Labeled probes with differentially cleavable linkers and their use in de-coding DNA and RNA molecules

Abstract

To provide a rapid and high-throughput process for decoding target DNA and RNA molecules with minimal repetition of hybridization and dehybridization rounds.SOLUTION: A method includes: a) containing a library of probes having specific structures and sequences, and a group of cleavable linker groups; b) removing unhybridized probes and detecting hybridized probes via the fluorophores as a first image; c) cleaving sequentially the linker groups from the hybridized probes and removing the linker groups, and then detecting the remaining hybridized probes as a second image; d) detecting the removed fluorophores by comparing the first and second image; e) obtaining a part of the sequence information of the target sequences via the sequence information of the probes associated with the removed fluorophores; and f) repeating the step c) until all groups of linkers are cleaved.SELECTED DRAWING: None

Description

Technical Field

[0001] Background of the Invention The present invention relates to a novel method for deciphering DNA and RNA molecules by hybridizing a labeled DNA probe conjugated to a fluorescent label or tag via a cleavable linker (CL). A library of DNA probes conjugated to labels by different linkers can be selectively cleaved under different conditions and in a predictable manner.

[0002] Labeled probes have many applications in modern biology and medicine. For example, labeled DNA probes are used for the detection of specific target sequences of DNA or RNA by hybridization. These are, to name a few, the basis of DNA microarray technology, in situ detection of specific sequences of DNA or RNA (e.g., FISH), and DNA sequencing by ligation. Similarly, labeled antibodies are used for the detection of specific antigen targets.

[0003] Conventionally, a labeled DNA probe having a fluorescent reporter unit is attached to the probe via a stable linker. When using such a labeled probe, the probe can only be removed under denaturation conditions such as higher temperature, low salt, use of formamide, high pH, etc. To probe for multiple targets, this method requires multiple rounds of hybridization and denaturation steps. Both steps are relatively time-consuming. Typically, each step requires a reaction time of 30 minutes to overnight depending on the complexity and length of the probe.

[0004] Furthermore, the relatively harsh conditions used to carry out these processes may damage the nucleic acid target and the substrate on which the target DNA or RNA molecule is immobilized, such as tissue or surface coating. Therefore, the number of repeatable hybridization-dehybridization cycles that can be performed may be limited. In this case, throughput depends on the number of dyes used and the number of hybridization and dehybridization rounds performed. Both can be limiting factors. The same problems still persist in the detection of other biomolecules such as antibodies, cellular carbohydrates, and peptides.

[0005] Therefore, an object of the present invention is to provide a rapid, high-throughput process for decoding target DNA and RNA molecules while minimizing the number of hybridization and dehybridization rounds.

[0006] Current advancements in RNA sequencing (SBS) techniques involve the use of labeled DNA probes with non-cleavable linkers and / or indiscriminately cleavable disulfide linkers. Therefore, current methods do not allow for pattern generation through stepwise and sequential cleavage steps to improve sequencing throughput. These methods rely solely on dehybridization or single-step cleavage to remove the fluorescent reporter from the target. Consequently, throughput and applications are naturally limited. These methods may be incompatible with certain surface chemicals or matrices, such as when used in in situ sequencing and spatial transcriptomics studies, due to tissue degradation caused by repeated processing using dehybridization steps.

[0007] For example, International Publication No. 2015054050 discloses a method for probing multiple targets in which the probes are cut, but each cutting step is performed in the same manner. Therefore, the cutting step is not used to distinguish between different probes.

[0008] Summary of the Invention It was found that when the library of labeled probes consists of different cleavable linkers between the probes and labels, the sequential removal of labeled molecules can be achieved in a predictable and controllable manner.

[0009] Therefore, the object of the present invention is a) General formula (I) P-(CL-D) x (I) [In the formula, P: a probe having at least 10 nucleotides or amino acids] CL: Cuttable linker D: Fluorescent dye X: an integer between 1 and 5. Hybridizing a library of probes having a target sequence of RNA, DNA, or protein, wherein the library comprises probes P having different sequences of nucleotides or amino acids and different groups of cleavable linkers CL that can be cleaved by different means. b) Remove the unhybridized probe and detect the hybridized probe in the first image using fluorophor D. c) Each group of chemical linker CL is sequentially cleaved from the hybridized probe by different means, the fluorophores D thus cleaved are removed, and the remaining hybridized probe is detected by those fluorophores D in the second image. d) Detect the removed fluorophore D by comparing the first and second images. e) Obtaining a portion of the target sequence information from the sequence information of probe P associated with the removed fluorophore D, f) Repeat step c) until all groups of the chemical linker CL are cleaved. This is a method for detecting target sequences of RNA, DNA, or proteins.

[0010] A key feature of the present invention is the use of a library of labeled probes to which a reporter molecule or tag is attached via a set or group of cleavable linkers (CLs) that enable rapid cleavage under different conditions by chemical, photochemical, electrochemical, and / or enzymatic means. Because the cleavage chemistry is much faster than the dehybridization of the DNA probe, this method is inherently a more rapid method for decoding RNA or DNA targets.

[0011] By sequentially removing labels, target sequences can be elucidated by comparing the fluorescent signatures before and after each cleavage step. The main advantage of this method is its ability to decipher more DNA and RNA targets in each round of hybridization. Furthermore, a combination of repeated hybridization cycles and sequential cleavage steps, along with re-hybridization using a new set of probes, allows for the decipherment of numerous target genes.

[0012] This new class of DNA probes is called differentially cleavable linker (DCL) DNA probes. In the method of the present invention, a library of DCL DNA probes is hybridized for sequence-specific binding to a target. After initial fluorescence imaging, the sample is sequentially treated with different cleavage reagents. Further fluorescence imaging is taken after each round of cleavage. The target sequence is decoded from the change in fluorescence signal before and after a cleavage step in which a particular fluorescence signal disappears. In conventional methods, the number of targets that can be detected is determined by the number of fluorescent dyes. In the method of the present invention, multiple targets can be detected from each individual dye due to coupling to different cleavable linkers and their different responses to cleavage reagents. Using this new method, a significantly larger number of targets could be detected with a limited number of dyes compared to conventional hybridization methods. [Brief explanation of the drawing]

[0013] [Figure 1]This diagram schematically illustrates how nine RNA targets can be detected by single-event hybridization using DNA barcodes labeled with only three different dyes (blue, yellow, and red) via differently cleavable linkers. Conventional methods can only distinguish three targets using three dyes. In this novel method, RNA1, RNA2, and RNA3 fluoresce in the same blue channel but are distinguished from each other by their response to cleavage reagents. The fluorescence of RNA2 and RNA3 disappears when treated with cleavage reagents 1 and 2, respectively. RNA1, on the other hand, remains unaffected because it is coupled to the dye by a non-cleavable linker (NC). Similarly, the other RNA targets (RNA4-9) can be detected in the yellow and red channels. [Figure 2a] This figure shows the general structure of a DCL-DNA probe. CL = cleavable linker, EnCL = enzymatically cleavable linker. D / T = fluorescent dye or molecular tag. [Figure 2b] This figure shows several cleavable functional groups, such as disulfide (-SS-), oxymethylene disulfide (-OCH2-SS-), azidomethine (-OCH-N3-), azoarene (-ArN=NAr-), diar (-CH(OH)-CH(OH)-), photocleavable nitrobenzyl, and enzymatically cleavable linkers. [Figure 2c] This figure shows some representative structures of DCL-DNA probes with cleavable linkers. It also shows how multiple DNA probes can be labeled by a single fluorescent dye via different cleavable linkers. [Figure 3] Figure 3(A) is a schematic diagram of a library set of ligated labeled DNA probes (DCL-DNA probe set) that can be cleaved in different ways, which can be prepared by combining various cleavable linkers and labeling them with one or more labels. Figure 3(B) shows some representative schematic structures. [Figure 4]This figure illustrates how 24 targets can be detected by one-step hybridization using a DCL-DNA probe with only three fluorescent dyes. Conventional methods can only probe three targets with a DNA probe labeled with three dyes, and cannot probe all 24 targets. [Figure 5] This figure shows substructures of two reversible nucleotide terminators that can be used after DNA probe hybridization methods to decipher further sequences of a target. The combination of hybridization and synthetic sequencing (SBS) chemistry can dramatically improve the throughput of target sequence detection. In this figure, CL represents a cleavable linker, and the 3'-OH capping groups are -CH2SSMe and -CH2N3. [Figure 6] This figure illustrates how three DNA or RNA targets can be decoded by single-event hybridization in four color patterns using DNA probes labeled with four fluorescent dyes. Target-1 retains its fluorescent signal under three different cleavage conditions because all bound probes are linked with an uncleavable linker (NC). Targets-2 and-3 sequentially lose three dyes during the cleavage process (three CL-linked and one NC-linked labeled DNA probe), but exhibit different patterns after treatment with the cleavage reagent. All three targets can be identified from these color patterns. In this figure, B = blue dye, G = green dye, Y = yellow dye, and R = red dye. [Figure 7]A diagram showing how to use combinations of labeled DCL-DNA probes, non-cleavable labeled DNA probes, and tags for decoding multiple targets by hybridization of a single event. For Target-1, all but one fluorescent dye are removed during the cleavage step, and its probing does not cause the reappearance of other fluorescent signatures. On the other hand, the fluorescent dyes of Target-2 and Target-3 are removed after treatment with a cleavage reagent (all probes are DCL-DNA probes), but the fluorescent signatures reappear in different patterns after probing with the tag. Target-2 reappears in green and red, and Target-3 reappears in yellow and red. By using various combinations, as well as cleavable, non-cleavable, and tag-labeled DNA probes, a number of target genes could be decoded. In this figure, B = blue dye, G = green dye, Y = yellow dye, R = red dye. [Figure 8] A diagram showing the chemical reaction steps for the synthesis of a DCL-DNA probe having an oxymethylenedisulfide (-OCH2SS-) linker that can be effectively cleaved by phosphine and thiol-based cleavage reagents (e.g., DMPS). [Figure 9] A diagram showing the chemical reaction steps for the synthesis of a DCL-DNA probe having a cleavable azidomethine (-OCH-N3-) linker. [Figure 10] A diagram showing probes suitable for the method of the present invention. [Figure 11] A diagram showing the general workflow of the present invention. [Figure 12] A diagram showing the staining results. [Figure 13] A diagram showing the staining results.

[0014] Detailed Description In conventional methods for decoding DNA and RNA targets such as seq-FISH and MERSISH, by using fluorescently labeled DNA barcodes, the maximum F NA number of targets can be sequenced, where F = number of fluorophores and N = number of hybridization steps. For 4 fluorophores and 5 rounds of hybridization, 4 5 = Only 1024 targets can be detected.

[0015] On the other hand, the method of the present invention uses a library of DCL-DNA probes having the same number of fluorescent dyes and four sequentially cleavable linkers (CL) to potentially (F×CL) N =(4×4) 5 = 1,048,576 targets can be detected, where F = number of fluorophores, N = number of hybridization steps, and CL = number of linkers that can be cleaved differently.

[0016] Furthermore, DCL-DNA probes can carry one or more fluorescent reporters that can be removed under the same or different conditions. Additionally, DCL-DNA probes may be used in combination with tagged DNA probes, such as biotin, hapten, or antigen labeling, which can be probed by secondary interactions. Given the number of such combinations, the number of detectable targets can increase dramatically. Moreover, this hybridization method, which optionally requires or does not require a dehybridization step, could accelerate the decoding process.

[0017] Furthermore, the rounds in the present invention's method, which include hybridization and sequential cleavage, allow the detection and re-hybridization steps to cover multiple probes, exponentially increasing the decoding power of this method. Unlike other conventional methods, this method can sequence a vast number of DNA and RNA targets.

[0018] Specific examples include, but are not limited to, fluorescence in situ hybridization (FISH) methods for sequencing RNA transcripts and their use in spatial transcriptomics studies. The same cleavable linker can also be used for multi-omics applications to detect other biomolecules such as proteins, antibodies, antigens, and carbohydrates.

[0019] This invention provides a faster method for detecting nucleic acid sequences using a minimal hybridization process. After hybridization, and sequentially after each cleavage reaction, the sample is imaged to generate a differential pattern of the target, which can then be used to decode the nucleic acid target. A general scheme is shown in Figure 1. Here, multiple RNA targets are hybridized with probes of the same color (three blue: RNA). 1~3 , three red colors: RNA 4~6 , three yellow: RNA 7~9 ), different linkers capable of cleaving are used. RNA1, RNA2, and RNA3 have the same blue dye, but respond differently to different cleavage reagents. RNA1 remains in the same state, while RNA2 and RNA3 disappear at different stages of the cleavage process. The same is true for other RNA targets. The response to the cleavage reagent allows these RNA targets to be read despite having the same dye. Without differently cleavable linkers, only three RNA targets could be decoded for three dyes, instead of nine targets.

[0020] With current technology, when using four-color labeled DNA probes, single-event hybridization yields only 16 decipherables, even with multiple dye combinations: 1 code with all four colors, 4 codes with three colors, 6 with two colors, 4 with one color, and finally 1 with no color.

[0021] On the other hand, using the DCL-DNA probe system with four colors and four differently cleavable linkers, the decoding capability can be increased from 16 (above) to potentially over 500 through single-event hybridization combining all possible probes, followed by four selective cleavage steps and molecular probing.

[0022] Figure 2 shows schematic diagrams of labeled DNA probes (DCL-DNA probes) with linkers that can be cleaved in different ways. Also shown are some representative structures of the cleavable linkers (Figure 2b), and a method (Figure 2c) in which cleavable linkers can be used to create a set of labeled DNA probes from a single fluorescent dye using a diverse set of linkers.

[0023] Figure 3 shows, but is not limited to, examples of methods for constructing large-scale library sets of DCL-DNA probes using various combinations of cleavable (CL), non-cleavable (NC), molecular tag (T), and labeling with multiple reporters on the DNA backbone.

[0024] Figure 4 shows how more than 20 targets can be read by a single hybridization event using only four dyes. Conventional methods can only detect four targets from four dyes using probes labeled with a single dye. However, by using differently cleavable linkers, multiple targets can be detected by each dye. In this figure, targets 5-8 are labeled with the same blue dye but are distinguished by different responses to different cleavage reagents (in this case, the dyes disappear under different cleavage conditions). Therefore, the present invention helps to increase each fluorescent dye to multiple distinguishable dyes. For example, by using seven differently cleavable linkers, seven different targets can be distinguished using a single blue dye.

[0025] In the method according to the present invention, after step f), probe P is optionally dehybridized from the target sequence of RNA, DNA, or protein.

[0026] Furthermore, steps a) to f) can be repeated until sequence information of the target RNA, DNA, or protein is obtained. The acquisition of sequence information of the target RNA, DNA, or protein includes not only complete (100%) sequence information but also partial information. The amount of information, and therefore the speed of the method, can be controlled by the selection of the probe, i.e., the selection of the nucleotide or protein probe. While the acquisition of information may range from 10% to 100%, a nearly complete sequence analysis (such as 90% to 100%) is preferred.

[0027] The hybridization-based method of the present invention using a cleavable linker can be further combined with synthetic sequencing chemistry (SBS chemistry). SBS chemistry can be initiated after reading with a DCL-DNA probe and cleaving the fluorescent dye. In this case, the DNA probe can be used as a sequencing primer without the need to remove it from the target. SBS chemistry relies on the use of reversibly terminating nucleotides having a fluorescent dye label via a cleavable linker. Two representative reversibly terminating nucleotides that can be used in combination with a DCL-DNA probe are shown in Figure 5. Here, CL represents a chemically cleavable linker that links the fluorescent dye to the nucleic acid base, and the capping group on the 3'-OH is preferably -CH2SSMe or -CH2N3. The cleavable linker may be the same as or different from the DCL linker.

[0028] DCL-DNA probes have an uncleavable reporter group and can be used in conjunction with DNA probes to which a molecular tag is attached. The tag enables target probing via secondary interactions. In this context, the uncleavable labeled DNA probe can be used not only to read specific target sequences but also as an internal control. The tag may be, for example, biotin, digitogenin, antigen, hapten, etc.

[0029] DNA probes may comprise one or more fluorescent dyes having the same or different cleavable linkers. They may also comprise a combination of fluorescent dye and a tag. The tag can be probed by secondary interactions, such as DNA-biotin probes detected by interaction with labeled streptavidin.

[0030] The terms "differently severable linkers," "different groups of linkers," and "diverse linkers" refer to a set of linkers that are interchangeable and capable of sequentially removing labels under different conditions, enabling them to be removed in a controllable and predictable manner.

[0031] The term "cleavable linker CL that can be cleaved by different means" refers to a cleavable linker CL that can be cleaved by a first method but not by a second method. Different means or methods for cleavage allow for sequential imaging of different groups of probes that stain the target.

[0032] In the method of the present invention, chemical, photochemical, or enzymatic means can be used to cleave the cleavable linker CL. For example, the library includes a probe having at least two different groups of cleavable linker CL that can be cleaved by two different means selected from the group consisting of two different photochemically activated radiations, two different enzymes, two different chemicals, one photochemically activated radiation and one enzyme, one photochemically activated radiation and one chemical, one enzyme and one chemical.

[0033] Naturally, the cleavable linker CL may be cleavable by three, four, or five different means without departing from the spirit of the present invention. For example, it is possible to obtain four different destaining images by using two different enzymes and two different chemicals, or four different chemicals.

[0034] The cleavable linker CL can be selected from the group consisting of, for example, the following formulas a) to g), disulfide linkers (-SS-, (a)), oxymethylene disulfide (-OCH2SSCR1R2-, (b)), oxymethine azide (-CR1,R2OCH(N3)R3,R4)-, (c)), azoarene (-ArN=NAr-, (d)), nitrobenzyl derivatives (e), allyl derivatives (f), and thiocarbamate (g), where R1, R2, R3, and R4 can independently be H, methyl, ethyl, propyl, t-butyl, C5-C10 alkyl, alkene, alkyne, hydroxyl, halogen, amine, amide, carboxylate, and polyethylene glycol.

[0035] [ka]

[0036] The cleavage reaction / chemistry is determined by the linker (CL) that can be cleaved, and can be selected from the following as needed.

[0037] Disulfide linkers can be selectively cleaved by treating them with thiols or phosphines while leaving other linkers intact.

[0038] Oxymethine azide (-OCH(N3)-) can be selectively cleaved when treated with phosphine (e.g., TCEP) in the presence of azoarenes, photocleavable or other enzymatically cleavable linkers.

[0039] Alternatively, both disulfide and oxymethine azide linkers can be cleaved using TCEP. Furthermore, azoarene linkers can be selectively cleaved with sodium dithionite (Na2S2SO4), a very mild reagent in aqueous buffer.

[0040] The cleavage reagents may be thiols (e.g., dithiothreitol-DTT, dimercaptopropanesulfonate-DMPS, etc.), phosphines (e.g., TCEP, THPP, etc.), mild reducing agents (e.g., Na2S2O4), light of a specific wavelength, electrical potential, or enzymes (e.g., peptidase, dextranase, esterase, phosphatase, proteinase, etc.).

[0041] Enzymatically cleavable linkers can be any molecule that can be cleaved or digested by a specific enzyme, such as a peptidase. For example, polysaccharides, proteins, peptides, depsipeptides, polyesters, nucleic acids, and their derivatives are suitable as enzymatically cleavable linkers. Suitable polysaccharides include, for example, dextran, pullulan, inulin, amylose, cellulose, hemicellulose, xylan or glucomannan, pectin, chitosan, or chitin. These can be derivatized to obtain covalent or non-covalent functional groups for the linker. Proteins, peptides, and depsipeptides used as enzymatically cleavable linkers can be functionalized via amino acid side-chain functional groups. Polyesters, polyacrylamides, and polyesteramides used as enzymatically cleavable linkers can be synthesized using comonomers that provide side-chain functional groups, or functionalized afterward. In the case of branched polyesters, functionalization may be via carboxyl, amine, or hydroxyl terminal groups. Functionalization of polymer chains after polymerization may be carried out, for example, by addition to unsaturated bonds, i.e., by thiolene reactions, or by azidoalkyne or tetrazine alkyne click reactions, or by introduction of functional groups by radical reactions.

[0042] Enzymatically degradable linkers are broken down by the addition of a suitable enzyme. The selection of the enzyme as the cleavage reagent is determined by the chemical properties of the enzymatically degradable linker and may be a single enzyme or a mixture of different enzymes. Such enzymes may be hydrolases, lyases, reductases, esterases, peptidases, etc. Preferred enzymes can be selected from the group consisting of glycosidases, dextranases, pullulanases, amylases, inulinases, cellulases, hemicellulases, pectinases, chitosanases, chitinases, proteinases, esterases, glycosidases, pyrophosphatases, lipases, phosphatases, and nucleases.

[0043] The method of the present invention can be used in conjunction with a multi-probe hybridization method in which two or more probes bind to a specific target. Figure 6 shows how three DNA targets can be decoded by single-event hybridization and sequential cleavage and imaging steps in a four-color scheme using DNA probes labeled with four fluorescent dyes. Calling of a specific gene is obtained from the fluorescent response to the cleavage reagent and by comparing the initial image with the fluorescence images of subsequent steps exposed to the cleavage reagent. Target-1 retains its fluorescent signal under three different cleavage conditions because all of its DNA probes are linked to an uncleavable linker (NC). Targets-2 and-3 sequentially lose the three dyes during the cleavage reaction, but in different patterns.

[0044] In another aspect of the present invention, the DNA probe may comprise two or more fluorescent dyes or tags, or a combination thereof (Figure 7). This configuration allows for selective cleavage of the target dye and subsequent probing of the target by secondary interaction with the tag. The use of such a combination is shown in Figure 7. Figure 7 shows how three different targets can be distinguished by probing with a mixture containing a labeled DNA probe linked to a DCL and an uncleavable (NC) tag. The three targets respond differently to cleavage and molecular probing of the tag. Target-1 loses all but one of its fluorescent signals upon treatment with the cleavage reagent and does not respond to molecular probing. Targets 2 and 3, on the other hand, lose all of their fluorescent signatures and respond differently to subsequent probing of the attached tag. Using various combinations and DNA probes labeled with cleavable, uncleavable, and tagged DNA, numerous target genes could be sequenced.

[0045] In another aspect of the present invention, after removing the fluorescent dye, the free 3'-OH of the probe DNA can be used for single-base extension and additional sequencing by synthetic chemistry using labeled reversible-terminated nucleotides. The reversible-terminated nucleotide stops complementary strand synthesis by single-base extension but can be further extended after the 3'-OH capping group is removed. The reversible-terminated nucleotide can be 3'-OH capped with methylmethylenedisulfide (-CH2SSMe) or azidomethyl (-CH2N3) having the general structure shown in Figure 5. These nucleotides may also contain a DCL linker, as in the DNA probe design. These can be used independently or in combination with DCL-probes.

[0046] In another aspect of the present invention, hybridization involves the use of different probes, each having a specific target, and is accompanied by multicolor fluorescence measurement. Each hybridization event may include two or more cycles of cleavage and imaging steps, or one or more steps of hybridization-imaging-cleavage-imaging and rehybridization cycles with an optional dehybridization step between each cycle. It may also include one or more steps of probing by secondary interactions, two specific examples of which are biotin-streptavidin, DIG-anti-DIG interaction and subsequent imaging.

[0047] Probes, labels, and tags The probe may be ordinary natural or non-natural DNA or RNA and their modified analogs, i.e., consisting of natural or non-natural nucleotides. The probe may also be a protein, i.e., consisting of natural or non-natural amino acids. DNA probes may be lock nucleic acids that provide better double-strand stability. These may have modifications at the 3' or 5' end, or modifications on any of the bases on the main backbone, such as dUTP with amino modifications. The label may be a fluorescent molecule (rhodamine dye (R6G, ROX), cyanine dye (Cy3, Cy5), Alexa dye, ATTO dye, etc.), or an energy transfer dye or quencher. The molecular tag may be biotin, hapten, antigen, DIG, fluorescein, nitrophenyl, etc., or any modification identified by secondary interactions (e.g., biotin-STV with fluorescent labeling, antibody-antigen, DIG-anti-DIG interaction). The DCL linker may be on the probing protein.

[0048] Target sequence and method The target sequence may be DNA or RNA containing mRNA. This can be used in fluorescence in situ hybridization (FISH) or sequential FISH (seqFISH). Sequencing may be performed on clone-amplified DNA clones on beads, surface-bound rolling cycle amplification (RCA) products or tissues, single molecules, or spatially resolved single cells. These can be used for in situ or ex situ sequencing. The sequencing method may include multiple rounds of hybridization to enhance sequencing efficiency. The sequencing method may include imaging following hybridization with a mixture of labeled DNA probes, sequential cleavage steps and imaging, and probing of molecular tags via secondary interactions. Targets can be proteins, peptides, carbohydrates, antibodies, and other biomolecules.

[0049] fluorescent dyes Suitable fluorescent dyes are those known in the field of fluorescence technology, such as flow cytometry or fluorescence microscopy. For example, fluorescent dyes are xanthene dyes such as fluorescein, or rhodamine dyes, coumarin dyes, cyanine dyes, pyrene dyes, oxazine dyes, pyridyloxazole dyes, cascade dyes, polymer dyes, pyromethene dyes, acridine dyes, oxadiazole dyes, carbopyronin dyes, benzopyrylene dyes, fluorene dyes, or organometallic complexes such as Ru, Eu, and Pt complexes. In addition to single molecular entities, clusters of small organic molecule dyes, fluorescent oligomers, or fluorescent polymers, such as polyfluorene, can also be used as the fluorescent moiety. Furthermore, fluorescent dyes may be protein-based, such as phycobiliproteins, nanoparticles such as quantum dots, upconversion nanoparticles, gold nanoparticles, or dye polymer nanoparticles.

[0050] Examples To establish proof of concept for the use of DCL-DNA probes in mRNA sequencing, several probe sets were synthesized. One was an oxymethylene disulfide linker cleavable with thiols, and the other was an azidomethyl linker cleavable with phosphines. Their synthesis required multi-step reactions and purification. The cleavable linkers were first prepared and then conjugated with amino-modified DNA probes. In the final step, the linkers were labeled with a fluorescent dye using the amino terminus of the cleavable linker.

[0051] Synthesis of a DCL-DNA probe (5) having a cleavable oxymethylenedisulfide linker The synthesis process is shown in Figure 8. Activated linker 2 was obtained as described in the U.S. Patent No. 10,301,346 (Mong Marma et al., 2016) and the method for synthesizing nucleotide analogs having disulfide linkers (Mong Marma et al., U.S. Patent No. 10,336,785, 2016). The synthesis includes the following steps (Figure 8): (a) Introduction of a linker and deprotection of the Fmoc group: Amino-modified DNA barcode (BC-NH2, 1) (approximately 700 nmol, source IDT DNA) was dissolved in 1.4 mL of DI water and transferred to a 50 mL Falcon tube. 0.3 mL of 0.5 M Na2HPO4 was added to this to adjust the base concentration to approximately 0.1 mM. In a separate 2.0 mL centrifuge tube, approximately 6 mg of activated linker (NHS-ARA-Fmoc linker, compound 2) was dissolved in 0.5 mL of DMF. This was then added to the BC-NH2 solution and stirred at room temperature for 45 minutes. The same amount was added again in 0.5 mL of DMF and stirred for another 45 minutes.

[0052] Next, this was quenched by adding 2.0 mL of DI water. The mixture was then treated with 0.3 mL of piperidine for 20 minutes. The product was immediately purified by prep-HPLC (X-bridge, 19 × 250 mm column, method: 100% A for 0-3 minutes, followed by 40% B for 40 minutes, then a uniform gradient up to 50 minutes). The target product usually eluted in about 43 minutes. The target fraction was lyophilized, and the solid product was suspended in 1.0 mL of DI water. The concentration was determined by UV at 260 nm to obtain 160 nmol of compound 4 (1.0 mL × 0.160 mM).

[0053] (b) Labeling with fluorescent dyes: BC-ARA-NH2 (compound 4) obtained from the previous step was treated with Na2HPO4 salt (the salt was dissolved directly to avoid further dilution) to a final concentration of 0.1 mM. This was then treated with approximately 10 equivalents of NHS dye in 200 μL of DI water (add 100 μL of DMF for low-polarity dyes such as AF532-NHS). After stirring the mixture for 30 minutes, it was immediately purified by prep-HPLC (X-bridge, 19 × 250 mm column, method: 100% A for 0-3 minutes, followed by 40% B for 40 minutes, then a uniform gradient up to 50 minutes). Target product 5 eluted in approximately 40 minutes. The target fraction was then lyophilized, and the final product was dissolved in 1.0 mL of RNA-free water, and the concentration was determined by UV spectroscopy (based on the characteristics of the dye). The concentration of the product was then adjusted to prepare a 0.1 mM stock solution.

[0054] Synthesis of a DCL-DNA probe (11) having a cleavable oxymethine azid linker: The synthesis of a DCL-DNA probe with a cleavable oxyazidomethin linker begins with commercially available compound 6, as shown in Figure 9. The following steps are included: (a) Synthesis of Fmoc protection linker (7) (a) Activation of the linker (9) (b) Coupling to amino-modified DNA probe (10) (c) Coupling to fluorescent dye (11)

[0055] The synthesis process of the Fmoc-protected linker (7) and the activation of the linker for coupling to the DNA probe (9) are described below. The coupling reaction between the linker and the DNA probe (10) and the final labeling reaction for preparing the labeled probe (11) were carried out in the same manner as with oxymethylene disulfide described above.

[0056] DIPEA (0.66 mL, 3.8 mmol, 2 equivalents) was added to a solution of Fmoc-NHS (760 mg, 2.3 mmol, 1.2 equivalents) and compound 6 (734 mg, 1.9 mmol, 1 equivalent) in anhydrous DMF (12 mL). The resulting reaction mixture was stirred at room temperature for 2 hours. This was then diluted with siRNA (20 mL) and quenched with 1 M HCl (20 mL). The aqueous layer was separated and extracted with EtOAC (3 × 20 mL). The combined organic layers were washed with water (5 × 20 mL) and brine (saturated, 20 mL), dried over Na₂SO₄, and concentrated under reduced pressure. The crude product was purified by flash chromatography (ISCO Silica 24 g Gold; gradient: MeOH / DCM (0-30%), 35 mL / min) to obtain the desired product 7 as a white solid (488 mg, 44%). Next, DIPEA (0.22 mL, 1.3 mmol, 1.5 equivalents) and DSC (318 mg, 1.25 mmol, 1.5 equivalents) were added to a solution of compound 7 (488 mg, 0.83 mmol, 1 equivalent) in anhydrous DMF (8.3 mL). The resulting reaction mixture was stirred at room temperature for 2 hours. This was then concentrated under reduced pressure, and the residue was purified by flash chromatography (ISCO Silica 24 g Gold; gradient: Â1 / hexane (30-100%), 35 mL / min) to obtain the desired product 8 as a syrup (408 mg, 72%).

[0057] Dyeing experiment We designed probes targeting four transcripts with six unique hybridization domains. Two of the probes contained sequences that hybridized with oligonucleotides having an azide linker (-OCHN3-) conjugated to Alexa Fluor 647 or an oligonucleotide having a disulfide linker (-OCH2-SS-) conjugated to Alexa Fluor 532 (Figure 1A). For control, two of the probes contained two different binding regions that hybridized with two oligonucleotides (Figure 1A). One probe contained sequences that hybridized with oligonucleotides having a disulfide linker containing Alexa Fluor 647 and oligonucleotides having an uncleavable Alex Fluor 532. The other probe contained sequences that hybridized with oligonucleotides having an azide linker containing Alexa Fluor 532 and oligonucleotides having an uncleavable Alex Fluor 647. Having two independent fluorophores across the hybridization domain allowed this technique to be evaluated at multiple fluorescence wavelengths.

[0058] The probe used had the structure shown in Figure 10.

[0059] Next, the probes were hybridized onto FFPE mouse brain sections, followed by rolling circle amplification to generate lorony, or DNA nanoballs, and then amplified for in situ detection of the signal (Figure 11). All six complementary probes, i.e., readout probes, were hybridized simultaneously after generating the lorony.

[0060] Next, a series of sequential chemical cleavage steps were performed. The first cleavage step utilized sodium 2,3-dimercaptopropanesulfonate monohydrate (DMPS) to selectively cleave oligonucleotides containing a disulfide linker. Figure 12 shows the first of two sequential cleavage rounds using DMPS to selectively remove fluorophores attached to the oligonucleotide via the azid linker. All images are pseudocolored to provide sufficient contrast to determine which points have signals removed between rounds. At Alexa Fluor 532 wavelength, points cleaved by DMPS appear yellow in the merged image (yellow arrows). At Alexa Fluor 647 wavelength, points cleaved by DMPS appear purple in the merged image. Objects unaffected by DMPS cleavage appear white in the merged image at both wavelengths.

[0061] Following this, a second cleavage was performed using tris(2-carboxyethyl)phosphine hydrochloride (TCEP) to cleave the oligonucleotide containing the azid linker. Figure 13 shows the second of two consecutive cleavage rounds using TCEP to selectively remove fluorophores attached to the oligonucleotide via the disulfide linker. All images are pseudo-colored to provide sufficient contrast to determine which points have signals removed between rounds. At Alexa Fluor 532 wavelength, points cleaved by TCEP appear purple in the merged image (yellow arrows). At Alexa Fluor 647 wavelength, points cleaved by TCEP appear blue in the merged image. After TCEP cleavage, only uncleavable oligonucleotides remain detectable and appear white in the merged image at both wavelengths.

[0062] After both cutting rounds, only the uncuttable points remained.

Claims

1. a) General formula (I) P-(CL-D) ｘ (I) [Wherein, P: a probe having at least 10 nucleotides or amino acids CL: cleavable linker D: fluorescent dye X is an integer of 1 to 5. to a target sequence of RNA, DNA or protein, wherein the library comprises probes P having different sequences of nucleotides or amino acids and different groups of cleavable linkers CL that can be cleaved by different means; b) removing unhybridized probes and detecting said hybridized probes in a first image by fluorophore D; c) sequentially cleaving each group of chemical linkers CL from the hybridized probes by different means, removing the fluorophores D thus cleaved, and detecting the remaining hybridized probes by their fluorophores D in a second image; d) detecting the removed fluorophore D by comparing the first and second images; e) obtaining part of the sequence information of the target sequence by the sequence information of the probe P associated with the removed fluorophore D; f) repeating step c) until all groups of the chemical linker CL are cleaved. A method for detecting a target sequence of RNA, DNA or protein by

2. 2. The method of claim 1, wherein after step f) said probe P is dehybridized from said RNA, DNA or protein target sequence.

3. 3. The method according to claim 1, wherein steps a) to f) are repeated until the sequence information of the target sequence of the RNA, DNA or protein is obtained.

4. 3. The method according to claim 1 or 2, characterized in that the cleavable linker CL is cleavable by chemical, photochemical or enzymatic means.

5. 3. The method according to claim 1 or 2, characterized in that the cleavable linker CL is an enzymatically cleavable linker selected from the group consisting of polysaccharides, proteins, peptides, depsipeptides, polyesters, nucleic acids and derivatives thereof.

6. 6. The method of claim 5, wherein the polysaccharide is selected from the group consisting of dextran, pullulan, inulin, amylose, cellulose, hemicellulose, xylan, glucomannan, pectin, chitosan and chitin.

7. The cleavable linker CL is a disulfide linker (-SS-, (a)), an oxymethylene disulfide (-OCH ２ SSCR １ R ２ -, (b)), oxymethine azide (-CR １ , R ２ OCH (N ３ ) R ３ , R ４ )-, (c)), azoarenes (-ArN=NAr-, (d)), nitrobenzyl derivatives (e), allyl derivatives (f), and thiocarbamates (g), wherein R １ , R ２ , R ３ , R ４ The method of claim 1 or 2, wherein each of the groups can be independently H, methyl, ethyl, propyl, t-butyl, C5-C10 alkyl, alkene, alkyne, hydroxyl, halogen, amine, amide, carboxylate, polyethylene glycol.

8. 3. The method of claim 1 or 2, characterized in that the library comprises probes having at least two different groups of cleavable linkers CL that are cleavable by two different means selected from the group consisting of two different photochemically activating radiations, two different enzymes, two different chemicals, one photochemically activating radiation and one enzyme, one photochemically activating radiation and one chemical, one enzyme and one chemical.

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