large to diffuse in situ to subcellular biological targets to reveal high-resolution spatial information in single cells. In addition, combinatorial encoding can be applied to iterative binding or spectral encoding to exponentially improve multiplexity (Chen, KH, Boettiger, AN, Moffitt, JR, Wang, S. & Zhuang, X. RNA imaging. Spatially resolved, highly multiplexed RNA profiling in single cells. Science 348, aaa6090 (2015) and Eng, CL et al., Transcriptome-scale super-resolved imaging in tissues by RNA seq FISH. Nature 568, 235-239 (2019)).

[0006] Therefore, there remains a need in the art for reagents and methods for improved multiplexed detection of target molecules, such as proteins and RNAs. The present disclosure satisfies some of these needs. Summary of the invention

[0007] The methods, compositions, and kits described herein are based in part on the generation of labeled nucleic acid probes having regions of distinct melting / denaturation temperatures such that changing the temperature to a given temperature channel can release inhibition of a reporter molecule, thereby allowing visualization of a given nucleic acid in a cell or tissue.

[0008] In one aspect, the present invention provides a method for detecting a target molecule in a sample. The method includes providing a detection probe group. The detection probe group includes at least three nucleic acid chains-target binding chain, reporting chain and quenching chain. The first chain (also referred to as the target binding chain) includes a target binding agent for binding to the target, and the target binding agent is connected to a hybridization domain for hybridizing with the reporting chain. The second chain (also referred to as the reporting chain herein) includes a first hybridization domain for hybridizing with the hybridization domain of the target binding chain, and the first hybridization domain is connected to a second hybridization domain for combining with the quenching chain. The reporting chain further includes a reporter molecule capable of generating a detectable signal. The first hybridization domain of the reporting chain includes a nucleotide sequence that is substantially complementary to the nucleotide sequence of the hybridization domain of the target binding chain. The third chain (also referred to as the quenching chain) includes a hybridization domain for hybridizing with the second hybridization domain of the reporting chain. The quenching chain includes a quencher molecule. The hybridization domain of the quenching chain includes a nucleotide sequence that is substantially complementary to the nucleotide sequence of the second hybridization domain of the reporting chain. The reporter molecule in the reporter strand and the quencher molecule in the quencher strand are arranged so that when the reporter strand and the quencher strand hybridize to each other (e.g., through a hybridization domain), the quencher molecule quenches the detectable signal from the reporter molecule. When the quencher strand and the reporter strand are not hybridized to each other, the detectable signal from the reporter molecule is not quenched.

[0009] The target binding agent can be selected from the group consisting of nucleic acids, proteins, peptides, peptidomimetics, amino acids, disaccharides, trisaccharides, oligosaccharides, polysaccharides, lipopolysaccharides, lectins, nucleosides, nucleotides, vitamins, steroids, hormones, cofactors, receptors, and receptor ligands. In some embodiments, the target binding agent is a nucleic acid, an antibody, an antigen-binding fragment of an antibody, an antibody mimetic, a receptor, or a ligand of a receptor. For example, the target binding molecule is a nucleic acid. In another non-limiting example, the target binding molecule is an antibody or an antigen-binding fragment of an antibody.

[0010] The target molecule can be a nucleic acid, a protein, a sugar, a lipid, a small molecule or an antigen. In some embodiments, the target molecule is a nucleic acid. For example, the target molecule is RNA, DNA or a mixture of RNA and DNA. In some embodiments, the target molecule is a protein. In some embodiments, the target molecule is a primary metabolite or a secondary metabolite.

[0011] The melting temperatures of different hybridization domains are different to allow for the release of a large portion of the quencher strand from the reporter strand without substantially releasing the reporter strand from the target binding strand and / or releasing the target binding strand from the target molecule. Typically, the melting temperature (T) of the quencher strand hybridized to the reporter strand is about 100°C. m) is lower than the melting temperature of the reporter strand hybridized to the target binding strand, and the melting temperature of the quencher strand hybridized to the reporter strand is lower than the melting temperature of the target binding strand bound to the target molecule. Typically, the melting temperature of the reporter strand hybridized to the target binding strand is lower than the target binding agent bound to the target molecule.

[0012] Probe groups are contacted with the target under the condition that the probe groups are bound to the target by the target binding agent. The probe groups are analyzed for binding to the target by evaluating the detectable signal generated as a function of temperature. For example, detectable signals are detected or measured at a temperature higher than the melting temperature of the quencher chain hybridized with the reporter chain and lower than the melting temperature of the reporter chain hybridized with the target binding chain. At a temperature higher than the melting temperature of the quencher chain hybridized with the reporter chain but lower than the melting temperature of the target binding agent combined with the target, the quencher chain and the reporter chain are no longer hybridized with each other, and the detectable signal from the reporter molecule is not quenched. On the contrary, when the detectable signal is measured at a temperature lower than the melting temperature of the quencher chain hybridized with the reporter chain, the quencher chain and the reporter chain remain hybridized with each other and the detectable signal from the reporter molecule is quenched. Therefore, the detectable signals at two temperatures are different.

[0013] In some embodiments of the various aspects described herein, the target binding strand further comprises a second hybridization domain. The second hybridization domain of the target binding strand can be used to hybridize with the reference strand. The reference strand comprises a hybridization domain comprising a nucleotide sequence that is substantially complementary to the nucleic acid strand of the second hybridization domain of the target binding strand. The reference strand also comprises a reporter molecule capable of generating a detectable signal. The melting temperature of the reference strand hybridized with the target binding strand is higher than the melting temperature of the reporter strand hybridized with the target binding strand.

[0014] In some embodiments of any aspect described herein, the detection probe group comprises at least four nucleic acid chains-target binding strand, reporter strand, quencher strand and reference strand.When using the detection probe group comprising four chains (target binding strand, reporter strand, quencher strand and reference strand) for detection, the method includes detecting or measuring the detectable signal from the reporter molecule at a first temperature and a second temperature.The first temperature is a temperature higher than the melting temperature of the quencher strand hybridized with the reporter strand but lower than the melting temperature of the reporter strand hybridized with the target binding strand.The second temperature is a temperature higher than the melting temperature of the reporter strand hybridized with the target binding strand but lower than the melting temperature of the reference strand hybridized with the target binding strand.The detection of the detectable signal from the reporter molecule attached to the reference strand can be used as a control or reference.

[0015] In some embodiments, the method further comprises detecting or measuring a detectable signal from the reporter molecule at a temperature below the melting temperature of the reporter strand hybridized to the target binding strand.

[0016] Different reporter chains and quencher chains can be prepared so that they have different melting temperatures. Since different quencher chains and reporter chains denature or melt at different temperatures, the detectable signals from different reporter molecules will not be quenched at different temperatures. Therefore, the binding of different probe groups to different targets can be evaluated simultaneously at different temperatures. Therefore, on the other hand, a method for multiple detection of target molecules is provided herein. The method includes providing a plurality of detection probe groups, wherein each detection probe group comprises three chains as described herein-target binding chain, reporter chain and quencher chain.

[0017] In a plurality of probe sets, the melting temperature of the quencher strand hybridized with the reporter strand in one detection probe set is different from the melting temperature of the quencher strand hybridized with the reporter strand in at least one other probe set. In addition, the melting temperature of the second strand in the probe set with the third strand having a lower melting temperature is substantially the same as or lower than the melting temperature of the second strand in the probe set with the third strand having a higher melting temperature. It should be noted that the quencher strand in one detection probe set may or may not be hybridized with the reporter strand in another detection probe set in the plurality of detection probe sets.

[0018] The multiple detection probe groups are contacted with the target molecule to allow the detection probe groups to be bound to their respective targets by the combination of the target binding agent and the target. The detection probe groups are analyzed for the combination of the target by evaluating the detectable signal produced as a function of temperature. For example, the detectable signal from the first probe group bound to its target is measured at a temperature higher than the melting temperature of the quencher chain hybridized with the reporter chain and lower than the melting temperature of the reporter chain hybridized with the target binding chain. The temperature makes the quencher chain in the second detection probe group bonded to the target keep hybridizing with the reporter chain in the second detection probe group. The detectable signal from the second probe group bound to its target is measured at a temperature higher than the melting temperature of the quencher chain of the second probe group hybridized with the reporter chain of the second probe group and lower than the melting temperature of the reporter chain of the second probe group hybridized with the target binding chain of the second probe group. Because the detectable signal from different probe groups is generated at different temperatures, it is possible to distinguish the combination of different detection probe groups to their respective targets, and detect the target in a multiplex manner. This allows the detection of multiple target molecules in one reaction, i.e., multiple detection. In some embodiments, at least two target molecules are detected, for example, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more different target molecules are detected.

[0019] It is noteworthy that the reporter molecules in different members of the multiple detection probe groups can be the same or different. Therefore, in some embodiments of various aspects described herein, the reporter molecules in one detection probe group in the multiple detection probe groups are different from the reporter molecules in at least one other detection probe group in the multiple detection probe groups. Without being bound by theory, using different reporter molecules can provide additional multiple detection capabilities. For example, the following probe groups can be used: the melting temperature of the quencher chain hybridized with the reporter chain is similar, but different probe groups have different reporter molecules. The detectable signal from a reporter molecule is distinguishable from the detectable signal from at least one reporter molecule. The distinguishable detectable signal can be measured at a temperature to detect the combination of different probe groups to their respective targets, for example, a temperature higher than the melting temperature of the quencher chain hybridized with the reporter chain. This allows the detection of multiple target molecules in one reaction, i.e., multiple detection. In some embodiments, at least two target molecules are detected, for example, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more different target molecules are detected. In some embodiments, each target molecule is detected with a detection probe set comprising distinguishable reporter molecules. These can be used with other quencher and reporter strand pairs that denature or melt at different temperatures for further multiplexing.

[0020] In another aspect, a detection probe set is provided herein. The detection probe set comprises at least three nucleic acid strands as described herein - a target binding strand, a reporter strand, and a quencher strand.

[0021] In yet another aspect, a kit is provided herein. The kit comprises a detection probe set as described herein. In some embodiments, the kit comprises a plurality of detection probe sets as described herein.

[0022] It is noteworthy that the detection of detectable markers can be carried out by fluorescence microscopy and fluorescently labeled reporters. Or by colorimetric detection and, for example, nanoparticle labeling reporters. Detection can also be used for multiple imaging of biological species in fixed cells, organoids or tissue samples of prokaryotic, eukaryotic, other or mixed sources. Reading can also be carried out on a surface (such as a glass slide or other substrate), for example, for sequencing the barcoded beads on the surface or for sequencing the fixed oligonucleotides on the surface for nucleic acid data storage applications.

[0023] There are a number of ways to heat the sample to the correct temperature. One method is through a Peltier stage system, such as VAHEAT. Another method is to have a buffer flow continuously through the chamber, where an external heater controls the buffer temperature. Another method to control the temperature is to use lasers or light coupled to metal nanomaterials to convert light into heat energy. See, for example, www.cell.com / trends / biochemical-sciences / fulltext / S0968-0004(19)30236-1?dgcid. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1A-Figure 1C Thermal multiple imaging scheme according to an exemplary embodiment. Figure 1A ) Imaging concept and scheme of DNA thermal-scope; Figure 1B ) A scheme for a thermal control platform having cells or tissues; ( Figure 1C )Thermal spectrum of DNA thermal display.

[0025] Figure 2A-2C .Programmable thermal spectrum of DNA thermal display. Figure 2A ) Thermographic signal yields with different combinations of melting temperatures of quencher and binder. Figure 2B ) Thermographic signal yields with different combinations of melting temperatures of quencher and binder. Figure 2C ) Thermal spectra of the five best thermal channels with minimal crosstalk and high signal yield.

[0026] Figure 3A-3E .Robustness and fast channel switching speed of DNA thermal display. ( Figure 3A ) RNA imaging process using reference probe and thermal probe at different temperature channels. Figure 3B ) In situ imaging of APC RNA transcripts in HeLa cells. Figure 3C )Puncta analysis based on imaging of reference and thermal probes. Figure 3D ) Fluorescent signals of spots detected by DNA thermal imaging at different heating times in three temperature channels. Figure 3E ) FISH images of the APC gene after heating at different temperature channels for 5 seconds.

[0027] Figure 4A-4B . Five hot channel design with minimal signal crosstalk. ( Figure 4A )FISH images of different designed DNA thermal probes under different temperature channels. Figure 4B ) Average RNA quantity per cell detected by different DNA thermal probes at different temperature channels.

[0028] Figure 5A-5C . 15-plex RNA imaging in fixed HeLa cells using a combination of thermal and fluorophore channels. ( Figure 5A )The imaging process of DNA thermal imaging device in different temperature channels. Figure 5B )Single RNA spot in different combinations of DNA thermal and fluorescent channels. Figure 5C ) Overlapping images of 15 imaged RNA species in a single cell.

[0029] Figure 6A-6B .15-plex RNA imaging in retinal tissue using thermal-plex. Fig. 6A ) The workflow of the DNA thermal imager under different temperature channels. Figure 6B ) Individual RNA spots of 15 imaged RNA species in a single cell under different imaging conditions.

[0030] Figure 7A-7B .Resolved 15-fold RNA information. Fig. 7A )15-plex RNA imaging reconstructed in retinal tissue. Figure 7B ) Comparison between smFISH and thermal-plex imaging of 15 RNA targets.

[0031] Figure 8A-8E This is a schematic diagram of thermal-plex imaging. Fig. 8A The thermal-plex imaging concept is based on the gradual melting of DNA thermal probes from in situ targets. After labeling the target biomolecule (e.g., DNA, RNA, or protein) with a DNA barcode, a thermal probe set containing a quencher strand and an imaging agent strand is hybridized to the DNA barcode attached to the target. At temperatures below the melting temperature of the quencher, the fluorescent signal is quenched. When the temperature is heated to the signal temperature, the quencher melts and the fluorescent signal is emitted and can be imaged, including after the sample is cooled to room temperature. The signal is then removed by heating to a temperature substantially above the melting temperature of the barcode domain (to which the imaging agent is bound). ( Figure 8B ) Thermal profile of an exemplary thermal-plex probe set. The melting curves of the quencher and imager pair are used to determine the thermal profile of the DNA thermal probe set. s is the temperature that produces the maximum fluorescence signal. mq is the melting temperature of the quencher. mb is the melting temperature of the barcode domain. Width is the distance between the half-peaks of the signal. ( Figure 8C)thermal-plex imaging configuration. Cells are plated on a slide embedded in an on-scope temperature control unit that rapidly changes the slide temperature in seconds. Volumetric buffer allows for efficient DNA strand dissociation, thereby activating or deactivating the fluorescent signal after a heating spike. Fig.8D ) Temperature profile of the slide during heating and cooling. It takes about 5 seconds to reach the desired signal temperature (57°C) to melt the DNA strands. After the temperature cools to about 30°C, the sample is imaged. Fig. 8E ) Example process of multiplex fluorescence imaging of three targets (Ta, Tb, and Tc) using thermal-plex. (1) Single-stranded DNA barcodes (a1, b1, and c1) are attached to different target molecules. (2) Orthogonal DNA thermal probe sets (a, b, c) are hybridized to the DNA barcodes. (3) Heat to the signal temperature T sa After activating the fluorescent signal, the target Ta is visualized at room temperature. sb 、T sc ) After that, multiple other targets are visualized in sequence. (4) The images are computationally aligned and reconstructed to overlap all targets.

[0032] Fig. 9 Schematic diagram showing multiplex protein imaging in tissue samples using thermal-plex imaging according to an exemplary embodiment. The target protein is first bound to its corresponding antibody, which is conjugated with a DNA barcode. DNA thermal probes encoding different thermal channels will be used to bind to the DNA barcodes on the antibodies. Multiple rounds of heating spikes and imaging at different temperatures are applied to activate the signal of the DNA thermal probe.

[0033] Fig.10 is a schematic diagram showing a thermal-plex for protein imaging according to an exemplary embodiment. First, a DNA barcode-conjugated antibody is applied to bind the target protein. Then a DNA thermal probe is bound to the DNA barcode. Heating and imaging are used to visualize the target protein. DETAILED DESCRIPTION

[0034] It should be understood that the foregoing general description and the following detailed description are merely exemplary and explanatory and do not limit the claimed invention. Herein, unless otherwise expressly stated, the use of the singular includes the plural. As used herein, the use of "or" means "and / or" unless otherwise expressly stated. In addition, the use of the term "includes" and other forms (such as "comprises" and "containing") is not restrictive. In addition, unless otherwise expressly stated, terms such as "element / element" or "component / component" include both elements / elements and components / components comprising one unit, and also include elements and components comprising more than one subunit.

[0035] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents or portions of documents cited in this application (including but not limited to patents, patent applications, articles, books, and treatises) are expressly incorporated herein by reference in their entirety for any purpose.

[0036] Provided herein are nucleic acid probe compositions that allow for differential detection of targets in single cells or tissues using temperature-sensitive melting probes that are detectable only when exposed to a specific threshold temperature or temperature range (e.g., a thermal channel).

[0037] Embodiments of various aspects described herein include detection probe groups. The detection probe group comprises at least three nucleic acid chains--target binding strand, reporter strand and quencher strand. As described herein, the quencher strand and the reporter strand can hybridize with each other, and the reporter strand and the target binding strand can hybridize with each other. Therefore, in some embodiments of any aspect described herein, the quencher strand and the reporter strand hybridize with each other and / or the reporter strand and the target binding strand hybridize with each other.

[0038] As used herein, the term "hybridization" refers to the interaction or annealing of two single-stranded nucleic acids into double-stranded nucleic acids (e.g., duplexes) under specific hybridization conditions. Typically, when two single-stranded nucleic acids have sufficient complementarity to each other under a given set of conditions, they will form a duplex. Such conditions can be, for example, stringent conditions, wherein stringent conditions can include: 400mMNaCl, 40mM PIPES pH 6.4, 1mM EDTA, 50°C or 70°C for 12-16 hours, followed by cleaning. Other conditions can be applied, such as physiologically relevant conditions that may be encountered in vivo. The technician will be able to determine the most suitable set of conditions for testing the complementarity of the two sequences based on the final application of the hybridized nucleotides. Target binding strand

[0039] Embodiments of the various aspects described herein include a target binding strand. Typically, the target binding strand comprises a target binding agent linked to a first hybridization domain.

[0040] The length of the first hybridization domain of the target binding chain can be, for example, in the range of about 10 to about 100 nucleotides, or about 15 to about 90 nucleotides, about 20 to about 80 nucleotides, or about 25 to about 75 nucleotides, or about 30 to about 50 nucleotides. Typically, the length of the first hybridization domain is selected so that the melting temperature of the first hybridization domain hybridized with the complementary chain is lower than the temperature at which the target binding agent dissociates from its target. For example, the melting temperature of the first hybridization domain hybridized with the complementary chain (e.g., the reporter chain) is at least 2°C, at least 3°C, at least 4°C, at least 5°C, at least 6°C, at least 7°C, at least 8°C, at least 9°C, at least 10°C, at least 11°C, at least 12°C, at least 13°C, at least 14°C, or at least 15°C, compared to the temperature at which the target binding agent dissociates from its target.

[0041] In some embodiments, the melting temperature of the first hybridization domain that hybridizes to the complementary strand (eg, the reporter strand) is at least 5°C lower than the temperature at which the target binding agent dissociates from its target.

[0042] The target binding strand also includes a target binding agent, also referred to herein as a target binding domain. It is noteworthy that the target binding agent and the first hybridization domain can be arranged in the target binding strand in any desired direction. For example, the target binding agent can be at the 5' end of the first hybridization domain or at the 3' end of the first hybridization domain. In some embodiments, the target binding agent is at the 5' end of the first hybridization domain. In some other embodiments, the target binding agent is at the 3' end of the first hybridization domain. In some embodiments, the target binding agent is a nucleic acid. For example, the target binding agent is a single-stranded nucleic acid comprising a nucleotide sequence substantially complementary to at least a portion of the target nucleic acid. When the target binding agent is a nucleic acid, the length of the target binding agent nucleic acid chain can be, for example, about 25 to about 200 nucleotides, or about 30 to about 150 nucleotides, about 35 to about 125 nucleotides, or about 40 to about 100 nucleotides, or about 50 to about 75 nucleotides long.

[0043] In some embodiments, the target binding agent is an antibody or an antigen binding portion of an antibody. Typically, the target binding agent is selected so that the melting temperature of the target binding agent that is bound or hybridized to the target is higher than the melting temperature of the hybridization domain hybridized to the reporter chain. For example, compared with the melting temperature of the hybridization domain hybridized to the reporter chain, the melting temperature of the target binding agent that is bound or hybridized to the target is at least 2°C, at least 3°C, at least 4°C, at least 5°C, at least 6°C, at least 7°C, at least 8°C, at least 9°C, at least 10°C, at least 11°C, at least 12°C, at least 13°C, at least 14°C or at least 15°C.

[0044] In some embodiments of any of the aspects described herein, the melting temperature of the target binding agent that binds or hybridizes to the target is at least 5°C higher than the melting temperature of the hybridization domain that hybridizes to the reporter strand.

[0045] In some embodiments of any of the aspects described herein, when the target binding agent is a nucleic acid, the length of the target binding nucleic acid is longer than the length of the hybridization domain of the target binding strand.

[0046] In some embodiments, the target binding strand further comprises a second hybridization domain, such as a hybridization domain for hybridizing with a reference nucleic acid strand. The domain is also referred to herein as a reference binding domain. The length of the reference binding domain can be, for example, in the range of about 25 to about 200 nucleotides, or about 30 to about 150 nucleotides, about 35 to about 125 nucleotides, or about 40 to about 100 nucleotides, or about 50 to about 75 nucleotides. Typically, the length of the reference binding domain is selected so that the melting temperature of the reference binding domain hybridized with the complementary reference strand is higher than the melting temperature of the hybridization domain of the target binding strand hybridized with the reporter strand. For example, the melting temperature of the reference binding domain hybridized to the complementary reference strand is at least 2°C, at least 3°C, at least 4°C, at least 5°C, at least 6°C, at least 7°C, at least 8°C, at least 9°C, at least 10°C, at least 11°C, at least 12°C, at least 13°C, at least 14°C or at least 15°C higher than the melting temperature of the hybridization domain of the target binding strand hybridized to the reporter strand.

[0047] In some embodiments of any of the aspects described herein, the melting temperature of the reference binding domain hybridized to the complementary reference strand is at least 5°C higher than the melting temperature of the hybridization domain of the target binding strand hybridized to the reporter strand.

[0048] The reference binding domain can be located at any position in the target binding strand. In some embodiments, the reference binding domain is at the 5' end of the target binding strand. In some other embodiments, the reference binding domain is at the 3' end of the target binding strand. In some embodiments, the target binding strand comprises a reference binding domain in a 5'->3' direction, the reference binding domain is connected to a target binding agent, and the target binding agent is connected to a first hybridization domain. In some other embodiments, the target binding strand comprises a first hybridization domain in a 5'->3' direction, the first hybridization domain is connected to a target binding agent, and the target binding agent is connected to the reference binding domain. Reporting chain

[0049] Embodiments of various aspects described herein include a reporter nucleic acid strand. Typically, the reporter nucleic acid strand comprises a first hybridization domain connected to a second hybridization domain, and the reporter molecule is connected to the reporter strand. The first hybridization domain comprises a nucleotide sequence that is substantially complementary to the nucleotide sequence of the first hybridization domain of the target binding strand.

[0050] The length of the first hybridization domain of the reporter chain can be the same or different from the length of the first hybridization domain of the target binding chain. For example, the length of the first hybridization domain of the reporter chain can be, for example, about 10 to about 100 nucleotides, or about 15 to about 90 nucleotides, about 20 to about 80 nucleotides, or about 25 to about 75 nucleotides, or about 30 to about 50 nucleotides in length. Typically, the length of the first hybridization domain of the reporter chain is selected so that the melting temperature of the reporter chain hybridized with the target binding chain is lower than the temperature at which the target binding agent dissociates from its target. For example, compared with the temperature at which the target binding agent dissociates from its target, the melting temperature of the first hybridization domain of the reporter chain hybridized with the first hybridization domain of the target binding chain is at least 2°C, at least 3°C, at least 4°C, at least 5°C, at least 6°C, at least 7°C, 8°C, at least 9°C, at least 10°C, at least 11°C, at least 12°C, at least 13°C, at least 14°C or at least 15°C.

[0051] In some embodiments of any of the aspects described herein, the melting temperature of the first hybridization domain of the reporter strand hybridized to the first hybridization domain of the target binding strand is at least 5°C lower than the temperature at which the target binding agent dissociates from its target.

[0052] The length of the second hybridization domain of the reporting chain can be the same or different from the length of the first hybridization domain of the reporting chain. For example, the length of the second hybridization domain of the reporting chain can be, for example, about 10 to about 100 nucleotides, or about 15 to about 90 nucleotides, about 20 to about 80 nucleotides, or about 25 to about 75 nucleotides, or about 30 to about 50 nucleotides in length. Typically, the length of the second hybridization domain of the reporting chain is selected so that the melting temperature of the reporting chain hybridized with the quenching chain is lower than the melting temperature of the reporting chain hybridized with the target binding chain. For example, compared with the melting temperature of the reporting chain hybridized with the target binding chain, the melting temperature of the second hybridization domain of the reporting chain hybridized with the complementary chain (such as the quenching chain) is low by at least 2°C, at least 3°C, at least 4°C, at least 5°C, at least 6°C, at least 7°C, 8°C, at least 9°C, at least 10°C, at least 11°C, at least 12°C, at least 13°C, at least 14°C or at least 15°C.

[0053] In some embodiments of any of the aspects described herein, the melting temperature of the second hybridization domain of the reporter strand hybridized to the complementary strand (eg, the quencher strand) is at least 5°C lower than the melting temperature of the reporter strand hybridized to the target binding strand.

[0054] It is noteworthy that the first and second hybridization domains of the reporting strand can be arranged in any desired direction. For example, the first hybridization domain of the reporting strand can be at the 5' end or 3' end of the second hybridization domain of the reporting strand. Therefore, in some embodiments, the first hybridization domain of the reporting strand is at the 5' end of the second hybridization domain of the reporting strand. In some other embodiments, the first hybridization domain of the reporting strand is at the 3' end of the second hybridization domain of the reporting strand.

[0055] The reporter strand also comprises a reporter molecule capable of generating a detectable signal. Typically, the reporter molecule is located in or near the second hybridization domain of the reporter strand, such that hybridization of the reporter strand to the quencher strand quenches the detectable signal. Quenching chain

[0056] Embodiments of various aspects described herein include a quenching nucleic acid strand. Typically, the quenching nucleic acid strand comprises a first hybridization domain and a quencher molecule. The first hybridization domain comprises a nucleotide sequence that is substantially complementary to the nucleotide sequence of the second hybridization domain of the reporter strand.

[0057] The length of the first hybridization domain of the quencher chain can be the same or different from the length of the second hybridization domain of the reporter chain. For example, the length of the first hybridization domain of the quencher chain can be, for example, about 10 to about 100 nucleotides, or about 15 to about 90 nucleotides, about 20 to about 80 nucleotides, or about 25 to about 75 nucleotides, or about 30 to about 50 nucleotides in length. Typically, the length of the first hybridization domain of the quencher chain is selected so that the melting temperature of the quencher chain hybridized with the reporter chain is lower than the melting temperature of the reporter chain hybridized with the target binding chain. For example, compared with the melting temperature of the reporter chain hybridized with the target binding chain, the melting temperature of the first hybridization domain of the quencher chain hybridized with the second hybridization domain of the reporter chain is low by at least 2°C, at least 3°C, at least 4°C, at least 5°C, at least 6°C, at least 7°C, 8°C, at least 9°C, at least 10°C, at least 11°C, at least 12°C, at least 13°C, at least 14°C or at least 15°C.

[0058] In some embodiments of any of the aspects described herein, the melting temperature of the first hybridization domain of the quencher strand hybridized to the second hybridization domain of the reporter strand is at least 5°C lower than the melting temperature of the reporter strand hybridized to the target binding strand.

[0059] The quencher chain also includes a quencher molecule that can quench the detectable signal from the reporter molecule. Typically, the quencher molecule in the quencher chain is placed so that when the quencher chain and the reporter chain hybridize to each other, the detectable signal from the reporter molecule is quenched. When the quencher chain and the reporter chain do not hybridize to each other, the detectable signal is not quenched. In some embodiments, the quencher molecule is a member of a FRET pair. For example, the quencher can be a FRET receptor. Reference Chain

[0060] Embodiments of various aspects described herein include a reference nucleic acid strand. Typically, the reference nucleic acid strand comprises a first hybridization domain and a reporter molecule capable of generating a detectable signal. The first hybridization domain comprises a nucleotide sequence that is substantially complementary to the nucleotide sequence of the second hybridization domain of the target binding strand.

[0061] The length of the first hybridization domain of the reference chain can be the same or different from the length of the second hybridization domain of the target binding chain. The length of the first hybridization domain of the reference chain can be, for example, about 25 to about 200 nucleotides, or about 30 to about 150 nucleotides, about 35 to about 125 nucleotides, or about 40 to about 100 nucleotides, or about 50 to about 75 nucleotides in length. Typically, the length of the first hybridization domain of the reference chain is selected so that the melting temperature of the reference chain hybridized with the target binding chain is higher than the melting temperature of the target binding chain hybridized with the reporter chain. For example, compared with the melting temperature of the target binding chain hybridized with the reporter chain, the melting temperature of the first hybridization domain of the reference chain hybridized with the second hybridization domain of the target binding chain is at least 2°C, at least 3°C, at least 4°C, at least 5°C, at least 6°C, at least 7°C, at least 8°C, at least 9°C, at least 10°C, at least 11°C, at least 12°C, at least 13°C, at least 14°C or at least 15°C.

[0062] In some embodiments of any of the aspects described herein, the melting temperature of the first hybridization domain of the reference strand hybridized to the second hybridization domain of the target binding strand is at least 5°C higher than the melting temperature of the target binding strand hybridized to the reporter strand.

[0063] As described herein, the melting temperatures of the various hybridization domains used to hybridize their complementary strands can be different. Typically, the melting temperature of the quencher strand hybridized to the reporter strand is lower than the melting temperature of the reporter strand hybridized to the target binding strand, and the melting temperature of the reporter strand hybridized to the target binding strand is lower than the temperature at which the target binding agent dissociates from the target, i.e., lower than the melting temperature of the target binding strand hybridized to the target molecule. Reporter

[0064] Embodiments of the various aspects described herein rely on reporter molecules capable of producing a detectable signal. For example, the reporter strand comprises a reporter molecule. As used herein, the terms "reporter molecule" and "detectable label" are used interchangeably and may refer to any chemical moiety attached to a nucleotide, a nucleotide polymer, or a nucleic acid binding factor, wherein the attachment may be covalent or non-covalent and allows detection of signals in a single cell or tissue. The reporter molecule may include a luminescent molecule, a chemiluminescent molecule, a fluorochrome, a fluorescence quencher, a colored molecule, a radioisotope, or a scintillator. The reporter molecule may also include any useful linker molecule (e.g., biotin, avidin, streptavidin, HRP, protein A, protein G, an antibody or fragment thereof, Grb2, polyhistidine, Ni 2+ , FLAG tags, myc tags), heavy metals, enzymes (examples include alkaline phosphatase, peroxidase and luciferase), electron donors / acceptors, acridinium esters, dyes and calorimetric substrates. One skilled in the art will readily recognize useful reporter molecules not mentioned above that can be used in the methods and compositions described herein. In one embodiment, the reporter molecule comprises a fluorophore portion of a fluorescence resonance energy transfer (FRET) pair.

[0065] In some embodiments of any aspect, the reporter molecules provided herein are selected from the group consisting of: fluorescent molecules / fluorophores, radioisotopes, chromophores, enzymes, enzyme substrates, chemiluminescent moieties, bioluminescent moieties, echogenic substances, non-metallic isotopes, optical reporter molecules, paramagnetic metal ions, and ferromagnetic metals.

[0066] In some embodiments of any aspect described herein, the reporter molecule is a fluorescent compound. When the fluorescently labeled reagent is exposed to light of the appropriate wavelength, its presence can be detected due to fluorescence. In some embodiments of any aspect, the detectable label can be a fluorescent dye molecule or a fluorophore. A wide variety of fluorescent reporter dyes are known in the art. Typically, the fluorophore is an aromatic or heteroaromatic compound, and can be pyrene, anthracene, naphthalene, acridine, stilbene, indole, benzindole, oxazole, thiazole, benzothiazole, cyanine, carbocyanine, salicylate, anthranilate, coumarin, fluorescein, rhodamine or other similar compounds.

[0067] Exemplary fluorophores include, but are not limited to, 1,5IAEDANS; 1,8-ANS; 4-methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-carboxyfluorescein (5-FAM); 5-carboxynaphthofluorescein (5-Carboxynapthofluorescein) (pH 10); 5-carboxytetramethylrhodamine (5-TAMRA); 5-FAM (5-carboxyfluorescein); 5-hydroxytryptamine (HAT); 5-ROX (carboxy-X-rhodamine); 5-TAMRA (5-carboxytetramethylrhodamine); 6-carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7-amino-4-methylcoumarin; 7-aminoactinomycin D (7-AAD); 7-hydroxy-4-methylcoumarin; 9-amino-6-chloro-2-methoxyacridine; ABQ; acid fuchsin; ACMA (9-amino-6-chloro-2-methoxyacridine); acridine orange; acridine red; acridine yellow; acriflavin; acriflavin Feulgen SITSA; aequorin (luminescent protein); Alexa Fluor 350 TM Alexa Fluor 430 TM ;Alexa Fluor 488 TM Alexa Fluor 532 TM ; Alexa Fluor 546 TM ; Alexa Fluor 568 TM Alexa Fluor 594 TM ;Alexa Fluor 633 TM ; Alexa Fluor 647 TM ;AlexaFluor 660 TM Alexa Fluor 680 TM ; Alizarin aminocarboxylate complex; Alizarin red; Allophycocyanin (APC); AMC, AMCA-S; AMCA (aminomethylcoumarin); AMCA-X; Aminactinomycin D; Aminocoumarin; Aniline blue; Anthrocyl stearate; APC-Cy7; APTS; Astrazon Brilliant Red 4G; Astrazon OrangeR; Astrazon Red 6B; Astrazon Yellow 7GLL; Atrazon; ATTO-TAG TM CBQCA; ATTO-TAG TMFQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); berberine sulfate; β-lactamase; BFP blue-shifted GFP (Y66H); BG-647; Bimane; bisbenzamide; Blancophor FFG; Blancophor SV; BOBO TM -1;BOBO TM -3; Bodipy 492 / 515; Bodipy 493 / 503; Bodipy 500 / 510; Bodipy 505 / 515; Bodipy 530 / 550; Bodipy 542 / 563; 581 / 591; Bodipy 630 / 650-X; Bodipy 650 / 665-X; Bodipy 665 / 676; Bodipy Fl; Bodipy FL ATP; Bodipy Fl-ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; BodipyTR-X SE; BO-PRO TM -1; BO-PRO TM -3; Brilliant Sulphoflavin FF; Calcein; Calcein Blue; Calcium Crimson TM ; Calcium green; Calcium green-1Ca 2+ Dye; Calcium Green-2Ca 2+ ; Calcium Green-5N Ca 2+ ; Calcium Green - C18Ca 2+ ; Calcium Orange; Calcofluor White; Carboxy-X-Rhodamine (5-ROX); Cascade Blue TM ); Cascade Yellow; Catecholamine; CFDA; CFP-cyan fluorescent protein; Chlorophyll; Chromomycin A; Chromomycin A; CMFDA; Coelenterazine; Coelenterazine cp; Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazine hcp; Coelenterazine ip; Coelenterazine O; Coumarin phalloidin; CPM methylcoumarin; CTC; Cy2 TM ; Cy3.1 8; Cy3.5 TM ;Cy3 TM ;Cy5.1 8;Cy5.5 TM ;Cy5 TM;Cy7 TM ; cyan GFP; cyclic AMP fluorescent sensor (FiCRhR); d2; Dabcyl; Dansyl; Dansylamide; Dansylcadaverine; Dansyl chloride; Dansyl DHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3; DCFDA; DCFH (dichlorodihydrofluorescein diacetate); DDAO; DHR (dihydrorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS (non-ratiometric); DiA (4-Di-16-ASP); DIDS; dihydrorhodamine 123 (DHR); DiO (DiOC18 (3)); DiR; DiR (DiIC18 (7)); dopamine; DsRed; DTAF; DY-630-NHS; DY-635-NHS; EBFP; ECFP; EGFP; ELF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidium homodimer-1 (EthD-1); Euchrysin; Europium (III) chloride; Europium; EYFP; Fast Blue; FDA; Feulgen (pararosaniline); FITC; FL-645; Flazo Orange; Fluo-3; Fluo-4; Fluorescein diacetate; Fluoro-Emerald; Fluoro-Gold (Fluoro-Ruby); FluorX; FM 1-43 TM ;FM 4-46; Fura Red TM(high pH); Fura-2, high calcium; Fura-2, low calcium; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow 5GF; GFP (S65T); red-shifted GFP (rsGFP); wild-type GFP, non-UV excited (wtGFP); wild-type GFP, UV excited (wtGFP); GFPuv; Gloxalic Acid; Granular Blue; hematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; hydroxycoumarin; hydroxystilbene (fluorescent gold); hydroxytryptamine; indole dicarbocyanine (DiD); indole tricarbocyanine (DiR); Intrawhite Cf; JC-1; JO-JO-1; JO-PRO-1; LaserPro; Laurodan; LDS 751; Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine rhodamine; Lissamine rhodamine B; LOLO-1; LO-PRO-1; Lucifer Yellow; Mag Green; Sudan Red (Magdala Red) (Root Bark Red B); Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; Maxilon Brilliant Flavin 10GFF; Maxilon Brilliant Flavin 8GFF; Merocyanin; Methoxycoumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green Pyroxadiazole); NBD; NBD Amine; Nile Red; Nitrobenzoxadidole; Norepinephrine; Nuclear Fast Red; Nuclear Yellow; Nylosan Brilliant Iavin E8G; Oregon Green TM ; Oregon Green 488-X; Oregon Green TM 488; Oregon Green TM 500; Oregon Green TM514; Pacific Blue; Feulgen; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-TexasRed (Red 613); Radix Red B (Sudan Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist; Phycoerythrin B [PE]; Phycoerythrin R [ΡΕ]; PKH26; PKH67; PMIA; Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-PRO-3; Primuline; Procion Yellow; Propidium Iodide (PI); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine; Resorufin; RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5GLD; Rhodamine 6G; Rhodamine B 540; Rhodamine B 200; Rhodamine Bextra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine Phalloidin; Rhodamine Red; Rhodamine WT; Bengal Rose Red; R-phycoerythrin (PE); red-shifted GFP (rsGFP, S65T); S65A; S65C; S65L; S65T; Sapphire GFP; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron Brilliant Red B; Sevron Orange; Sevron Yellow L; sgBFP TM ;sgBFP TM (superluminescent BFP); sgGFP TM ;sgGFP TM (superluminescent GFP); SITS; SITS (primrose yellow); SITS (stilbene isothiosulfonate); SPQ (6-methoxy-N-(3-sulfopropyl)-quinolinium; stilbene; sulforhodamine B can C; sulforhodamine G Extra; tetracycline; tetramethylrhodamine; Texas Red TM ;Texas Red-X TMConjugates; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TCN; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TMR; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC (tetramethylrhodamine isothiocyanate); True Blue; TruRed; Ultralite; Uranine B; Uvitex SFC; wt GFP; WW 781; XL665; X-rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO-3; YOYO-1; and YOYO-3. A variety of suitable forms of these fluorescent compounds are available and can be used.

[0068] A variety of suitable forms of these fluorescent compounds are available and can be used. Examples of other fluorophores include, but are not limited to, fluorescein, phycoerythrin, phycocyanin, o-phthalaldehyde, fluorescamine, Cy3™, Cy5™, allophycocyanin, Texas Red, peridinin, chlorophyll, cyanine, tandem conjugates (e.g., phycoerythrin-Cy5™), green fluorescent protein, rhodamine, fluorescein isothiocyanate (FITC) and Oregon Green™, rhodamine and derivatives (e.g., Texas Red and tetramethylrhodamine isothiocyanate (TRITC)), biotin, phycoerythrin, AMCA, CyDyes™, 6-carboxyfluorescein (commonly abbreviated as 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), benzylamine dyes (e.g., Hoechst 33258), phenanthridine dyes (e.g., Texas Red), ethidium dyes, acridine dyes, carbazole dyes, phenoxazine dyes, porphyrin dyes, polymethine dyes (e.g., cyanine dyes such as Cy3, Cy5, etc.), BODIPY dyes and quinoline dyes.

[0069] Other exemplary detectable labels include luminescent and bioluminescent markers (e.g., biotin, luciferase (e.g., bacteria, fireflies, click beetles, etc.), luciferin, and aequorin); radioactive labels (e.g., 3H, 125I, 35S, 14C, or 32P); enzymes (e.g., β-galactosidases, glucorinidases, phosphatases (e.g., alkaline phosphatase), peroxidases (e.g., horseradish peroxidase), and cholinesterase); and calorimetric labels, e.g., colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, and latex) beads. Patents that teach the use of such labels include U.S. Pat. Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241, each of which is incorporated herein by reference.

[0070] In some embodiments of any aspect, the detectable label may be radioactive, including but not limited to 3H, 125I, 35S, 14C, 32P and 33P. Suitable non-metallic isotopes include but are not limited to 11C, 14C, 13N, 18F, 123I, 124I and 125I. Suitable radioisotopes include but are not limited to 99mTc, 95Tc, 111In, 62Cu, 64Cu, Ga, 68Ga and 153Gd. Suitable paramagnetic metal ions include but are not limited to Gd (III), Dy (III), Fe (III) and Mn (II). Suitable X-ray absorbers include but are not limited to Re, Sm, Ho, Lu, Pm, Y, Bi, Pd, Gd, La, Au, Au, Yb, Dy, Cu, Rh, Ag and Ir.

[0071] In some embodiments, the detectable label is a fluorophore or quantum dot. Without wishing to be bound by theory, the use of fluorescent agents can reduce the signal-to-noise ratio in imaging / readout, thereby maintaining sensitivity. Quencher molecules

[0072] The advantage of the probes described herein is that the inhibition of the reporter molecule is released only within a specific thermal channel (eg, temperature range) due to the denaturation-mediated removal of the quencher molecule. Multiple reporter molecules and their corresponding quencher pairs can be designed to denature and induce fluorescence at multiple different thermal channels.

[0073] Therefore, embodiments of various aspects described herein include quencher molecules.For example, the quencher chain includes a quencher molecule.Quencher can play a role in reducing the detectable characteristics (for example, intensity, color, etc.) of the detectable signal from the reporter molecules provided herein.In some embodiments, the quencher molecule is at the 5' end of the quencher chain.In some embodiments, the quencher molecule is at the 3' end of the quencher chain.In some embodiments, the quencher molecule is at the internal position of the quencher chain.Usually, the quencher molecule is located in the quencher chain so that when the quencher chain and the reporter chain are hybridized with each other, the detectable characteristics (for example, intensity, color, etc.) of the detectable signal from the reporter molecule are reduced or quenched.And, when the quencher chain and the reporter chain are not hybridized with each other, the detectable characteristics (for example, intensity, color, etc.) of the detectable signal from the reporter molecule are not quenched or reduced.For example, multiple quencher molecules can be used to quench multiple different reporter molecules.In some embodiments, the nucleic acid probe comprises 2, 3, 4, 5 or more quencher molecules, which can be the same or different from each other.

[0074] In some embodiments of any aspect, the quenching is partial quenching or complete quenching. As used herein, the term "complete quenching" refers to the inability to detect any signal from the reporter molecule, i.e., 100% quenched or 0% detectable signal (e.g., fluorescence). As used herein, the term "partial quenching" refers to a decrease in the detectable signal from the reporter molecule compared to the total detectable signal from the reporter molecule. In some embodiments of any aspect, "partial quenching" refers to a decrease in the signal from the reporter molecule by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23% , at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least %, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least %, at least 99%, at least 99.1%, at least 99.5%, at least 99.9%, or more.

[0075] In some embodiments of any aspect, at least one quencher molecule quenches a specific wavelength of fluorescence emitted by a reporter molecule in a reporter probe. As a non-limiting example, some fluorophores (e.g., TET, HEX, and FAM) with an emission range between 500 nm and 550 nm are quenched by quenchers (e.g., Black hole quencher 1 (BHQ1) and Dabcyl) with an absorption range of 450 nm to 550 nm. Similarly, TMR, Texas Red, ROX, Cy3, and Cy5 are quenched by BHQ2. See, e.g., Marras, Selection of fluorophore and quencher pairs for fluorescent nucleic acid hybridization probes, Methods Mol Biol. 2006; 335: 3-16; the contents of which are incorporated herein by reference in their entirety.

[0076] In some embodiments of any aspect, the quencher molecule is a dark quencher. A dark quencher (also called a dark sucker) is a substance that absorbs excitation energy from a reporter molecule (e.g., a fluorophore) and dissipates the energy as heat; whereas a typical (fluorescent) quencher would re-emit most of this energy as light. Non-limiting examples of quencher molecules (e.g., non-fluorescent or dark quenchers that dissipate energy absorbed from a fluorescent dye) include Black Hole Quenchers TM (Biosearch Technologies TM ), Iowa Black quencher (e.g., Iowa Black FQ TM (“3IABkFQ”) and Iowa Black RQ TM (e.g., "3IAbRQSp")), Dark Quencher (Epoch Biosciences TM ), Zen TM Quencher (Integrated DNA Technologies TM , for example, "ZEN"), TAO TM Quencher (Integrated DNA Technologies TM , for example, "TAO"), Dabcyl (4-(4'-dimethylaminophenylazo)benzoic acid), Qxl TM Quencher, Quencher and QC-1. Other non-limiting examples of quenchers are also provided in U.S. Patent Nos. 6,465,175, 7,439,341, 12 / 252,721, 7,803,536, 12 / 853,755, 7,476,735, 7,605,243, 7,645,872, 8,030,460, 13 / 224,571, 8,916,345, the contents of each of which are incorporated herein by reference in their entirety.

[0077] In some embodiments of any aspect, the quencher molecule is Iowa In some embodiments of any aspect, Iowa The quencher is preferably located at the 5' or 3' position of the nucleic acid probe. In some embodiments of any aspect, the quencher molecule is Iowa FQ, which has a broad absorption spectrum ranging from 420nm to 620nm with a peak absorption at 531nm (i.e., the green-yellow region of the visible light spectrum). FQ (e.g., "3IABkFQ") is used to quench fluorescein or other fluorescent dyes that emit in the green to pink spectral range. In some embodiments of any aspect, the quencher molecule is Iowa RQ, which has a broad absorbance spectrum ranging from 500nm to 700nm with a peak absorption at 656nm (i.e., the orange-red region of the visible light spectrum). RQ (e.g., "3IAbRQSp") is used to quench Texas A Cy5 or other fluorescent dyes.

[0078] In some embodiments of any aspect, the quencher molecule is a ZEN quencher. In some embodiments of any aspect, the ZEN quencher is preferably located at an internal position of the nucleic acid probe. See, for example, Lennox et al., Mol Ther Nucleic Acids. 2013 Aug; 2(8): e117; U.S. Patents 8916345, 9506059; the contents of each of which are incorporated herein by reference in their entirety. ZEN can quench the Iowa In some embodiments, the nucleic acid probe comprises a fluorophore of a similar FQ range (e.g., FAM, SUN, JOE, HEX, or MAX). FQ and a reporter molecule (eg, FAM).

[0079] In some embodiments of any aspect, the quencher molecule is a TAO quencher. In some embodiments of any aspect, the TAO quencher is preferably located at an internal position of the nucleic acid probe. TAO can quench the Iowa In some embodiments, the nucleic acid probe comprises TAO, Iowa RQ and reporter molecule (eg Cy5).

[0080] In some embodiments of any aspect, the quencher molecule is a black hole quencher. TM It is a structure comprising at least three free radicals selected from substituted or unsubstituted aryl or heteroaryl compounds or combinations thereof, wherein at least two of the residues are linked by an exocyclic diazo bond (see, e.g., International Publication No. WO2001086001). Black Hole quenchers (BHQ) are capable of quenching over the entire visible spectrum. Non-limiting examples of Black Hole quenchers include BHQ-0 (430-520 nm); BHQ-1 (480-580 nm, absorbance (abs) maximum 534 nm); BHQ-2 (520-650 nm, abs maximum 544 nm); BHQ-3 (620-730 nm, abs maximum 672 nm); and BHQ-10 (480-550 nm, abs maximum 516 nm; water soluble).

[0081] In some embodiments of any aspect, the quencher molecule is Dabcyl (4-(4'-dimethylaminophenylazo)benzoic acid) or a derivative thereof. Dabcyl absorbs in the green region of the visible spectrum (e.g., 346-489 nm, with a peak absorbance at 474 nm) and can be used with fluorescein or other fluorophores that emit in the green region.

[0082] In some embodiments of any aspect, the quencher molecule is Dark Quencher. The maximum absorbance of Eclipse Quencher is located at 522nm compared to 479nm for Dabcyl. In addition, the structure of Eclipse Quencher is substantially more electron deficient than that of Dabcyl, and this allows for better quenching over a wider range of dyes, particularly those with emission maxima at longer wavelengths (red shifted) (e.g. Redmond Red and Cyanine 5). Furthermore, with an absorption range from 390nm to 625nm, Eclipse Quencher is able to effectively quench a wide range of fluorophores.

[0083] In some embodiments of any aspect, the quencher molecule is Quenchers. Non-limiting examples of QSY quenchers include QSY35 (410-500 nm, abs max 475 nm), QSY7 (500-600 nm, abs max 560 nm), QSYS21 (590-720 nm, abs max 661 nm), and QSY9 (500-600 nm, abs max 562 nm).

[0084] In some embodiments of any aspect, the quencher molecule is Qxl TM Quencher. Qxl TM The quenchers span the entire visible spectrum. Non-limiting examples of QXL quenchers include QXL490 (abs max 495 nm, useful as a quencher for EDANS, AMCA, and most coumarin fluorophores), QXL520 (abs max ~520 nm, useful as a quencher for FAM), QXL570 (abs max 578 nm, useful as a quencher for rhodamines (such as TAMRA, sulforhodamine B, ROX) and Cy3 fluorophores), QXL610 (abs max ~610 nm, useful as a quencher for ROX), and QXL670 (abs max 668 nm, useful as a quencher for Cy5 and Cy5-like fluorophores (such as HiLyte TM Fluor 647) quencher).

[0085] In some embodiments of any aspect, the quencher molecule is IRDye QC- 1. IRDye QC- 1 quenches dyes from the visible to near infrared range (500-900 nm, abs max 737 nm).

[0086] It is worth noting that the quencher molecule does not have to be a specially designed quencher modification. For example, a G base will typically quench a nearby fluorescent signal. Therefore, in some embodiments of any aspect, the quencher is a modified or classical nucleic acid base that can suppress a fluorescent signal. Nucleic acid modification

[0087] Various nucleic acid chains described herein (e.g., target binding strands, reporter strands, quencher strands, and / or reference strands) can independently include one or more nucleic acid modifications known in the art. For example, target binding strands, reporter strands, quencher strands, and / or reference strands can independently include non-naturally occurring nucleic acids and / or non-naturally occurring nucleotides and / or nucleotide analogs and / or chemical modifications. Non-naturally occurring nucleic acids can include, for example, a mixture of naturally occurring and non-naturally occurring nucleotides. Non-naturally occurring nucleotides and / or nucleotide analogs can be modified at ribose, phosphate, and / or base moieties.

[0088] Exemplary nucleic acid modifications include, but are not limited to, nucleobase modifications, sugar modifications, intersugar linkage modifications, conjugates (e.g., ligands), and combinations thereof. Nucleic acid modifications are known in the art, see, for example, US20160367702, US20190060458, U.S. Pat. No. 8,710,200, and U.S. Pat. No. 7,423,142, which are incorporated herein by reference in their entirety.

[0089] Exemplary modified nucleobases include, but are not limited to, thymine (T), inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidine; and substituted or modified analogs of adenine, guanine, cytosine, and uracil, such as 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and Cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-aminoallyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenine and guanine, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 Substituted purines (including 2-aminopropyladenine), 5-propynyl uracil and 5-propynyl cytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyl uracil, 7-alkyl guanine, 5-alkyl cytosine, 7-deazaadenine, N6,N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted 1,2,4-triazoles, 2-pyridones, 5-nitroindole, 3-nitropyrrole, 5- Methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino-3-carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N4-acetylcytosine, 2-thiocytosine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanine or O-alkylated base. Further, purines and pyrimidines include those disclosed in U.S. Pat. No. 3,687,808; Concise Encyclopedia of Polymer Science and Engineering, pp. 858-859, Kroschwitz, JI, ed., John Wiley & Sons, 1990; and Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613.

[0090] Exemplary sugar modifications include, but are not limited to, 2'-fluoro, 3'-fluoro, 2'-OMe, 3'-OMe, and acyclic nucleotides, such as peptide nucleic acids (PNA), unlocked nucleic acids (UNA), or glycol nucleic acids (GNA).

[0091] In some embodiments, nucleic acid modifications may include replacement or modification of intersugar linkages. Exemplary intersugar linkage modifications include, but are not limited to, phosphotriesters, methylphosphonates, phosphoramidates, phosphorothioates, methylenemethylimino, thiodiesters, thiocarbamates, siloxanes, N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-), amide-3 (3'-CH2-C(=O)-N(H)-5') and amide-4 (3'-CH 2 -N(H)-C(=O)-5'), hydroxylamino, siloxane (dialkylsiloxane), carboxamide, carbonate, carboxymethyl, carbamate, carboxylate, thioether, ethylene oxide linker, sulfide, sulfonate, sulfonamide, sulfonate, thioformal (3'-S-CH 2 -O-5'), methylal (3'-O-CH 2 -O-5'), oxime, methylene imino, methylene carbonyl amino, methylene methyl imino (MMI, 3'-CH 2 -N(CH 3 )-O-5'), methylenehydrazono, methylenedimethylhydrazono, methyleneoxymethylimino, ether (C3'-O-C5'), thioether (C3'-S-C5'), thioacetamide (C3'-N(H)-C(=O)-CH 2 -S-C5'),C3'-OP(O)-O-SS-C5',C3'-CH 2 -NH-NH-C5', 3'-NHP(O)(OCH 3 )-O-5' and 3'-NHP(O)(OCH 3 )-O-5'.

[0092] In some embodiments of any aspect, the 2'-modified nucleoside comprises a modification selected from the group consisting of 2'-halo (e.g., 2'-fluoro), 2'-alkoxy (e.g., 2'-O-methyl, 2'-O-methylmethoxy, and 2'-O-methylethoxy), 2'-aryloxy, 2'-O-amine, or 2'-O-alkylamine (amine NH 2 ; alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino, diheteroarylamino, ethylenediamine or polyamino), O-CH 2 CH 2 (NCH 2 CH2 NMe 2 ) 2 , methyleneoxy (4′-CH 2 -O-2′)LNA, ethyleneoxy (4′-(CH 2 ) 2 -O-2′)ENA, 2′-amino (e.g., 2′-NH 2 , 2'-alkylamino, 2'-dialkylamino, 2'-heterocyclicamino, 2'-arylamino, 2'-diarylamino, 2'-heteroarylamino, 2'-diheteroarylamino and 2'-amino acid); NH(CH 2 CH 2 NH) n CH 2 CH 2 -AMINE(AMINE=NH 2 , alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diheteroarylamino), -NHC(O)R (R = alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), 2'-cyano, 2'-thiol, 2'-alkyl-thio-alkyl, 2'-thioalkoxy, 2'-thioalkyl, 2'-alkyl, 2'-cycloalkyl, 2'-aryl, 2'-alkenyl and 2'-alkynyl.

[0093] In some embodiments, nucleic acid modifications can include peptide nucleic acids (PNA), bridged nucleic acids (BNA), morpholinos, locked nucleic acids (LNA), glycol nucleic acids (GNA), threose nucleic acids (TNA), or other xenonucleic acids (XNA) described in the art.

[0094] When present, nucleic acid modifications may be present at any position of the chain comprising the modification. For example, when the target binding strand comprises nucleic acid modifications, such modifications may be present in the target binding nucleic acid of the target binding strand, in the first hybridization domain, and / or in the reference domain. Alternatively, the nucleic acid modifications in the target binding strand may be located outside the domain of the target binding strand. Similarly, when the reporter strand comprises nucleic acid modifications, such modifications may be present in the first hybridization domain of the reporter strand, in the second hybridization domain, or outside the first and second domains. Similarly, when the quencher strand comprises nucleic acid modifications, such modifications may be present in the first hybridization domain of the quencher strand or outside the first hybridization domain. Similarly, when the reference strand comprises nucleic acid modifications, such modifications may be present in the first hybridization domain of the reference strand or outside the first hybridization domain.

[0095] The nucleic acid strands described herein (e.g., target binding strand, reporter strand, quencher strand, and reference strand) can independently comprise at least one thermal destabilizing modification or thermal stabilizing modification to optimize denaturation of a hybridization domain from its complementary domain within a thermal channel.

[0096] Thus, in some embodiments, at least one of the second hybridization domain of the reporter strand and the first hybridization domain of the quencher strand comprises at least one thermal destabilizing modification.

[0097] In some embodiments of any of the aspects described herein, at least one of the first hybridization domain of the reporter strand and the first hybridization domain of the target binding strand comprises at least one thermal destabilizing modification.

[0098] In some embodiments of any of the aspects described herein, the target binding agent is a nucleic acid and comprises at least one thermal destabilizing modification.

[0099] In some embodiments of any of the aspects described herein, at least one of the second hybridization domain of the target binding strand and the first hybridization domain of the reference strand comprises at least one thermal destabilizing modification.

[0100] In some embodiments, at least one of the second hybridization domain of the reporter strand and the first hybridization domain of the quencher strand comprises at least one thermostabilizing modification.

[0101] In some embodiments of any of the aspects described herein, at least one of the first hybridization domain of the reporter strand and the first hybridization domain of the target binding strand comprises at least one thermostabilizing modification.

[0102] In some embodiments of any of the aspects described herein, the target binding agent is a nucleic acid and comprises at least one thermostabilizing modification.

[0103] In some embodiments of any of the aspects described herein, at least one of the second hybridization domain of the target binding strand and the first hybridization domain of the reference strand comprises at least one thermostabilizing modification.

[0104] In some embodiments of the various aspects described herein, at least one of the second hybridization domain of the reporter chain and the first hybridization domain of the quencher chain comprises at least one thermal destabilizing modification, and at least one of the first hybridization domain of the reporter chain and the first hybridization domain of the target binding chain comprises at least one thermal stabilizing modification.

[0105] In some embodiments of the various aspects described herein, at least one of the second hybridization domain of the reporter strand and the first hybridization domain of the quencher strand comprises at least one thermal destabilizing modification, and the target binding domain of the target binding strand comprises at least one thermal stabilizing modification. Target molecule

[0106] The methods described herein, probe groups and / or kits can be used to detect any desired molecule. For example, any target of interest can be detected using the methods described herein, probe groups and / or kits. Exemplary target molecules that can be detected by the methods described herein, probe groups and / or kits include, but are not limited to, nucleic acids (e.g., DNA, RNA, microRNA), proteins, sugars (e.g., polysaccharides), lipids, small molecules and antigens. In some embodiments, the target molecule is a biomolecule. As used herein, "biomolecule" is any molecule produced by a living organism, including large macromolecules and small molecules, such as nucleic acids (e.g., DNA and RNA, such as mRNA), proteins (e.g., antibodies), polysaccharides, lipids, and small molecules such as primary metabolites, secondary metabolites and natural products. The examples of describing nucleic acid detection included in this disclosure are only for illustrative purposes and are not intended to limit the scope of the present invention.

[0107] In some embodiments of any of the aspects, the target molecule is a nucleic acid.

[0108] In some embodiments, the target is a nucleic acid, such as a nucleic acid in a cell environment. For example, the target is a nucleic acid, such as DNA or RNA. In some embodiments, the target can be a DNA product of genomic DNA, cDNA, mRNA, RNA that undergoes reverse transcription. In some embodiments, the target is a nucleic acid amplification product.

[0109] In some embodiments of any of the aspects, the target molecule is a protein.

[0110] In some embodiments, the target may be a protein target, such as a protein of the cellular environment (e.g., an intracellular protein or a membrane protein). Examples of proteins include, but are not limited to, fibrous proteins, such as cytoskeletal proteins (e.g., actin, arp2 / 3, coronin, dystrophin, FtsZ, keratin, myosin, nebulin, spectrin, tau, titin, tropomyosin, tubulin, and collagen) and extracellular matrix proteins (e.g., collagen, elastin, f-spondin, pikachurin, and fibronectin); globular proteins, such as plasma proteins (e.g., serum amyloid P component and serum albumin), coagulation factors (e.g., complement proteins, C1 inhibitor and C3 convertase, factor VIII, factor XIII, fibrin, protein C, protein S, protein Z, protein Z-related protease inhibitor, thrombin, von Willebrand factor (Von Willebrand factor), and fibronectin. Factor) and acute phase proteins, such as C-reactive protein; heme proteins; cell adhesion proteins (e.g., cadherins, ependymin, integrins, Ncam, and selectins); transmembrane transporters (e.g., CFTR, glycophorin D, and scramblase), such as ion channels (e.g., ligand-gated ion channels, such as nicotinic acetylcholine receptors and GABAa receptors; and voltage-gated ion channels, such as potassium, calcium, and sodium channels), synport / antiport proteins (e.g., glucose transporters); hormones and growth factors (e.g., epidermal growth factor (EGF), fibroblast growth factor (GFFA), and mitochondrial growth factor (MGF). factors (FGF), vascular endothelial growth factor (VEGF), peptide hormones such as insulin, insulin-like growth factor and oxytocin, and steroid hormones such as androgens, estrogens and progesterone); receptors, such as transmembrane receptors (e.g., G protein-coupled receptors, rhodopsin) and intracellular receptors (e.g., estrogen receptor); DNA binding proteins (e.g., histones, protamines, CI proteins); transcriptional regulators (e.g., c-myc, FOXP2, FOXP3, MyoD and P53); immune system proteins (e.g., immunoglobulins, major histocompatibility antigens and T cell receptors); nutrient storage / transport proteins (e.g., ferritin); chaperone proteins; and enzymes.

[0111] In some embodiments of any of the aspects, the target molecule is in a cell. Sample / Test piece

[0112] Methods, probe sets and kits for detecting targets in samples are described herein. As used herein, the term "sample" or "test sample" may refer to a sample extracted or separated from a biological organism (e.g., a subject to be tested). Exemplary biological samples include tissue samples, such as liver, spleen, kidney, lung, intestine, thymus, colon, tonsil, testis, skin, brain, heart, muscle and pancreatic tissue. Other exemplary biological samples include, but are not limited to, biopsies, bone marrow samples, organ samples, skin fragments and organisms. Materials obtained from clinical or forensic environments are also within the intended meaning of the term biological sample. In one embodiment, the sample is derived from a human, animal or plant. In one embodiment, the biological sample is a tissue sample, preferably an organ tissue sample. In one embodiment, the sample is a human. For example, a sample can be obtained from an autopsy, a biopsy, a muscle punch or an operation. It can be a solid tissue or solid tumor, such as parenchyma, connective tissue or adipose tissue, heart or skeletal muscle, smooth muscle, skin, brain, nerve, kidney, liver, spleen, breast, carcinoma (e.g., intestine, nasopharynx, breast, lung, stomach, etc.), cartilage, lymphoma, meningioma, placenta, prostate, thymus, tonsil, umbilical cord or uterus. The tissue can be a tumor (benign or malignant), cancerous or precancerous tissue. The sample can be obtained from an animal or human subject that is affected by disease or other pathology, or suspected of being affected by disease or other pathology (normal or diseased), or is considered normal or healthy.

[0113] In some embodiments of any aspect, the biological sample is a sputum sample, a pharyngeal sample, or a nasal sample. In some embodiments of any aspect, the biological sample is a cell, or a tissue, or peripheral blood, or a body fluid. In some embodiments, the biological sample is a biopsy, a tumor sample, a biological fluid sample; blood; serum; plasma; urine; semen; mucus; tissue biopsy; organ biopsy; synovial fluid; bile; cerebrospinal fluid; mucosal secretions; effusion; sweat; saliva; interstitial fluid; or a tissue sample. The term also includes mixtures of the above samples. The term "test sample" also includes untreated or pretreated (or preprocessed) biological samples.

[0114] In some embodiments of any aspect, the test sample can be an untreated test sample. As used herein, the phrase "untreated test sample" refers to a test sample that has not undergone any prior sample pretreatment except dilution and / or suspension in a solution. Example methods for processing a test sample include, but are not limited to, centrifugation, filtration, ultrasound, homogenization, heating, freezing and thawing, and combinations thereof. In some embodiments of any aspect, the test sample can be a frozen test sample. Before using the methods, assays, and systems described herein, the frozen sample can be thawed. After thawing, the frozen sample can be centrifuged and then subjected to the methods, assays, and systems described herein. In some embodiments of any aspect, the test sample is a clarified test sample, for example, by centrifugation and collecting a supernatant containing the clarified test sample. In some embodiments of any aspect, the test sample can be a pre-processed test sample, for example, a supernatant or filtrate produced from a treatment selected from the group consisting of: centrifugation, homogenization, ultrasound, filtration, thawing, purification, and any combination thereof. In some embodiments of any aspect, the test sample can be treated with chemical and / or biological reagents. For example, chemical and / or biological reagents can be used to protect and / or maintain the stability of the sample, including the biomolecules (e.g., nucleic acids and proteins) therein during processing. Those skilled in the art are fully aware of methods and processes for pre-processing of biological samples suitable for detecting targets (e.g., nucleic acids) as described herein. Generate Probe

[0115] The nucleic acid chains described herein can be synthesized using any probe or oligonucleotide synthesis method known in the art, including but not limited to solid phase oligonucleotide synthesis, RNA synthesis, or by using an automated DNA / RNA synthesizer (e.g., Sierra Biosystems (Sonora, CA), Biolytic (Fremont, CA)). Alternatively, probes can be synthesized by one or more commercial sources, including but not limited to Bio Basic (Amherst, New York), Integrated DNA technologies (Coralville, Iowa), Trilink Biotechnologies (San Diego, CA), etc. Reagent test kit

[0116] In one aspect, the present invention provides a kit for detecting a target molecule. The kit may include any probe set described herein and packaging and materials therefor. application

[0117] The methods, detection probe sets and kits described herein can be used with any method known in the art for detecting target molecules (e.g., gene sequences or RNA substances) in single cells or tissues. Non-limiting examples of applications that can be used with nucleic acid encoding probes and compositions described herein.

[0118] In one embodiment, the detection probe groups described herein can be used for transcriptional profiling in single cells. In some embodiments, the transcriptional state of the cell is imaged by detecting and distinguishing each mRNA. Fluorescence in situ hybridization (FISH) allows labeling and imaging of single mRNA molecules in fixed cells. This is achieved by hybridizing mRNA with a nucleic acid encoding probe having a sequence complementary to the mRNA sequence.

[0119] In another embodiment, nucleic acid encoding probes described herein can be used in methods for mapping chromosome structure. Chromosome rearrangements are involved in various forms of cancer, and recent studies have revealed that the chromosomes of eukaryotic organisms are packaged in a nonlinear and complex manner. Nucleic acid encoding probes can be used to image chromosome structure and determine its conformation in a single cell. Physical images of chromosomes and the addressing of individual genes in cells will enable unprecedented observations of how genomes are compressed, compare the organization of transcriptionally active and repressed regions, and detect subtle changes in genome structure in tumor cells.

[0120] In another embodiment, nucleic acid encoding probes can be used in methods for imaging transcription factor binding in single cells. Transcription factors (TFs) control genes in transcription networks through binding sites on DNA and interactions with regulatory proteins. The location distribution and binding state of a specific TF on a chromosome determine the transcriptional program it accesses in a cell. By incorporating transcription factor binding domains into nucleic acid encoding probes, the physical location of each TF can be determined. Those skilled in the art will appreciate that it may be necessary to achieve a higher melting point for the DNA binding domain or to make the interaction irreversibly modified nucleosides to allow temperature changes in the hot channel, which allows, for example, at least one fluorophore / quencher pair to denature, to allow detection of transcription factors.

[0121] Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, etc. may be made without departing from the spirit of the invention, and these are therefore considered to fall within the scope of the invention as defined in the claims hereinafter. In addition, to the extent not yet indicated, it will be understood by those of ordinary skill in the art that any of the various embodiments described and illustrated herein may be further modified to incorporate the features shown in any other embodiment disclosed herein.

[0122] Some implementations of the technology described herein may be defined according to any of the following numbered paragraphs:

[0123] Paragraph 1: A method for detecting a target molecule in a sample, the method comprising: (a) providing a detection probe set, wherein the detection probe set comprises: (i) a first nucleic acid chain, the first nucleic acid chain comprising a target binding domain connected to a first hybridization domain; (ii) a second nucleic acid chain, the second nucleic acid chain comprising a second hybridization domain connected to a third hybridization domain, wherein the second hybridization domain comprises a nucleotide sequence substantially complementary to the nucleotide sequence of the first hybridization domain, and wherein the second nucleic acid chain comprises a reporter molecule capable of generating a detectable signal; and (iii) a third nucleic acid chain, the third nucleic acid chain comprising a fourth hybridization domain, wherein The fourth hybridization domain comprises a nucleotide sequence that is substantially complementary to the nucleotide sequence of the third hybridization domain, wherein the third nucleic acid chain comprises a quencher molecule, and wherein, when the nucleic acid probe is not hybridized to the amplicon, the quencher molecule quenches the detectable signal from the reporter molecule, and wherein the melting temperature of the third chain hybridized to the second chain is lower than the melting temperature of the second chain hybridized to the first chain, and wherein the melting temperature of the third chain hybridized to the second chain is lower than the melting temperature of the first chain bound to the target molecule; and (b) analyzing the binding of the probe set to the target by evaluating the detectable signal generated as a function of temperature.

[0124] Paragraph 2: A method for multiplex detection of target molecules in a sample, the method comprising: (a) providing a plurality of detection probe sets, wherein each detection probe set comprises: (i) a first nucleic acid chain, wherein the first nucleic acid chain comprises a target binding domain connected to a first hybridization domain; (ii) a second nucleic acid chain, wherein the second nucleic acid chain comprises a second hybridization domain connected to a third hybridization domain, wherein the second hybridization domain comprises a nucleotide sequence substantially complementary to the nucleotide sequence of the first hybridization domain, and wherein the second nucleic acid chain comprises a reporter molecule capable of generating a detectable signal; and (iii) a third nucleic acid chain, wherein the third nucleic acid chain comprises a fourth hybridization domain, wherein the fourth hybridization domain comprises a reporter molecule capable of generating a detectable signal. wherein the third nucleic acid strand comprises a nucleotide sequence that is substantially complementary to the nucleotide sequence of the target molecule, wherein the third nucleic acid strand comprises a quencher molecule, and wherein the quencher molecule quenches the detectable signal from the reporter molecule when the nucleic acid probe is not hybridized to the amplicon, and wherein the melting temperature of the third strand hybridized to the second strand is lower than the melting temperature of the second strand hybridized to the first strand, and wherein the melting temperature of the third strand hybridized to the second strand is lower than the melting temperature of the first strand bound to the target molecule, and wherein the melting temperature of the third strand hybridized to the second strand in at least one probe set is different from the melting temperature of the third strand hybridized to the second strand in at least one other probe set; and (b) analyzing binding of the probe set to the target by evaluating the detectable signal generated as a function of temperature.

[0125] Paragraph 3: The method of Paragraph 2, wherein the melting temperature of the third strand hybridized to the second strand in at least one probe set is at least 5°C lower than the melting temperature of the third strand hybridized to the second strand in at least one other probe set.

[0126] Paragraph 4: The method of any one of Paragraphs 2-3, wherein the reporter molecules in different probe sets are the same.

[0127] Paragraph 5: The method of any of Paragraphs 2-3, wherein the reporter molecule in at least one probe set is different from the reporter molecule in at least one other probe set.

[0128] Paragraph 6: The method of any of paragraphs 1-5, wherein the melting temperature of the third strand hybridized to the second strand is at least 5°C lower than the melting temperature of the second strand hybridized to the first strand.

[0129] Paragraph 7: The method of any one of paragraphs 1-6, wherein the melting temperature of the third strand hybridized to the second strand is at least 5°C lower than the melting temperature of the first strand bound to the first strand of the target molecule.

[0130] Paragraph 8: The method of any of paragraphs 1-7, wherein at least one of the first strand, the second strand, or the third strand comprises a nucleic acid modification.

[0131] Paragraph 9: The method of any one of paragraphs 1-8, wherein the first hybridization domain of the first chain and / or the second hybridization domain of the second chain comprises a duplex stabilizing modification.

[0132] Paragraph 10: The method of any one of paragraphs 1-9, wherein the third hybridization domain and / or the fourth hybridization domain of the second chain comprises a duplex destabilizing modification.

[0133] Paragraph 11: The method of any of Paragraphs 1-10, wherein the reporter molecule and the quencher molecule are a FRET pair.

[0134] Paragraph 12: A method according to any one of paragraphs 1-11, wherein the reporter molecule is selected from the group consisting of: fluorescent molecules, radioactive isotopes, chromophores, enzymes, enzyme substrates, chemiluminescent moieties, bioluminescent moieties, echogenic substances, non-metallic isotopes, optical reporter molecules, paramagnetic metal ions and ferromagnetic metals.

[0135] Paragraph 13: A method according to any one of paragraphs 1-12, wherein the quencher molecule is a dark quencher.

[0136] Paragraph 14: The method of any one of paragraphs 1-13, wherein the first chain further comprises a reporter domain linked to the target binding domain.

[0137] Paragraph 15: The method according to paragraph 14, wherein the probe group further comprises a fourth nucleic acid chain, wherein the fourth nucleic acid chain comprises a fifth hybridization domain, wherein the fifth hybridization domain comprises a nucleotide sequence substantially complementary to the nucleotide sequence of the reporter domain of the first chain, and optionally, the fourth chain comprises a reporter molecule.

[0138] Paragraph 16: A method according to any one of paragraphs 1-15, wherein the binding of the analytical probe group to the target includes detecting a detectable signal at a first temperature and a second temperature, wherein one of the first temperature or the second temperature is lower than the melting temperature of the third chain hybridized to the second chain, and the other of the first temperature or the second temperature is higher than the melting temperature of the third chain hybridized to the second chain.

[0139] Paragraph 17: A probe set for detecting a target molecule, the probe set comprising: (i) a first nucleic acid chain, the first nucleic acid chain comprising a target binding domain connected to a first hybridization domain; (ii) a second nucleic acid chain, the second nucleic acid chain comprising a second hybridization domain connected to a third hybridization domain, wherein the second hybridization domain comprises a nucleotide sequence substantially complementary to the nucleotide sequence of the first hybridization domain, and wherein the second nucleic acid chain comprises a reporter molecule capable of generating a detectable signal; and (iii) a third nucleic acid chain, the third nucleic acid chain comprising a fourth hybridization domain, wherein the fourth hybridization domain comprises a nucleotide sequence substantially complementary to the nucleotide sequence of the third hybridization domain, wherein the third nucleic acid chain comprises a quencher molecule, and wherein, when the nucleic acid probe is not hybridized with the amplicon, the quencher molecule quenches the detectable signal from the reporter molecule.

[0140] Paragraph 18: A probe set for multiplex detection of target molecules, the probe set comprising a plurality of detection probe sets, wherein each detection probe set comprises: (i) a first nucleic acid chain, the first nucleic acid chain comprising a target binding domain connected to a first hybridization domain; (ii) a second nucleic acid chain, the second nucleic acid chain comprising a second hybridization domain connected to a third hybridization domain, wherein the second hybridization domain comprises a nucleotide sequence substantially complementary to the nucleotide sequence of the first hybridization domain, and wherein the second nucleic acid chain comprises a reporter molecule capable of generating a detectable signal; and (iii) a third nucleic acid chain, the third nucleic acid chain comprising a fourth hybridization domain, wherein the fourth hybridization domain The domain comprises a nucleotide sequence that is substantially complementary to the nucleotide sequence of the third hybridization domain, wherein the third nucleic acid strand comprises a quencher molecule, and wherein, when the nucleic acid probe is not hybridized to the amplicon, the quencher molecule quenches the detectable signal from the reporter molecule, and wherein the melting temperature of the third strand hybridized to the second strand is lower than the melting temperature of the second strand hybridized to the first strand, and wherein the melting temperature of the third strand hybridized to the second strand is lower than the melting temperature of the first strand bound to the target molecule, and wherein the melting temperature of the third strand hybridized to the second strand in at least one probe set is different from the melting temperature of the third strand hybridized to the second strand in at least one other probe set.

[0141] Paragraph 19: The probe set of Paragraph 18, wherein the melting temperature of the third strand hybridized to the second strand in at least one probe set is at least 5°C lower than the melting temperature of the third strand hybridized to the second strand in at least one other probe set.

[0142] Paragraph 20: The probe set described in any of Paragraphs 18-19, wherein the reporter molecules in different probe sets are the same.

[0143] Paragraph 21: The probe set of any of Paragraphs 18-19, wherein the reporter molecule in at least one probe set is different from the reporter molecule in at least one other probe set.

[0144] Paragraph 22: The probe set of any of paragraphs 17-21, wherein the melting temperature of the third strand hybridized to the second strand is at least 5°C lower than the melting temperature of the second strand hybridized to the first strand.

[0145] Paragraph 23: The probe set of any of paragraphs 17-22, wherein the melting temperature of the third strand hybridized to the second strand is at least 5°C lower than the melting temperature of the first strand bound to the first strand.

[0146] Paragraph 24: The probe set of any of paragraphs 17-23, wherein at least one of the first strand, the second strand, or the third strand comprises a nucleic acid modification.

[0147] Paragraph 25: The probe set described in any of paragraphs 17-24, wherein the first hybridization domain of the first chain and / or the second hybridization domain of the second chain comprises a duplex stabilizing modification.

[0148] Paragraph 26: The probe set as described in any of paragraphs 17-25, wherein the third hybridization domain and / or the fourth hybridization domain of the second strand comprises a duplex destabilizing modification.

[0149] Paragraph 27: The probe set of any of Paragraphs 17-26, wherein the reporter molecule and the quencher molecule are a FRET pair.

[0150] Paragraph 28: A probe group as described in any of paragraphs 17-27, wherein the reporter molecule is selected from the group consisting of: a fluorescent molecule, a radioactive isotope, a chromophore, an enzyme, an enzyme substrate, a chemiluminescent moiety, a bioluminescent moiety, an echogenic substance, a non-metallic isotope, an optical reporter molecule, a paramagnetic metal ion, and a ferromagnetic metal.

[0151] Paragraph 29: The probe set of any of Paragraphs 17-28, wherein the quencher molecule is a dark quencher.

[0152] Paragraph 30: The probe set of any of paragraphs 17-29, wherein the first strand further comprises a reporter domain linked to the target binding domain.

[0153] Paragraph 31: A probe group as described in any of paragraphs 17-30, wherein the probe group further comprises a fourth nucleic acid chain, wherein the fourth nucleic acid chain comprises a fifth hybridization domain, wherein the fifth hybridization domain comprises a nucleotide sequence substantially complementary to the nucleotide sequence of the reporter domain of the first chain, and optionally, the fourth chain comprises a reporter molecule.

[0154] Paragraph 32: A kit for detecting a target molecule, the kit comprising the probe set described in any one of Paragraphs 17-31. Some selected definitions

[0155] For convenience, certain terms used herein in the application documents, examples and appended claims are collected herein. Unless otherwise specified or implied in the context, the following terms and phrases include the meanings provided below. Unless otherwise explicitly stated or obvious from the context, the following terms and phrases do not exclude the meanings that the term or phrase has acquired in the field to which it belongs. These definitions are provided to help describe specific embodiments and are not intended to limit the claimed invention, as the scope of the invention is limited only by the claims. In addition, unless the context requires otherwise, singular terms shall include plural forms and plural terms shall include singular forms.

[0156] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art to which the invention belongs. Although any known methods, devices and materials can be used in the practice or testing of the present invention, the methods, devices and materials in this regard are described herein.

[0157] In addition, unless otherwise indicated, the practice of the present invention can adopt the conventional techniques of molecular biology (including recombinant technology), microbiology, cell biology, biochemistry and immunology, which are within the scope of those skilled in the art. Such technology has been fully explained in the literature, such as "Molecular Cloning: A Laboratory Manual", Second Edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (MJ Gait, 1984); "Animal Cell Culture" (RI Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (FM Ausubel, 1987, and periodic updates); "PCR: The Polymerase Chain Reaction", (Mullis et al., 1994); "A Practical Guide to Molecular Cloning" (Perbal Bernard V., 1988); "Phage Display: A Laboratory Manual" (Barbas et al., 2001).

[0158] When providing a range of values, it is understood that unless the context clearly indicates otherwise, each intermediate value (to one-tenth of the lower limit unit) between the upper and lower limits of the range and any other indicated or intermediate values ​​in the indicated range are included in the present invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also included in the present invention, except for any specifically excluded limits in the range. When the indicated range includes one or two limits, the range excluding one or two of these included limits is also included in the present invention.

[0159] Certain ranges are given herein with the term "about" preceding a numerical value. The term "about" is used herein to provide literal support for the exact number that it precedes, as well as numbers that are close to or approximately the number that the term precedes. In determining whether a number is close to or approximately a specifically recited number, a close or approximate unrecited number may be a number that, in the context in which it is presented, provides a substantial equivalence to the specifically recited number.

[0160] As used herein, the terms "comprising" or "comprises" are used to refer to compositions, methods and respective components thereof that are essential to the invention, but are open to the inclusion of unspecified elements, whether essential or not.

[0161] 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. It is further noted that the claims may be written to exclude any optional elements. Therefore, this statement is intended to serve as antecedent basis for use of exclusive terminology such as "solely," "only," or use of a "negative" limitation in conjunction with the recitation of claim elements.

[0162] As used herein, the term "hybridization" refers to the interaction or annealing of two single-stranded nucleic acids into double-stranded nucleic acids (e.g., duplexes) under specific hybridization conditions. Typically, when two single-stranded nucleic acids have sufficient complementarity to each other under a given set of conditions, they will form a duplex. Such conditions can be, for example, stringent conditions, wherein stringent conditions can include: 400mMNaCl, 40mM PIPES pH 6.4, 1mM EDTA, 50°C or 70°C for 12-16 hours, followed by cleaning. Other conditions can be applied, such as physiologically relevant conditions that may be encountered in vivo. The technician will be able to determine the most suitable set of conditions for testing the complementarity of the two sequences based on the final application of the hybridized nucleotides.

[0163] As used herein, the term "thermal channel" refers to different temperature ranges, under which a given double-stranded labeled region of a probe is denatured while other double-stranded labeled regions (including double-stranded regions formed by the binding of the probe to the target nucleic acid) remain annealed. For example, under a specific thermal channel, the double-stranded labeled region denatures, releasing, for example, a quenched fluorophore and allowing detection of targets in cells or tissues. When the term "thermal channel" is used to describe a double-stranded region formed by the binding of a probe to a target nucleic acid, the temperature range is a temperature range that will denature the probe from the target nucleic acid sequence. In some embodiments, the temperature channel includes a temperature range of less than 5°C (e.g., 50°C-55°C), less than 10°C (e.g., 50°C-60°C), less than 15°C (e.g., 50°C-65°C), less than 20°C (e.g., 50°C-70°C), or greater.

[0164] As used herein, the term "substantially complementary" refers to two nucleic acid chains that are sufficiently complementary in sequence to anneal and form a stable duplex, provided that the complementarity is sufficient to produce a melting point within the desired range. Complementarity does not need to be complete; for example, there may be any number of base pair mismatches between two nucleic acids. However, if the number of mismatches is so large that hybridization cannot occur even under the least stringent hybridization conditions, the sequence is not a substantially complementary sequence. When two sequences are referred to herein as "substantially complementary", it means that the sequences are sufficiently complementary to each other to hybridize under the selected reaction conditions. The relationship between hybridization stringency sufficient to achieve specificity and nucleic acid complementarity is well known in the art. Two substantially complementary chains may be, for example, fully complementary, or may contain 1 to multiple mismatches, as long as the hybridization conditions are sufficient to allow, for example, to distinguish between paired and unpaired sequences. Therefore, a "substantially complementary" sequence may refer to a sequence in which the base pair complementarity in the double-stranded region is 100%, 95%, 90%, 80%, 75%, 70%, 60%, 50% or lower or any number therebetween. In one embodiment, a single-stranded region of a DNA or RNA encoding probe is 100% complementary (eg, contains no mismatches) to a given region of a target sequence to allow generation of a binding region with a higher melting point than the double-stranded region of the probe.

[0165] The term "thermal destabilizing modification" includes modifications to a nucleic acid sequence such that the sequence has a lower overall melting temperature (Tm) (preferably, a Tm that is one, two, three or four degrees lower than the Tm of the nucleic acid sequence without such modification). Exemplary thermal destabilizing modifications can include, but are not limited to, abasic modifications, mismatches with the opposite nucleotide in the opposite strand (e.g., G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, and U:T), acyclic nucleotides (e.g., unlocked nucleic acids (UNA) and glycol nucleic acids (GNA), α-nucleotides, nucleotides with impaired Watson-Crick hydrogen bonding, nucleotides with non-classical bases (e.g., inosine, gouache, 2-aminopurine, 2,4-difluorotoluene, 5-nitroindole, 3-nitropyrrole, 4-fluoro-6-methylbenzimidazole, and 4-methylbenzimidazole), universal nucleobases with reduced or absent ability to form hydrogen bonds with the opposite base, and phosphate modifications (e.g., phosphorothioates and alkylphosphonates). Exemplary thermal destabilizing modifications are described, for example, in WO 2011 / 133876 and WO 2010 / 0011895, the contents of both of which are incorporated herein by reference in their entirety.

[0166] In contrast, the term "thermostable modification" includes modifications to a nucleic acid sequence such that the sequence has a higher overall melting temperature (Tm) (preferably, a Tm that is one, two, three, or four degrees higher than the Tm of a nucleic acid sequence without such modifications). In the context of the methods and compositions described herein, thermostabilizing modifications may be included in any region of the probe to achieve a higher melting temperature, thereby allowing the probe to melt within a desired thermal channel. Exemplary thermostabilizing modifications include, but are not limited to, 2'-fluoro modifications, locked nucleic acid (LNA) bases, minor groove binders (MGBs), 5-hydroxybutynyl-2'-deoxyuridine (SuperT), 5-Me-pyridine, 2-amino-deoxyadenosine, trimethoxystilbene, RNA bases, methylated RNA bases, 2'fluoro bases, and pyrene.

[0167] It should be understood that the present disclosure is not limited to the specific methods, protocols, reagents, etc. provided herein, as these may vary. The terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present disclosure, which is limited only by the claims. The present invention is further illustrated by the following examples, which should not be construed as further limiting. Example Example 1: Multiplexed fluorescence cell imaging using DNA-encoded thermal channels and their uses

[0168] Optical fluorescence microscopy is a powerful tool for visualizing biomolecules at both the single cell and tissue levels to reveal complex cell signaling networks and disease states. However, the ability to visualize multiple biological targets is fundamentally limited by the range of the palette. This article provides an excellent and effective method to expand the palette by adding a thermal channel dimension to fluorescence spectral imaging. The inventors have engineered DNA thermal probes that emit fluorescent signals only in the desired temperature channel and remain dark in other temperature channels. This article demonstrates five temperature channels that have minimal crosstalk and can be applied to biological imaging. The temperature channel switching time only takes 5 seconds, which is typically more than 100 times faster than the buffer exchange method in traditional imaging. Through the combination of thermal channels and fluorophore channels, the inventors demonstrated RNA imaging of 15 colors in fixed cells. Thermal multiplexing methods have wide applications in the field of space biology.

[0169] In order to fundamentally solve the color limitation of fluorescence imaging, the inventors developed a DNA thermal display system, which uses in-situ programmable melting of DNA probes induced by thermal fields to generate fluorescent signals in predetermined thermal channels ( Figure 1A). This article demonstrates that thermal fluorescence imaging works robustly in fixed HeLa cells. Channel switching time takes only 5 seconds, which is much faster than the typical microfluidic iterative cleaning system (2min) for traditional imaging. The inventors demonstrated five thermal channels with no observable crosstalk. By combining thermal plex with three widely used fluorescent channels, 15-plex RNA profiling in single cells was successfully demonstrated in situ. DNA thermal visualizers based on DNA-encoded thermal channels will become a powerful tool to expand the space biology toolbox. result

[0170] Design principles for fluorescence imaging with DNA thermal probes. The core part of the thermal-plex method is to design thermal DNA probes that allow efficient signal generation in the desired temperature channel and avoid signal crosstalk between different temperature channels. DNA thermal probes are designed to have two regions (i.e., signal region and binding region) to achieve these goals. The signal region contains a DNA duplex, whose ends are labeled with a fluorophore and a quencher pair. Unless the quencher chain is melted from the probe by thermal field induction at the desired signal temperature channel, the fluorescence of the dye is quenched. The binding region is designed to bind to a target DNA chain that has been attached in situ to a target biomolecule ( Figure 1A The binding region usually has a higher thermal stability than the signal region to ensure signal strength. After the probe binds to the in situ labeled DNA on the biological target, a thermal field is applied to induce a signal ( Figure 1B ). At the desired signal temperature, the quencher chain melts away from the probe, while the imaging agent is held together with the target biomolecule through the binding region. Therefore, the target biomolecule can be visualized by the fluorescent signal released by the imaging agent chain. Figure 1C The thermal curve of the quencher attached to the spot is shown. When switching to a higher temperature channel, the binding region also melts away and the fluorescent signal disappears, as shown in Figure 1C The intersection of the thermal curves of the quenching region and the binding region will give the thermal spectrum of the designed DNA thermal probe, as shown in Figure 1C The signal temperature (Ts) is determined at the peak region of the thermal spectrum.

[0171] Simulation-guided design of thermal channels of DNA thermal probes for multiplex imaging. To realize DNA thermal-plex for cell imaging, DNA thermal probes need to have two key properties: (1) efficiency sufficient to generate fluorescent signals at their signal temperature, and (2) as many channels as possible with non-overlapping thermal spectra for multiplex imaging. To achieve these two goals, the inventors used computer simulation to explore the design space under all combinations of melting temperatures of the binding region and the quenching region. The resulting thermal maps of signal yield and signal temperature are shown in Figure 2. Figure 2A and Figure 2BAs shown. Since the melting temperature of the binding region in the upper right triangle area is higher than that of the quenching region, the signal yield is also higher compared with the lower right triangle area. The design located in the upper right corner produces a relatively wide thermal spectrum, resulting in a small temperature bandwidth for multiplex imaging. To select a design for multiplex imaging, the parameters of the probe should be located in the upper right triangle area but close to the diagonal area of ​​the square space to ensure relatively high signal yield and high multiplex channels. Based on theoretical analysis, such as Figure 2C As shown, five thermal channels (39°C, 48°C, 57°C, 65°C, and 72°C) can be created with minimal crosstalk for space biological applications.

[0172] Validation of in situ RNA imaging using DNA thermal probes: To validate the above methods and principles, a DNA thermal probe was designed to target APC RNA transcripts in Hela cells. The melting temperatures of the signal region and the binding region were designed to be 53°C and 61°C, respectively. The optimal signal temperature obtained was about 57°C. 4 Design the primary probe for APC RNA. After the primary probe sequence was generated, a reference probe binding region was attached to the 5' end of the primary APC probe, and a DNA hot probe binding region was attached to the 3' end. The attached sequences of both the reference and hot probes were checked by blast to avoid alignment with any sequence of the human transcriptome. 5 .

[0173] For in situ RNA imaging, HeLa cells were cultured and fixed on a substrate with a temperature control module. The designed primary probe was incubated with the fixed cells overnight to fully bind to the target RNA, and then the reference probe and the thermal probe were bound to the primary probe ( Figure 3A ). The reference probe is labeled with Atto565 dye, while the hot probe is encoded with Alex647 dye. Then a set of thermal induction temperatures are applied to the substrate to generate a fluorescent signal by melting the DNA strands in situ. The signal is read using a typical fluorescence imaging procedure. Figure 3B As shown in the figure, at a temperature below the signal temperature (e.g., 48°C), only the 565nm channel with the reference probe shows RNA spot signals. At the signal temperature, both the 565nm and 647nm channels show RNA spot signals. When the temperature is switched to 72 degrees, only the 565nm channel shows spot signals again, indicating that the imaging agent chain melts away from the in situ binding site. Figure 3CAs shown, the spot information was analyzed at different temperatures and fluorophore channels, and 90% of the spot positions were very well aligned with the two fluorophore channels. Other unaligned spots may be attributed to the nonspecific binding of the fluorescent probe. The high consistency of the signal between the reference probe and the hot probe indicates that the hot probe can accurately illuminate the target RNA at the designed signal temperature. In the channels with lower or higher temperatures, the signal is completely removed, thus preventing signal crosstalk.

[0174] Fast dissociation kinetics for channel switching: Since the thermal channel color relies on the dissociation of the quencher strand at the signal temperature, the speed of color generation is very fast and can be achieved within seconds based on kinetic analysis. To test the thermal channel switching time, fluorescence imaging data were collected after heating the substrate for different lengths of time at temperatures below, at, and above the signal temperature, e.g. Figure 3E 3F. It was demonstrated that 5 seconds was sufficient for the quencher chain to completely melt to generate a signal at the signal temperature and to remove the imaging agent chain above the signal temperature.

[0175] In iterative binding methods for multiplex fluorescence imaging, washing steps are often time-consuming and may take about 30 minutes or even hours to remove the imaging agent from the previous round and allow the binding of the current imaging agent. DNA thermal imaging can speed up the imaging process by more than 100 times.

[0176] Design of five thermal channels for fluorescence imaging: The inventors have verified that DNA thermal probes can release fluorescence signals at the designed signal temperature while remaining dark in other temperature channels, so they attempted to engineer five thermal channels without signal crosstalk. The designed signal temperatures of the five DNA thermal probes are approximately 39°C, 48°C, 57°C, 65°C, and 72°C. After binding five different DNA thermal probes to the APC primary probe, a thermal field is applied to observe the fluorescence signals under the five designed temperature channels. Figure 4A As shown in the figure, all samples incubated with the designed DNA thermal probes showed fluorescent RNA spot signals only in the corresponding temperature channels, while remaining dark in other channels. The inventors also analyzed the number of RNA spots per cell for each designed thermal probe, and the number of RNA spots per cell detected by each thermal probe was at the same level ( Figure 4B ).

[0177] Multiplexed Cellular RNA Imaging: The thermal-plex approach provides additional signal channels beyond the fluorophore channel, which is powerful for improving the multiplex capability of biological imaging. It is expected that the combination of five engineered thermal channels and three fluorophore channels will provide the ability to increase the multiplexity of fluorescence imaging from 3 to 15. To demonstrate the multiplexed imaging capability, the inventors designed 15 sets of thermal probes to target 15 RNA targets in Hela cells. Each of the three thermal probes is encoded with three different fluorophores, Atto-488, Atto-565, and Alex-647. Figure 5A As shown in the scheme, during the imaging process, images will be taken at 488nm, 565nm, and 647nm channels after each round of temperature channel switching. Figure 5B Fifteen images of each designed thermal probe are shown. Figure 5C Overlay images of a single cell with fifteen colors are shown. The total imaging time took less than 10 min.

[0178] The multiplex fluorescence imaging method based on thermal field-induced sequential melting provides a convenient and effective method for monitoring in situ spatial biology. The previously described multiplex fluorescence imaging method uses spectroscopy 6 ,geometry 7 and iterative encoding 8 Implementation. Although spectral encoding is a simple and convenient method for multiplex imaging, it requires multiple fluorophores, which is fundamentally limited to 3-4 selectable colors. Geometric encoding requires a relatively large-sized bracket to place the fluorophores so that there is enough space for geometric distinction under a diffraction-limited microscope. Iterative binding of imaging agents is powerful for increasing multiplexity through unlimited washing rounds, but it requires setting up complex microfluidics, and washing the imaging agents of the previous round and then incubating the imaging agents of the current round to ensure signal fidelity is very time-consuming. In contrast, the thermal-plex method presented in this article only requires a simple temperature control unit and a designed DNA thermal probe.

[0179] The number of signal channels in thermal-plex has the potential to be highly scalable. The current number of channels is limited by the temperature bandwidth of the DNA thermal probe melting process (usually 8-10 degrees) to ensure that the signal crosstalk between adjacent channels is minimal. 9 Incorporating thermal probe designs, the bandwidth could potentially be reduced to 3-4 degrees, which would double or triple the current number of channels.

[0180] Furthermore, even with only the current five thermal channels, the combined encoding of fluorophore channels and thermal channels effectively enables profiling of hundreds or even thousands of RNAs in a single cell. 2,3Thermal-plex is a general imaging method that can be applied to other current imaging platforms, such as buffer exchange-based multiplex imaging, expansion microscopy, and super-resolution microscopy. The inventors have demonstrated its application in in situ RNA profiling. In the following steps, thermal-plex is used for in situ immunofluorescence imaging. 10，11 and chromosome imaging 12，13 The application will be easy to implement.

[0181] Given the convenience and robustness of thermal-plex for multiplexed fluorescence imaging, it will enable many direct applications in the field of space biology. References 1. Lewis, SMet al. Spatial omics and multiplexed imaging to explore cancer biology. Namre methods, 1-16 (2021) 2. Chen, KH, Boettiger, AN, Moffitt, JR, Wang, S. & Zhuang, X. RNAimaging. Spatially resolved, highly multiplexed RNA profiling in single cellsScience 348, aaa6090 (2015). 3. Eng, CLet al. Transcriptome-scale super-resolved imaging in tissues by RNA seqFISH. Nature 568, 235-239 (2019). 4. Beliveau, BJet al. OligoMiner provides a rapid, flexible environment for the design of genome-scale oligonucleotide in sim hybridization probesProc Natl Acad Sci USA 115, E2183-E2192 (2018). 5. Ye, J., McGinnis, S. & Madden, T L. BLAST: improvements for better sequence analysis. Nucleic Acids Res 34, W6-9 (2006). 6.Lubeck,E.&Cai,L.Single-cell systems biology by super-resolutionimaging and combinatorial labeling Nat Methods 9,743-748(2012). 7.Lin,C.et al.Submicrometre geometrically encoded fluorescentbarcodes self-assembled from DNA.Nat Chem 4,832-839(2012). 8.Jungmann,R.et al.Multiplexed 3D cellular super-resolution imagingwith DNA-PAINT and Exchange-PAINT.Nat Methods 11,313-318(2014). 9.Hunter,C.A.&Anderson,H.L.What is cooperativity?Angew Chem Int EdEngl 48,748877499(2009). 10.Saka.S.K.et al.Immuno-SABER enables highly multiplexed andamplified protein imaging in tissues.Nat Biotechnol 37,1080-1090(2019). 11.Goltsev,Y.et al.Deep Profiling of Mouse Splenic Architecture withCODEX Multiplexed Imaging Cell 174,968-981 e915(2018). 12.Wang,S.et al.Spatial organization of chromatin domains andcompartments in single chromosomes.Science 353,598-602(2016). 13. Bintu, B. et al. Super-resolution chromatin tracing reveals domains and cooperative interactions in single cells. Science 362 (2018).

[0182] All patents and other publications mentioned in the specification and examples are expressly incorporated herein by reference for all purposes. These publications are provided only because they are disclosed earlier than the filing date of this application. In this regard, nothing should be regarded as an admission that the inventor is not eligible to advance such disclosure by virtue of prior invention or any other reason. All statements about the dates or contents of these documents are based on information available to the applicant and do not constitute any admission that the dates or contents of these documents are correct.

Claims

1. A method for detecting a target molecule in a sample, the method comprising: a. allowing a first nucleic acid strand to bind to a target molecule, wherein the first nucleic acid strand comprises a first hybridization domain and is linked to a target binding agent capable of binding to the target molecule; b. contacting the probe set with the target molecule from step (a), wherein the probe set comprises: i. a second nucleic acid strand, comprising a second hybridization domain connected to a third hybridization domain, wherein the second hybridization domain comprises a nucleotide sequence that is substantially complementary to the nucleotide sequence of the first hybridization domain, and wherein the second nucleic acid strand comprises a reporter molecule capable of generating a detectable signal; and ii. a third nucleic acid strand comprising a fourth hybridization domain, wherein the fourth hybridization domain comprises a nucleotide sequence that is substantially complementary to the nucleotide sequence of the third hybridization domain, wherein the third nucleic acid strand comprises a quencher molecule, and wherein the quencher molecule quenches the detectable signal from the reporter molecule when the nucleic acid probe is not hybridized to the amplicon, and wherein the melting temperature of the third strand hybridized to the second strand is lower than the melting temperature of the second strand hybridized to the first strand, and wherein the melting temperature of the third strand hybridized to the second strand is lower than the melting temperature of the first strand bound to the target molecule; and c. Analyzing binding of the probe set to the target by evaluating the detectable signal generated as a function of temperature.

2. A method for multiplex detection of target molecules in a sample, the method comprising: a. allowing a first nucleic acid chain to bind to a plurality of target molecules, wherein each first nucleic acid chain comprises a first hybridization domain and is linked to a target binding agent capable of binding to the target molecule, and wherein the first hybridization domains of the first nucleic acid chains linked to different target binding agents are different; b. contacting a plurality of detection probe sets with the target molecule from step (a), wherein each detection probe set comprises: i. a second nucleic acid strand, comprising a second hybridization domain connected to a third hybridization domain, wherein the second hybridization domain comprises a nucleotide sequence that is substantially complementary to the nucleotide sequence of the first hybridization domain, and wherein the second nucleic acid strand comprises a reporter molecule capable of generating a detectable signal; and ii. a third nucleic acid strand comprising a fourth hybridization domain, wherein the fourth hybridization domain comprises a nucleotide sequence that is substantially complementary to the nucleotide sequence of the third hybridization domain, wherein the third nucleic acid strand comprises a quencher molecule, and wherein the quencher molecule quenches the detectable signal from the reporter molecule when the nucleic acid probe is not hybridized to the amplicon, and wherein, in each probe set, the melting temperature of the third strand hybridized to the second strand is lower than the melting temperature of the second strand hybridized to the first strand, and wherein, in each probe set, the melting temperature of the third strand hybridized to the second strand is lower than the melting temperature of the first strand bound to the target molecule, and wherein the melting temperature of the third strand hybridized to the second strand in at least one probe set is different from the melting temperature of the third strand hybridized to the second strand in at least one other probe set, and the melting temperature of the second strand in the probe set having the third strand with the lower melting temperature is approximately the same as or lower than the melting temperature of the second strand in the probe set having the third strand with the higher melting temperature; and c. Analyze the binding of the probe set to the target by evaluating the detectable signal generated as a function of temperature.

3. The method of claim 2, wherein: The melting temperature of the third strand hybridized to the second strand in at least one probe set is at least 5°C lower than the melting temperature of the third strand hybridized to the second strand in at least one other probe set.

4. The method according to any one of claims 2 to 3, wherein: The reporter molecule in at least one probe set is different from the reporter molecule in at least one other probe set.

5. The method according to any one of claims 2 to 3, wherein: The reporter molecules in different probe sets are the same.

6. The method according to any one of claims 1 to 5, wherein: The target binding agent is selected from the group consisting of nucleic acids, proteins, peptides, peptidomimetics, amino acids, disaccharides, trisaccharides, oligosaccharides, polysaccharides, lipopolysaccharides, lectins, nucleosides, nucleotides, vitamins, steroids, hormones, cofactors, receptors and receptor ligands.

7. The method of claim 6, wherein: The target binding agent is a nucleic acid, an antibody, an antigen-binding fragment of an antibody, an antibody mimetic, a receptor, or a ligand of a receptor.

8. The method of claim 7, wherein: The target binding molecule is a nucleic acid.

9. The method of claim 7, wherein: The target binding molecule is an antibody or an antigen-binding fragment of an antibody.

10. The method according to any one of claims 1 to 9, wherein: The melting temperature of the third strand hybridized to the second strand is at least 5°C lower than the melting temperature of the second strand hybridized to the first strand.

11. The method according to any one of claims 1 to 10, wherein: The melting temperature of the third strand hybridized to the second strand is at least 5°C lower than the melting temperature of the first strand bound to the target molecule.

12. The method according to any one of claims 1 to 11, wherein: The melting temperature of the second strand hybridized to the first strand is at least 5°C lower than the melting temperature of the first strand bound to the target molecule.

13. The method according to any one of claims 1 to 12, wherein: At least one of the first strand, the second strand, or the third strand comprises a nucleic acid modification.

14. The method according to any one of claims 1 to 13, wherein: The first hybridization domain of the first chain and / or the second hybridization domain of the second chain comprises a duplex stabilizing modification.

15. The method according to any one of claims 1 to 14, wherein: The third hybridization domain and / or the fourth hybridization domain of the second chain comprises a duplex destabilizing modification.

16. The method according to any one of claims 1 to 15, wherein: The reporter molecule and the quencher molecule are a FRET pair.

17. The method according to any one of claims 1 to 16, wherein: The reporter molecule is selected from the group consisting of: a fluorescent molecule, a radioisotope, a chromophore, an enzyme, an enzyme substrate, a chemiluminescent moiety, a bioluminescent moiety, an echogenic substance, a non-metallic isotope, an optical reporter molecule, a paramagnetic metal ion, and a ferromagnetic metal.

18. The method according to any one of claims 1 to 17, wherein: The quencher molecule is a dark quencher.

19. The method of any one of claims 1 to 18, wherein: The first chain further comprises a reporter domain linked to the target binding domain.

20. The method of claim 19, wherein: The probe set further comprises a fourth nucleic acid strand, wherein the fourth nucleic acid strand comprises a fifth hybridization domain, wherein the fifth hybridization domain comprises a nucleotide sequence substantially complementary to the nucleotide sequence of the reporter domain of the first strand, and optionally, the fourth strand comprises a reporter molecule.

21. The method of any one of claims 1 to 20, wherein: Analyzing binding of the probe set to the target includes detecting a detectable signal at a first temperature and a second temperature, wherein one of the first temperature or the second temperature is below the melting temperature of the third strand hybridized to the second strand, and the other of the first temperature or the second temperature is above the melting temperature of the third strand hybridized to the second strand.

22. The method of any one of claims 1 to 21, wherein: The target molecule is selected from the group consisting of nucleic acids, proteins, sugars, lipids, small molecules and antigens.

23. The method of claim 22, wherein: The target molecule is a nucleic acid.

24. The method of claim 23, wherein: The target molecule is RNA.

25. The method of claim 23, wherein: The target molecule is DNA.

26. The method of claim 22, wherein: The target molecule is a protein.

27. The method of claim 22, wherein: The target molecule is a primary metabolite or a secondary metabolite.

28. The method of any one of claims 1 to 27, wherein: The target molecule is a biomolecule.

29. The method of any one of claims 1 to 28, wherein: The target molecule is in a cell.

30. The method of claim 29, wherein: The cells are fixed cells.

31. A probe set for detecting a target molecule, the probe set comprising: i. a first nucleic acid strand comprising a first hybridization domain and linked to a target binding agent capable of binding to the target molecule; ii. a second nucleic acid strand comprising a second hybridization domain connected to a third hybridization domain, wherein, The second hybridization domain comprises a nucleotide sequence that is substantially complementary to a nucleotide sequence of the first hybridization domain, and wherein the second nucleic acid strand comprises a reporter molecule capable of generating a detectable signal; and iii. a third nucleic acid chain, wherein the third nucleic acid chain comprises a fourth hybridization domain, wherein the fourth hybridization domain comprises a nucleotide sequence that is substantially complementary to the nucleotide sequence of the third hybridization domain, wherein the third nucleic acid chain comprises a quencher molecule, and wherein, when the nucleic acid probe is not hybridized with the amplicon, the quencher molecule quenches the detectable signal from the reporter molecule.

32. A probe set for multiplex detection of target molecules, the probe set comprising a plurality of detection probe sets, wherein: Each detection probe set contains: i. a first nucleic acid strand comprising a first hybridization domain and linked to a target binding agent capable of binding to the target molecule, wherein the first hybridization domains of the first nucleic acid strands linked to different target binding agents are different; ii. a second nucleic acid strand comprising a second hybridization domain connected to a third hybridization domain, wherein the second hybridization domain comprises a nucleotide sequence that is substantially complementary to the nucleotide sequence of the first hybridization domain, and wherein the second nucleic acid strand comprises a reporter molecule capable of generating a detectable signal; and iii. a third nucleic acid strand, the third nucleic acid strand comprising a fourth hybridization domain, wherein the fourth hybridization domain comprises a nucleotide sequence that is substantially complementary to the nucleotide sequence of the third hybridization domain, wherein the third nucleic acid strand comprises a quencher molecule, and wherein, when the nucleic acid probe is not hybridized to the amplicon, the quencher molecule quenches the detectable signal from the reporter molecule, and wherein the melting temperature of the third strand hybridized to the second strand is lower than the melting temperature of the second strand hybridized to the first strand, and wherein the melting temperature of the third strand hybridized to the second strand is lower than the melting temperature of the first strand bound to the target molecule, and wherein the melting temperature of the third strand hybridized to the second strand in at least one probe set is different from the melting temperature of the third strand hybridized to the second strand in at least one other probe set, and The melting temperature of the second strand in the probe set having a third strand with a lower melting temperature is approximately the same as or lower than the melting temperature of the second strand in the probe set having a third strand with a higher melting temperature.

33. The probe set according to claim 32, wherein: The melting temperature of the third strand hybridized to the second strand in at least one probe set is at least 5°C lower than the melting temperature of the third strand hybridized to the second strand in at least one other probe set.

34. The probe set according to any one of claims 32 to 33, wherein: The reporter molecules in different probe sets are the same.

35. The probe set according to any one of claims 32 to 34, wherein: The reporter molecule in at least one probe set is different from the reporter molecule in at least one other probe set.

36. The probe set according to any one of claims 31 to 35, wherein: The target binding agent is selected from the group consisting of nucleic acids, proteins, peptides, peptidomimetics, amino acids, disaccharides, trisaccharides, oligosaccharides, polysaccharides, lipopolysaccharides, lectins, nucleosides, nucleotides, vitamins, steroids, hormones, cofactors, receptors and receptor ligands.

37. The probe set according to claim 36, wherein: The target binding agent is a nucleic acid, an antibody, an antigen-binding fragment of an antibody, an antibody mimetic, a receptor, or a ligand of a receptor.

38. The probe set of claim 37, wherein: The target binding molecule is a nucleic acid.

39. The probe set according to claim 37, wherein: The target binding molecule is an antibody or an antigen-binding fragment of an antibody.

40. The probe set according to any one of claims 31 to 39, wherein The melting temperature of the third strand hybridized to the second strand is at least 5°C lower than the melting temperature of the second strand hybridized to the first strand.

41. The probe set according to any one of claims 31 to 40, wherein: The melting temperature of the third strand hybridized to the second strand is at least 5°C lower than the melting temperature of the first strand bound to the first strand.

42. The probe set according to any one of claims 31 to 41, wherein At least one of the first strand, the second strand, or the third strand comprises a nucleic acid modification.

43. The probe set according to any one of claims 31 to 42, wherein: The first hybridization domain of the first chain and / or the second hybridization domain of the second chain comprises a duplex stabilizing modification.

44. The probe set according to any one of claims 31 to 43, wherein The third hybridization domain and / or the fourth hybridization domain of the second chain comprises a duplex destabilizing modification.

45. The probe set according to any one of claims 31 to 44, wherein: The reporter molecule and the quencher molecule are a FRET pair.

46. ​​The probe set according to any one of claims 31 to 45, wherein: The reporter molecule is selected from the group consisting of: a fluorescent molecule, a radioisotope, a chromophore, an enzyme, an enzyme substrate, a chemiluminescent moiety, a bioluminescent moiety, an echogenic substance, a non-metallic isotope, an optical reporter molecule, a paramagnetic metal ion, and a ferromagnetic metal.

47. The probe set according to any one of claims 31 to 46, wherein: The quencher molecule is a dark quencher.

48. The probe set according to any one of claims 31 to 47, wherein The first chain further comprises a reporter domain linked to the target binding domain.

49. The probe set according to any one of claims 31 to 48, wherein The probe set further comprises a fourth nucleic acid strand, wherein the fourth nucleic acid strand comprises a fifth hybridization domain, wherein the fifth hybridization domain comprises a nucleotide sequence substantially complementary to the nucleotide sequence of the reporter domain of the first strand, and optionally, the fourth strand comprises a reporter molecule.

50. A kit for detecting a target molecule, the kit comprising the probe set according to any one of claims 31 to 49.

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