Markers, biomarkers, and methods for analyzing biological samples

Abstract

A marker for analyzing biological samples is provided. The marker includes a first marker portion and a second marker portion, the first marker portion including a first nucleic acid chain (106) and the second marker portion including a second nucleic acid chain (110). The first nucleic acid chain (106) and the second nucleic acid chain (110) are configured to form a double strand. The marker further includes at least one first marker portion (108) and at least one second marker portion (112), and the marker further includes at least one blocking nucleic acid chain (126, 128). In other aspects, markers (100, 500, 600, 700, 1000) including the marker and corresponding methods are provided.

Description

Technical Field

[0001] This invention relates to a marker for analyzing biological samples, said marker comprising at least one blocking nucleic acid strand. Alternatively, it provides a marker and method for analyzing biological samples. Background Technology

[0002] Labels and markers are frequently used in the analysis of biological samples, such as in fluorescence microscopy. Markers used in fluorescence microscopy typically consist of affinity reagents (such as antibodies) and a marker attached to the affinity reagent, enabling the marker to specifically (particularly via the affinity reagent) attach to a target analyte in the biological sample. This allows for the identification and / or localization of the target analyte within the biological sample.

[0003] The various applications of markers and biomarkers include multi-method approaches that require a large number of distinguishable markers and biomarkers, while biomarkers with bright markings are needed to detect analytes present in small quantities. In particular, when dealing with a large number of biomarkers (which can be assembled from individual components such as dyes and affinity reagents), it is important to reduce processing complexity while maintaining flexibility and reliability. Summary of the Invention

[0004] The purpose of this invention is to provide a marker and a biomarker to enable efficient processing when analyzing biological samples.

[0005] The above objectives are achieved through the subject matter of the independent claims. Advantageous embodiments are defined in the dependent claims and the following description.

[0006] In a first aspect, a marker for analyzing biological samples is provided. The marker includes a first marker portion and a second marker portion, the first marker portion having a first nucleic acid chain and the second marker portion having a second nucleic acid chain. The first and second nucleic acid chains are configured to form a double strand. The marker further includes at least one first marker portion and at least one second marker portion. The marker also includes at least one blocking nucleic acid chain, said at least one blocking nucleic acid chain being at least partially complementary to one of the first and second nucleic acid chains.

[0007] The blocking nucleic acid strand prevents unnecessary or uncontrolled hybridization between the first and second labeling portions. Accordingly, this enables efficient processing of the labeling portions, for example, in preparing biological samples for analysis, such as when analyzing the proximity between target analytes in biological samples using the label.

[0008] The label is preferably configured to be optically detectable at least when the first and second label portions are in close proximity. Specifically, the label portions are (covalently) attached to one of the first and second nucleic acid chains. For example, the first and second label portions can be a fluorescence resonance energy transfer (FRET) pair, which is optically detectable when they are in close proximity (particularly within 1 to 10 nm). This can be used for FRET-based proximity analysis, where the FRET efficiency between different label portions of the marker can be measured by readouts in the presence and absence of one or more blocking strands (i.e., before and after the removal of the blocking strand). FRET can be measured by readout intensity and / or fluorescence lifetime changes (e.g., by confocal microscopy).

[0009] Specifically, the first nucleic acid chain may include a first nucleotide sequence that is complementary to a second nucleotide sequence of the second nucleic acid chain. Preferably, the first and second nucleotide sequences are at least partially complementary to each other, particularly based on Watson-Crick base pairing. Therefore, the first and second sequences can form a double-stranded structure. This enables stable hybridization between the first and second nucleotide sequences. The double-stranded structure can be a double helix, such as B-type DNA.

[0010] In one embodiment, the marker comprises (first) a plurality of blocking nucleic acid chains, wherein the blocking nucleic acid chains are linked to each other by cleavable linkers, specifically to form cleavable linear strands of the blocking nucleic acid chains. In particular, the cleavable linkers can be selectively cleaved by applying a cleavage agent. This enables efficient and selective removal of the blocking nucleic acid chains. Typically, the plurality of blocking nucleic acid chains linked by the cleavable linkers form a duplex with the first or second nucleic acid chain, which is thermodynamically more stable than the shorter individual blocking nucleic acid chains formed after they are cleaved together. In particular, the individual blocking nucleic acid chains have a lower melting temperature or a reduced magnitude of the free energy of hybridization (ΔG), resulting in lower hybridization stability, thereby enabling easy removal of the individual blocking nucleic acid chains from the first or second nucleic acid chain.

[0011] In one embodiment, the cleavable linker is a photocleavable, chemically cleavable, or enzymatically cleavable linker. This allows for specific cleavage of the linker, particularly without affecting the remaining elements of the marker. In a particular embodiment, the cleavable linker is not a nucleotide or nucleic acid, or does not include nucleotides or nucleic acids.

[0012] For example, photolytically cleavable connectors can be cleaved by irradiation with (UV) light, which acts as the cleavage agent. A specific example of a photolytically cleavable connector is the BMN connector (Biomers).

[0013] The chemilytic linker can be cleaved by contact with a specific chemical that serves as the cleavage agent. For example, the chemilytic linker may comprise an arylboronic ester (which is cleavable by hydrogen peroxide) or a disulfide bond (which is cleavable by reduction with TCEP, DTT, or glutathione). Thiazole, thiazolidinyl, oxime, and hydrazone linkers are also chemilytically cleavable under conditions generally compatible with bioanalytical processes. For example, pH-sensitive linkers, such as hydrazone linkers, can be cleaved at pH 4–6.

[0014] The cleavable linker can be cleaved by contacting a specific enzyme used as the cleavage agent. For example, the valine-citrulline dipeptide linker can be cleaved by cathepsin B. Alternatively, the cleavable linker may comprise a sequence fragment that is cleaved by a naturally occurring or engineered cleavage enzyme configured to cleave only one strand of the DNA duplex. Examples of lyases and their sequence fragments include: Nt. BstNBI, NEB (5'-GAGTC-3'); Nb. BtsI, NEB (5'-GCAGTG-3'); Nb. BsrDI, NEB (5'-GCAATG-3'); Nt. BbvCI, NEB (5'-CCTCAGC-3'); Nt. CviPII, NEB (5'-CCD-3', where D = A, G, or T); Nt. CviQII, NEB (5'-RAG-3', where R = A or G); I-HmuI, NEB (recognizes degenerate sequences); I-BasI, NEB (recognizes degenerate sequences); Nb. Mva1269I, ThermoFisher Scientific (5'-GAATGC-3').

[0015] In one embodiment, all of the plurality of blocking nucleic acid strands are complementary to the corresponding nucleotide sequence of the first or second nucleic acid strand. This effectively blocks hybridization between the first and second nucleic acid strands. Specifically, adjacent blocking nucleic acid strands of the cleavable linear strand of the blocking nucleic acid strand are complementary to the corresponding adjacent nucleotide sequence of the first or second nucleic acid strand. Therefore, each of the plurality of blocking nucleic acid strands of the marker can hybridize with a different portion of the first nucleic acid strand or with a different portion of the second nucleic acid strand.

[0016] In one embodiment, adjacent blocking nucleic acid strands of the plurality of blocking nucleic acid strands are complementary to corresponding adjacent nucleotide sequences of the first or second nucleic acid strand. This enables the linked blocking nucleic acid strands to have high melting temperatures or increase their hybridization free energy (ΔG) to the order of magnitude, resulting in high hybridization stability. The linked blocking nucleic acid strands can form cleavable linear strands of the blocking nucleic acid strands, which are complementary to the corresponding nucleotide sequences along the first or second nucleic acid strand. Therefore, each of the plurality of blocking nucleic acid strands of the marker can hybridize with different portions of the first nucleic acid strand or different portions of the second nucleic acid strand.

[0017] In one embodiment, each of the at least one blocking nucleic acid chain, particularly each of the plurality of blocking nucleic acid chains, comprises a plurality of nucleotides, the number of which is 5 to 15, preferably 5 to 12, and more preferably 6 to 9. This enables each of the blocking nucleic acid chains to individually have a lower melting temperature or a reduced hybridization free energy (ΔG), thereby achieving lower hybridization stability. The nucleotides of each of the blocking nucleic acid chains can be linked together by phosphodiester bonds to form the corresponding nucleic acid chain.

[0018] Preferably, the melting temperature of the at least one blocking strand can be in the range of 28°C to 42°C, particularly between 28°C and 36°C. The at least one blocking strand may have a sequence that mismatches with its corresponding sequence on the first or second nucleic acid strand. Therefore, compared to the corresponding sequence on the first or second nucleic acid strand, the at least one blocking strand may have one or more mismatched nucleotides. This allows for modulation of the hybridization stability of the at least one blocking strand.

[0019] In one embodiment, the first nucleic acid chain and / or the second nucleic acid chain comprises, in particular, each comprising a plurality of nucleotides, the number of said nucleotides being 50 to 150.

[0020] In one embodiment, the at least one blocking nucleic acid strand is complementary to a portion of the first nucleic acid strand or a portion of the second nucleic acid strand, wherein the portion of the first nucleic acid strand and the portion of the second nucleic acid strand are complementary to each other. This effectively blocks hybridization between the first and second nucleic acid strands.

[0021] In one embodiment, the marker includes a second plurality of blocking nucleic acid strands, wherein the blocking nucleic acid strands of the second plurality of blocking nucleic acid strands are linked to each other by cleavable linkers, and wherein the blocking nucleic acid strands of the second plurality of blocking nucleic acid strands are at least partially complementary to the corresponding nucleotide sequences of the second nucleic acid strand. This enables the blocking of both the first and second nucleic acid strands using dedicated blocking nucleic acid strands. Preferably, in this case, the blocking nucleic acid strands of the first plurality of blocking nucleic acid strands may be at least partially complementary to the corresponding nucleotide sequences of the first nucleic acid strand.

[0022] The linked blocking nucleic acid strands of the second plurality of blocking nucleic acid strands can form a second cleavable linear strand of the blocking nucleic acid strand. The cleavable linker can be selectively cleaved by applying a cleavable agent. The cleavable linkers of the second plurality of blocking nucleic acid strands can be configured in a manner similar to that described for the first plurality of blocking nucleic acid strands.

[0023] In one embodiment, the at least one first labeling portion and / or the at least one second labeling portion is optically detectable. This enables efficient detection of the label, for example, by a microscope, particularly a fluorescence microscope. For instance, the first labeling portion and / or the second labeling portion may comprise a fluorophore, such as a fluorescent protein, an organic or inorganic fluorescent molecule, or fluorescent nanoparticles.

[0024] In one embodiment, the at least one first marking portion and / or the at least one second marking portion have the same optical characteristics, or the at least one first marking portion and / or the at least one second marking portion have different optical characteristics. The optical characteristics may include, for example, excitation or emission wavelength, or fluorescence lifetime.

[0025] In one embodiment, the at least one first labeled portion and the at least one second labeled portion are configured for nonradiative energy transfer between them, particularly when they are in close proximity. This enables efficient determination of whether the labeled portions are in close proximity to each other. For example, the first labeled portion and the second labeled portion can be configured to form an FRET pair with each other, one being an FRET donor and the other an FRET acceptor. FRET between the first labeled portion and the second labeled portion can typically occur when the labeled portions hybridize with each other and are therefore in close proximity.

[0026] In one embodiment, both the at least one first labeling portion and the at least one second labeling portion are attached, preferably covalently attached, to the first or second nucleic acid chain. For example, they may be attached to the phosphate backbone of the respective nucleic acid. In particular, the labeling portions may be attached to opposite ends of the respective first or second nucleic acid chains. For example, the at least one first labeling portion may be attached toward the 3' end of the respective nucleic acid chain, and the second labeling portion may be attached toward the 5' end of the respective nucleic acid chain.

[0027] In one embodiment, the at least one first labeling portion is preferably covalently attached to the first nucleic acid chain, for example, to the phosphate backbone of the nucleic acid chain, and the at least one second labeling portion may (covalently) be attached to the second nucleic acid chain, for example, to the phosphate backbone of the nucleic acid chain. In this case, when the first and second nucleic acid chains hybridize with each other, the optical properties of the first and second labeling portions may similarly change due to their proximity.

[0028] In one embodiment, the first nucleic acid chain extends along a first direction, and a plurality of first label portions are disposed on the first nucleic acid chain along the first direction; and / or the second nucleic acid chain extends along a second direction, and a plurality of second label portions are disposed on the second nucleic acid chain along the second direction. This enables the generation of an optically detectable signal that is proportional to the distance between the first label portions and the second label portions. Specifically, the corresponding label portions are disposed sequentially along the respective directions.

[0029] In one embodiment, the marker includes a third marker portion comprising a third nucleic acid strand, wherein the third nucleic acid strand is configured to form a triplet with the first and second nucleic acid strands. Preferably, the marker portion is disposed on or attached only to the third nucleic acid strand, and not on the other nucleic acid strands.

[0030] In particular, where the marker includes the third marker portion, the marker may include a third blocking nucleic acid strand that is at least partially complementary to the third nucleic acid strand. The third nucleic acid strand and the third blocking nucleic acid strand may additionally incorporate features described for the first nucleic acid strand or the second nucleic acid strand, or the first blocking nucleic acid strand or the second blocking nucleic acid strand. For example, the third blocking nucleic acid strand may include a cleavable linker. Specifically, there may be multiple third blocking nucleic acid strands, wherein the third blocking nucleic acid strands of the multiple blocking nucleic acid strands are connected to each other by cleavable linkers, particularly to form a cleavable linear strand of the blocking nucleic acid strand. The connected third blocking nucleic acid strands of the multiple third blocking nucleic acid strands can form a third cleavable linear strand of the blocking nucleic acid strand. The cleavable linker can be selectively cleaved by applying a cleavage agent.

[0031] Specifically, providing the marker having the third nucleic acid strand requires the three nucleic acid strands to specifically hybridize with each other to generate the marker. In particular, when the marker portion is attached to the third nucleic acid strand, the marker can only be detected if the three nucleic acid strands have hybridized with each other and formed the triploid structure. Furthermore, the triploid structure provides a robust and rigid structure to which the marker portion can attach.

[0032] Specifically, the third nucleic acid strand may have a third nucleotide sequence. This third nucleotide sequence may be at least partially complementary to the first and second nucleotide sequences in the hybridization, particularly based on Hoogsteen base pairing, to form the triplet structure. Therefore, the third nucleotide sequence can hybridize with the duplex of the first and second nucleotide sequences, particularly with the major groove of the duplex. This enables a stable triplet structure.

[0033] In one embodiment, each marker portion is equidistant from any adjacent marker portion. This enables the generation of an optically detectable signal that is proportional to the distance between the first and second marker portions. When the marker comprises multiple marker portions, each first marker portion is substantially equidistant from any adjacent first marker portion, and / or each second marker portion is substantially equidistant from any adjacent second marker portion. Preferably, the substantially equidistant spacing can be in the range of 0.33 to 33 nm.

[0034] On the other hand, a biomarker for analyzing biological samples having multiple target analytes is provided. The biomarker includes a marker, specifically, as described above, the marker includes a first marker portion and a second marker portion. The biomarker further includes: a first marker portion having a first affinity reagent and the first marker portion; and a second marker portion having a second affinity reagent and the second marker portion. Each of the first affinity reagent and the second affinity reagent is configured to specifically bind to one of the target analytes of the biological sample. Optionally, the biomarker may include a third marker portion, the third marker portion including the third marker portion having the third nucleic acid chain.

[0035] The biomarker can efficiently determine the distance or proximity between two target analytes. The biomarker can also accurately determine the presence and / or location of two or a single target analyte in a biological sample. Furthermore, the at least one blocking nucleic acid strand can process the first and second biomarker portions without the risk of unwanted uncontrolled hybridization between the first and second nucleic acid strands, for example, when preparing to analyze a biological sample containing the biomarker.

[0036] The biological sample may be a cell sample, such as a tissue section. Other examples include biopsy samples, such as liquid biopsy samples or tissue biopsy samples. The target analyte may be, for example, a protein or nucleic acid of the biological sample. The affinity reagent for the marker may be an antibody, an antibody fragment, an amino acid-based or nucleic acid-based aptamer, or a linear nucleic acid. This enables the detection of a wide variety of target analytes. In particular, the nucleic acid strands of the first and second marker portions are attached to the corresponding affinity reagent, preferably covalently attached to the corresponding affinity reagent.

[0037] The affinity reagents of the markers can bind to a single target analyte or different target analytes. In a first case, the first affinity reagent can be configured to specifically bind to a first region or epitope of the single target analyte, and the second affinity reagent can be configured to specifically bind to a second region or epitope of the single target analyte. By requiring both markers to partially bind to the single target analyte, the presence and / or location of the single target analyte can be precisely determined, particularly with improved specificity. In a second case, the first affinity reagent can be configured to specifically bind to a first target analyte, and the second affinity reagent can be configured to specifically bind to a second target analyte. This enables the determination of the distance or proximity between the first and second target analytes.

[0038] In particular, when the biomarker includes the third biomarker portion and the biomarker portion is attached to the third nucleic acid strand, the biomarker is thus detectable only if the third biomarker portion is present and the triplet is formed. This enables improved specificity in the detection of the single target analyte. Similarly, the third biomarker portion enables improved specificity in determining the proximity of the first and second target analytes.

[0039] On the other hand, a method for analyzing biological samples is provided. The method includes introducing at least one biomarker (particularly a biomarker as described herein) into the biological sample. Optionally, a first optical readout of the biological sample having the biomarker can be generated, particularly prior to the step of removing the blocking nucleic acid chain from the biomarker. In another step, the at least one blocking nucleic acid chain is removed from the biomarker. In a subsequent step, another optical readout of the biological sample having the biomarker is generated. In a subsequent optional step, the first optical readout and the other optical readout are compared, particularly to determine changes in the optical properties of the biomarker. Specifically, a fold change between the first and other optical readouts, particularly a fold change in the fluorescence emission of the biomarker, can be calculated.

[0040] For example, the optical readout can be generated using a microscope, such as a fluorescence microscope. The optical readout can determine the presence and / or location of a target analyte or the proximity of two target analytes.

[0041] The step of introducing the at least one biomarker may optionally include waiting for a predetermined amount of time to allow the at least one biomarker to bind to one or more corresponding target analytes of the biological sample. After the introduction of the at least one biomarker, any biomarkers not bound to the target analytes may optionally be removed from the biological sample, for example, by washing. When the at least one biomarker is introduced, the biomarker comprises the blocking nucleic acid strand, preferably hybridized to a first or second nucleic acid strand corresponding to a marker of the biomarker. The step of introducing the at least one biomarker may include adding individual portions of the at least one biomarker to the sample, particularly adding them individually over time. In this case, the biomarker is assembled in situ in the sample. Alternatively, the individual portions of the biomarker may be added together or simultaneously to the sample.

[0042] Following the introduction of the at least one marker, preferably after the at least one marker has bound to the corresponding target analyte of the biological sample, and particularly after any markers not bound to the target analyte have been removed from the biological sample (e.g., by washing), the first optical readout of the biological sample containing the marker can be generated. This enables the optical readout of the individual marker or label portion (e.g., the first and second markers or label portions) before proceeding to the next method step (i.e., removal of the blocking nucleic acid chain from the marker). While not always necessary, it is generally recommended to perform at least two optical readouts of the biological sample: one in which the first and / or second label portion is still hybridized with the blocking nucleic acid chain, and another after the blocking chain has been removed. This allows for the calculation of the fold change or proportion of fluorescence emission.

[0043] The step of removing the at least one blocking nucleic acid chain may include applying a cleavage agent to cleave the cleavable linkers of the at least one marker. This allows the blocking nucleic acid chain to be removed due to a decrease in its melting temperature. In particular, the individual blocking nucleic acid chains may be replaced by the first / second nucleic acid chain. Additionally, the step of removing the at least one blocking nucleic acid chain may include washing the sample to remove excess blocking chains. Furthermore, the step may include raising the sample temperature to a range of 37°C to 42°C.

[0044] The method for analyzing the biological sample and the marker described herein have the same advantages as the marker itself. Furthermore, the method and the marker can be supplemented with features that complement the marker described herein, particularly the features in the dependent claims of the marker. Attached Figure Description

[0045] The specific implementation scheme will be described below with reference to the attached drawings, in which: Figure 1 This is a schematic diagram of a marker that includes markers for analyzing biological samples. Figure 2 It is based on Figure 1 A schematic diagram of the logo. Figure 3 It is based on Figure 1 A schematic diagram of the logo. Figure 4 It is based on Figure 1 A schematic diagram of the logo. Figure 5 It is a schematic diagram of the marker according to the second implementation scheme. Figure 6 It is a schematic diagram of the marker according to the third implementation plan. Figure 7It is a schematic diagram of the marker according to the fourth implementation plan. Figure 8 It is based on Figure 7 A schematic diagram of the logo. Figure 9 A flowchart of a method for analyzing biological samples. Figure 10 This is a schematic diagram illustrating the use of biomarkers in flow cytometry. Figure 11 This is a schematic diagram illustrating the use of proximity hybridization assays (PHAs) for evaluating gene modifications. Figure 12 This is a schematic diagram illustrating the use of neighboring hybridization (PHA) analysis for quality control and monitoring in gene and cell therapy. Detailed Implementation

[0046] Figure 1 This is a schematic diagram of a biomarker 100 used for analyzing biological samples (not shown). Biomarker 100 includes a first biomarker portion 102 and a second biomarker portion 104. The first biomarker portion 102 may include a first label portion (having a first nucleic acid chain 106) and a plurality of first label portions 108. The first label portions 108 may (covalently) attach to the first nucleic acid chain 106.

[0047] The second marker portion 104 includes a second marker portion (having a second nucleic acid chain 110) and a plurality of second marker portions 112 (attached to the second nucleic acid chain 110). For example, the first nucleic acid chain 106 and the second nucleic acid chain 110 can be polynucleotides.

[0048] The first nucleic acid strand 106 and the second nucleic acid strand 110 are at least partially complementary to each other. This enables the first nucleic acid strand 106 and the second nucleic acid strand 110 to hybridize, particularly to form a double strand based on Watson-Crick base pairing.

[0049] The first biomarker portion 102 also includes a first affinity reagent 114 configured to specifically bind to the first target analyte 116. Similarly, the second biomarker portion 104 also includes a second affinity reagent 118 configured to specifically bind to the second target analyte 120.

[0050] The first labeled portion attaches to the first affinity reagent 114 via barcoded oligonucleotide 122. Similarly, the second labeled portion attaches to the second affinity reagent 118 via barcoded oligonucleotide 124. Each barcoded oligonucleotide 122, 124 (particularly its nucleotide sequence) is preferably specific to the corresponding affinity reagent 114, 118 and / or the corresponding labeled portion, particularly to the first and second nucleic acid chains 106, 110. In particular, the first and second affinity reagents 114, 118 are antibodies.

[0051] The first marker portion 102 binds to the first target analyte 116 via the first affinity reagent 114. Similarly, the second marker portion 104 binds to the second target analyte 120 via the second affinity reagent 118.

[0052] The marker 100 also includes a first plurality of blocking nucleic acid strands 126 and / or a second plurality of blocking nucleic acid strands 128. The blocking nucleic acid strands 126 and 128 are configured to hybridize with the first nucleic acid strand 106 or the second nucleic acid strand 110. Figure 1 The image shows the hybridization of the first blocking nucleic acid strand 126 with the first nucleic acid strand 106, and also shows the hybridization of the second blocking nucleic acid strand 128 with the second nucleic acid strand 110.

[0053] The first plurality of blocking nucleic acid strands 126 are interconnected by a cleavable linker 130. This forms a linear strand of blocking nucleic acid strand 126, which is complementary to the first nucleic acid strand 106. The cleavable linker 130 can form covalent links between adjacent blocking nucleic acid strands 126, 128.

[0054] The cleavable linker 130 is configured to be specifically cleaved by a cleaving agent. For example, the cleavable linker 130 may be configured to be a photocleavable linker that is cleaved by UV light. An example of such a photocleavable linker is the BMN linker (Biomers). Preferably, the cleavable linker is not a nucleotide or does not contain nucleotides. Alternatively, the cleavable linker 130 may be a chemically cleavable linker or an enzymatically cleavable linker.

[0055] Similar to the blocking nucleic acid strand 126, the second plurality of blocking nucleic acid strands 128 are connected to each other via a cleavable adapter 130.

[0056] The presence of the connecting blocking nucleic acid strands 126 and 128 and their hybridization with the corresponding nucleic acid strands 106 and 110 means preventing the first nucleic acid strand 106 and the second nucleic acid strand 110 from directly hybridizing with each other. Therefore, when processing the marker portions 102 and 104, for example when preparing to analyze biological samples, the marker portions 102 and 104 do not hybridize with each other due to the connecting blocking nucleic acid strands 126 and 128.

[0057] In an alternative implementation, marker 100 may include only one of the first or second plurality of blocking nucleic acid strands 126, 128. Such a single plurality of blocking nucleic acid strands 126, 128 can still achieve the above-mentioned effect, namely, preventing hybridization between the first nucleic acid strand 106 and the second nucleic acid strand 110.

[0058] exist Figure 1 In an exemplary embodiment, target analytes 116 and 120 are in contact with each other, for example, due to mutual affinity between target analytes 116 and 120. This causes marker portions 102 and 104 to come into proximity once bound to target analytes 116 and 120. As described above, although the first nucleic acid strand 106 and the second nucleic acid strand 110 are in proximity, they do not hybridize with each other due to blocking nucleic acid strands 126 and 128. Therefore, marker 100, particularly its marker portions 102 and 104, can be easily handled during the analysis of biological samples containing target analytes 116 and 120. For example, if marker portions 102 and 104 have not yet bound to target analytes 116 and 120 when marker 100 is introduced into a biological sample, the blocking nucleic acid strands 126 and 128 prevent unnecessary and uncontrolled hybridization of the first nucleic acid strand 106 and the second nucleic acid strand 110.

[0059] The first labeled portion 108 and the second labeled portion 112 can be fluorophores used as FRET donors or FRET acceptors, respectively. Therefore, each first labeled portion 108 can form an FRET pair with one second labeled portion 112. Because the first nucleic acid strand 106 and the second nucleic acid strand 110 are in... Figure 1 In the indicated state, no hybridization occurs, therefore the first nucleic acid strand 106 and the second nucleic acid strand 110 are not in close proximity to each other. Specifically, the first labeled portion 108 and the second labeled portion 112 are not within the distance that would allow FRET to occur. This distance is typically in the range of 1 to 10 nm. Therefore, the first labeled portion 108 and the second labeled portion 112 could be optically detectable (single) ordinary fluorescent dyes, but would not meet the characteristics of FRET.

[0060] In particular, Figure 1 A step in a method for analyzing biological samples is shown, according to which a marker 100 is introduced into the biological sample to be analyzed. Before the marker 100 is introduced into the biological sample and before affinity reagents 114, 118 have bound to their respective target analytes 116, 120, blocking nucleic acid strands 126, 128 prevent unwanted hybridization of the first and second nucleic acid strands 106, 110.

[0061] Figure 2This is a schematic diagram of marker 100 in the presence of lysis agent 200. Specifically, lysis agent 200 is UV light that irradiates marker 100, particularly the cleavable linker 130. Lysis agent 200 is capable of (specifically) cleaving the cleavable linker 130. This causes the individual blocking nucleic acid strands 126, 128 to separate, with only residues 202 of linker 130 remaining attached to the blocking nucleic acid strands 126, 128. In particular, lysis agent 200 can be applied to the biological sample to be analyzed as part of a method for analyzing biological samples.

[0062] Generally, hybridization between longer nucleic acids is more thermodynamically stable than hybridization between shorter nucleic acids. Therefore, compared with the hybridization of the respective cleaved blocking nucleic acid strands 126, 128 with the first or second nucleic acid strands 106, 110, the hybridization of the linked blocking nucleic acid strands 126, 128 with the first or second nucleic acid strands 106, 110 is thermodynamically more stable.

[0063] Preferably, each of the blocking nucleic acid strands 126, 128 has a plurality of nucleotides, the number of which is 5 to 15. The plurality of blocking nucleic acid strands 126, 128 can be linked to obtain corresponding linear strands of blocking nucleic acid strands 126, 128. Specifically, 5 to 30 blocking nucleic acid strands 126, 128 can be linked to each other via a connector 130.

[0064] Therefore, by cleaving the blocking nucleic acid chains 126 and 128 apart, the double strands formed between the blocking nucleic acid chains 126 and 128 and the corresponding nucleic acid chains 106 and 110 can be made unstable. This can cause the blocking nucleic acid chains 126 and 128 to dissociate from the nucleic acid chains 106 and 110 and / or cause the nucleic acid chains 106 and 110 to form double strands to replace the blocking nucleic acid chains 126 and 128.

[0065] In addition, in order to further destabilize the double strands formed between each blocking nucleic acid strand 126, 128 and the corresponding nucleic acid strands 106, 110, the temperature of the marker can be increased, especially to near or above the average melting temperature of the double strands between each blocking nucleic acid strand 126, 128 and the corresponding nucleic acid strands 106, 110.

[0066] Figure 3 This is a schematic diagram showing that marker 100 of the blocking nucleic acid chains 126 and 128 has been removed, for example, due to... Figure 2 The previously described addition of lysis agent 200. In the absence of blocking nucleic acid strands 126 and 128, the first and second nucleic acid strands 106 and 110 can freely hybridize with each other to form a double strand. Figure 3 The image shows the first and second nucleic acid chains 106 and 110 hybridizing with each other.

[0067] Hybridization of the first nucleic acid chain 106 with the second nucleic acid chain 110 brings the first labeled portion 108 and the second labeled portion 112 closer together, thereby allowing FRET to occur between the first labeled portion 108 and the second labeled portion 112. Therefore, the proximity of target analytes 116 and 120 can be determined, for example, by optical readout using a fluorescence microscope, through FRET between the first labeled portion 108 and the second labeled portion 112. Figure 3 In this context, FRET can occur between all the first marking portions 108 and the second marking portions 112.

[0068] In a method for analyzing biological samples, markers 100, particularly marker portions 102 and 104, can be initially introduced into the biological sample in the presence of blocking nucleic acid strands 126 and 128, such as... Figure 1 As shown. After a suitable period of time (for the biomarker portions 102 and 104 to bind), any biomarker portions 102 and 104 that remain unbound to the corresponding target analytes 116 and 120 can be removed, for example, by washing the biological sample.

[0069] Subsequently, as Figure 2 As described, blocking nucleic acid chains 126 and 128 can be cleaved, causing them to dissociate. Blocking nucleic acid chains 126 and 128 can then be further removed from the sample, for example, by washing the sample. Figure 3 The marker 100 is shown after the removal of blocking nucleic acid chains 126 and 128.

[0070] This enables hybridization of the first nucleic acid strand 106 with the second nucleic acid strand 110. Subsequently, an optical readout of the biological sample with marker 100 can be generated. The optical readout can be an image, such as an image generated by a fluorescence microscope.

[0071] Optical readout can determine whether target analytes 116 and 120 are in close proximity to each other. In particular, optical readout can be used to determine whether FRET has occurred between the first marker portion 108 and the second marker portion 112 by observing the corresponding changes in the optical properties of the first marker portion 108 and the second marker portion 112 caused by FRET.

[0072] Figure 4 This is a schematic diagram of marker 100 with target analytes 116 and 120, and... Figures 1 to 3 In comparison, the target analytes 116 and 120 are more widely spaced. Figure 3 Similarly, there are no blocking nucleic acid strands 126 and 128, and the first nucleic acid strand 106 and the second nucleic acid strand 110 can hybridize with each other. However, the greater distance between the target analytes 116 and 120 results in only partial hybridization between the first nucleic acid strand 106 and the second nucleic acid strand 110, especially with... Figure 3 Compared to the state shown (where target analytes 116 and 120 are in contact with each other), the degree of hybridization is lower.

[0073] Partial hybridization of the first nucleic acid strand 106 and the second nucleic acid strand 110 results in a greater distance between some of the labeled portions in the first labeled portion 108 and the second labeled portion 112. Specifically, the distance between these first labeled portions 108 and the second labeled portions 112 can prevent FRET from occurring between them. Therefore, the overall observable FRET efficiency is related to... Figure 3 The state shown is reduced compared to the previous state. This reduced FRET efficiency can be observed when generating optical readout of a biological sample with marker 100. Therefore, the first labeled portion 108 and the second labeled portion 112 that do not form FRET pairs (e.g., the labeled portions 108, 112 between barcode oligonucleotides 122, 124 and the hybrid portions of the first nucleic acid chain 106 and the second nucleic acid chain 110) may be optically detectable as (single) ordinary fluorescent dyes based on their fluorescent dye (spectral) emission characteristics; while the first labeled portion 108 and the second labeled portion 112 that form FRET pairs (e.g., the labeled portions 108, 112 of the hybrid portions of the first nucleic acid chain 106 and the second nucleic acid chain 110) can be detected based on the FRET characteristics of the FRET pairs and therefore based on the different spectral characteristics of the FRET pairs compared to the spectral characteristics of a single fluorescent dye. The ratio between the two different spectral characteristics (the spectral characteristics of the FRET pair and the spectral characteristics of the individual fluorescent dyes) can be used as a quantitative measure of the proximity of the affinity reagents 114 and 118, and therefore as the corresponding proximity of the target analytes 116 and 120.

[0074] In short, and specifically refer to Figure 3 and Figure 4 It can be seen that the distance between target analytes 116 and 120 directly affects the degree of hybridization between the first nucleic acid chain 106 and the second nucleic acid chain 110, and therefore directly affects the overall FRET efficiency between the first labeled portion 108 and the second labeled portion 112. Thus, the FRET efficiency between the first labeled portion 108 and the second labeled portion 112 is proportional to the distance between target analytes 116 and 120, and can be used to determine the distance or proximity between target analytes 116 and 120.

[0075] Figure 5 This is a schematic diagram of marker 500. Marker 500 includes a first marker portion 502 having an affinity reagent 503, which is a linear nucleic acid. Marker 500 also includes the second marker portion 104 described above. For simplicity, Figure 5The marking portion of marker 500 is not shown. As mentioned above, marker 500 may also include blocking nucleic acid chains 126 and 128. Figure 5 The image shows marker 500, which does not have blocking effect on nucleic acid chains 126 and 128.

[0076] The first marker portion 502 specifically binds to the nucleic acid target analyte 504. The second marker portion 104 specifically binds to the second target analyte 120, which may be a protein. Target analytes 504 and 120 may interact with or bind to each other. As described with respect to marker 100, such interaction or proximity between target analytes 504 and 120 can be determined by marker 500 in the same manner. In particular, the proximity of the marker portions can be determined by FRET occurring between the corresponding labeled portions (not shown) of marker portions 502 and 104, which serves as a measure of the distance between target analytes 504 and 120.

[0077] Figure 6 This is a schematic diagram of marker 600. Marker 600 includes a first marker portion 502 and a second marker portion 602, which respectively have first and second affinity reagents 503 and 604, and the first and second affinity reagents 503 and 604 are linear nucleic acids. For simplicity, Figure 6 The marking portion of marker 600 is not shown. As mentioned above, marker 600 may also include blocking nucleic acid chains 126 and 128. Figure 6 The image shows marker 600, which does not have blocking effect on nucleic acid chains 126 and 128.

[0078] Both affinity reagents 503 and 604 are configured to specifically bind to specific target sequences. Nucleic acid target analyte 504 includes these target sequences. The proximity between target sequences along target analyte 504 can be determined using marker 600, or, with high confidence, the presence of target analyte 504 can be determined by using two affinity reagents (which need to bind to target analyte 504 simultaneously). Marker 600 can be detected as described above for markers 100 and 500. Specifically, the proximity of the corresponding labeled portions (not shown) of marker portions 502 and 602 can be determined by FRET occurring between the corresponding labeled portions (not shown) of marker portions 502 and 602, which serves as a measure of the distance between target sequences or as a measure of the presence of target analyte 504 in a biological sample.

[0079] Figure 7 and Figure 8This is a schematic diagram of a marker 700 including a first marker portion 702, a second marker portion 704, and a third marker portion 706. The first marker portion 702 includes a first nucleic acid strand 106, and the second marker portion 704 includes a second nucleic acid strand 110. The third marker portion 706 includes a third nucleic acid strand 708, which is configured to form a triplet with the first and second nucleic acid strands 106 and 110, specifically based on Hoogsteen base pairing. Two marker portions 710 are attached to the third nucleic acid strand 708.

[0080] exist Figure 7 The diagram shows a marker 700 having a second plurality of blocking nucleic acid strands 128, which are interconnected by a connector 130 and hybridize with a second nucleic acid strand 110. This prevents hybridization between marker portions 702, 704, and 706. Specifically, double strands between the first and second nucleic acid strands 106 and 110, and triple strands between the third nucleic acid strand 708 and the first and second nucleic acid strands 106 and 110, cannot be formed.

[0081] exist Figure 8 The diagram illustrates marker 700, in which the linker 130 is cleaved by applying lysis agent 200, and the blocking nucleic acid strands 128 separate from each other. This enables the formation of a triplet among the first, second, and third nucleic acid strands 106, 110, and 708. Specifically, this associates the labeled portion 710 with affinity reagents 114 and 118, and thus with target analytes 116 and 120. In optical readout of the sample, this allows determination of the presence and / or location of target analytes 116 and 120. Simultaneously, close proximity of target analytes 116 and 120 is required to enable triplet formation. The labeled portion 710 may have the same or different optical properties.

[0082] Figure 9 This is a schematic flowchart of a method for analyzing biological samples. The method begins at step S900.

[0083] In step S902, at least one biomarker 100, 500, 600, 700 as described herein is introduced into the biological sample. Specifically, multiple biomarkers can be introduced into the sample, each biomarker including an affinity reagent configured to specifically bind to a different target analyte. In this step, the linked blocking nucleic acid strand is bound to the corresponding nucleic acid strand of the biomarker moiety. This allows the biomarker moiety to be introduced into the biological sample without the risk of hybridization between the biomarker moieties before they bind to their respective target analytes. Subsequently, step S902 may include removing biomarker moieties not bound to any target analyte, for example, by washing the sample.

[0084] In optional step S904, a first optical readout of the sample with the marker is generated, for example, by microscopy. This is particularly important when the marker 100 is introduced into the sample in step S902. The first optical readout can determine the fluorescence of the labeled portions 108 and 112 when the marker portions 102 and 104 are prevented from forming double strands due to the presence of the linked blocking nucleic acid strands 126 and 128. Therefore, the labeled portions 108 and 112 retain their inherent optical properties without undergoing FRET.

[0085] In step S906, the blocking nucleic acid chains are removed. This includes cleaving the cleavable linkers 130 between the individual blocking nucleic acid chains, for example by applying a cleavage agent, such as UV light. This cleavage step separates the blocking nucleic acid chains from each other and destabilizes the duplexes between the blocking nucleic acid chains and the corresponding nucleic acid chains of the marker portions.

[0086] Step S906 may optionally include altering the conditions for preserving the sample to further destabilize the duplex between the blocking nucleic acid strand and the corresponding nucleic acid strand of the marker moiety. Such altered conditions may be physical or chemical, such as the temperature of the sample or the concentration of the buffer / salt used to preserve the sample. Specifically, the sample temperature may be increased to a range of 37°C to 42°C.

[0087] As an alternative, cleaved blocking nucleic acid chains can be removed from the sample, for example, by washing the sample.

[0088] Removing the blocking nucleic acid strands allows the nucleic acid strands in the marker portion to form double strands.

[0089] In step S908, another optical readout of the sample with the markers is generated, for example, by a microscope. For example, when marker 100 is in close proximity to target analytes 116 and 120, marker portions 102 and 104 (specifically their corresponding nucleic acid chains 106 and 110) now hybridize with each other. This results in the formation of FRET pairs by the marker portions 108 and 112, which can be optically detected in the optical readout.

[0090] In step S910, the presence and / or location of the target analyte in the sample can be determined based on the optical signal of the marked portion present in at least one other optical readout. For example, in the case of marker 700, the optical signal of the marked portion 710 can be determined in another optical readout, and based on this, the presence and / or location of the target analytes 116 and 120 can be determined. Additionally, it can be determined that the target analytes 116 and 120 are close to each other.

[0091] Optionally, and in the case of marker 100, the first optical readout can be compared with another optical readout, and the differences in the optical properties of the marker portions 108, 112 can be used to determine the presence, location and / or proximity of the target analytes 116, 120.

[0092] The method ends at step S912.

[0093] Method steps S902 to S910 can be repeated iteratively, with each iteration using a different set of markers. In this case, the marked portion of the marker can preferably be removed after each iteration to remove the marked portion of the corresponding marker.

[0094] Figure 10 This is a schematic diagram illustrating the use of marker 1000 in flow cytometry. Marker 1000 includes marker portions 1002 and 1004, which bind to corresponding targets 1006 and 1008 present or expressed on the cell surface 1010. If both targets 1006 and 1008 are present on the cell surface 1010, both marker portions 1002 and 1004 bind to them, and marker 1000 generates an optically detectable signal 1012. If only one target 1008 is present on the cell surface 1010, only marker portion 1004 binds to it, and no optically detectable signal is generated. Therefore, prior to flow cytometry, cells can be contacted with marker 1000 to distinguish cells possessing one or both targets 1006 and 1008.

[0095] Marker 1000 can also be used to determine whether two types of cells (each with target 1006 or target 1008) are closely adjacent, especially whether targets 1006 and 1008 are close to each other on the surface of the cells.

[0096] Figure 11 This is a schematic diagram of PHA analysis, used to assess the integration of gene cassettes for gene and cell therapy. These analyses are based on the use of oligonucleotides as affinity reagents and single-molecule fluorescent in situ hybridization (smFISH) technology. Figure 11 In the diagram, the first and second marker portions are schematically shown as semi-circular shapes. The corresponding markers may include blocking nucleic acid strands to prevent the first and second marker portions from hybridizing with each other.

[0097] Figure 11 The top row of the diagram shows the integration of the gene cassette (white arrow) into the target site next to the exon (black rectangle), with the rest of the genomic DNA represented by black lines. Figure 11 The bottom row of the top figure shows random or ectopic integration. PHA analysis allows for accurate differentiation of different types of integration because it can detect target site integration with only the two labeled portions 1100 and 1102, which are the markers, whereas in random integration, only the first labeled portion 1100 or the second labeled portion 1102 is present. After removal of the blocking strand, target site integration is detectable as a fluorescent spot, which is brighter than the fluorescent spot produced by ectopic integration when using dequenching-based PHA analysis, and / or has higher FRET efficiency or different fluorescence lifetimes when using FRET-based PHA analysis.

[0098] Figure 11 The bottom diagram illustrates a schematic of a PHA analysis used to assess genomic sequence deletions. For example, in cell lines, partial genomic deletions can be induced to correct gene defects. This can be achieved, for example, using CRISPR / Cas9 or similar systems and utilizing homologous recombination. For example, if patient-derived cells are modified to delete specific portions of the genome, it may be necessary to assess the appropriate frequency of deletion occurrence. This can be achieved using a PHA analysis schematically shown in the bottom diagram. Due to the deletion of the target site, the two probes of the first and second marker portions are brought close together, and they can be read as corresponding smFISH spots, which are brighter than spots at sites without deletions when using dequenching-based PHA analysis, and / or have higher FRET efficiency or different fluorescence lifetimes when using FRET-based PHA analysis.

[0099] Figure 12 yes Figure 11 This is a schematic diagram of the hypothetical readout of the described analysis. Analysis is performed on various cell types 1200a, 1200b, 1200c, 1200d, and 1200e, which may be derived, for example, from blood samples, to monitor the evolution of the cell therapy product in a patient. Such monitoring can be performed to assess how different clones within the cell therapy product evolve in the patient during cell therapy. Such clones may not include genetic modifications (e.g., no insertion / integration or deletion), they may include only the desired target site modification, they may include only ectopic integration and / or deletion, and they may include a mixture of target site and ectopic modifications. Cells are stained and readouted before and after removal of the blocking chain, the spots are segmented, and the before-and-after images are registered to identify spots that change after removal of the blocking chain; changes in the spots represent a paired detection event.

[0100] As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items and may be abbreviated as “ / ”.

[0101] Although some aspects have been described in the context of the device, these aspects clearly also represent descriptions of the corresponding methods, where blocks or devices correspond to method steps or features of method steps. Similarly, aspects described in the context of method steps also represent descriptions of corresponding blocks, items, or features of the corresponding device.

[0102] Figure Labels

Claims

1. A marker for analyzing biological samples, comprising: The first marker portion includes a first nucleic acid chain (106), and The second marker portion includes a second nucleic acid strand (110), and The first nucleic acid strand (106) and the second nucleic acid strand (110) are configured to form a double strand. The marker further includes at least one first marking portion (108, 710) and at least one second marking portion (112, 710), and The marker further includes at least one blocking nucleic acid strand (126, 128) that is at least partially complementary to one of the first nucleic acid strand (106) and the second nucleic acid strand (110).

2. The marker according to claim 1, comprising a plurality of blocking nucleic acid strands (126, 128), wherein the blocking nucleic acid strands (126, 128) are connected to each other by a cleavable adapter (130).

3. The marker according to claim 2, wherein the cleavable connector (130) is a photocleavable connector, a chemically cleavable connector, or an enzyme-cleavable connector.

4. The marker according to claim 2 or 3, wherein all of the plurality of blocking nucleic acid chains (126, 128) are complementary to the corresponding nucleotide sequence of the first nucleic acid chain (106) or the second nucleic acid chain (110).

5. The marker according to any one of claims 2 to 4, wherein the adjacent blocking nucleic acid chains (126, 128) of the plurality of blocking nucleic acid chains are complementary to the corresponding adjacent nucleotide sequences of the first nucleic acid chain (106) or the second nucleic acid chain (110).

6. The marker according to any one of claims 2 to 5, wherein each of the at least one blocking nucleic acid chain (126, 128) comprises a plurality of nucleotides, the number of said nucleotides being in the range of 5 to 15, preferably 5 to 12, more preferably 6 to 9.

7. The marker according to any one of the preceding claims, wherein the first nucleic acid chain (106) and / or the second nucleic acid chain (110) comprises a plurality of nucleotides, the number of said nucleotides being in the range of 50 to 150.

8. The marker according to any one of the preceding claims, comprising a second plurality of blocking nucleic acid chains (128), wherein the blocking nucleic acid chains (128) of the second plurality of blocking nucleic acid chains are connected to each other by cleavable linkers (130), and wherein the blocking nucleic acid chains (128) of the second plurality of blocking nucleic acid chains are complementary to the corresponding nucleotide sequences of the second nucleic acid chain (110).

9. The marker according to any one of the preceding claims, wherein the at least one blocking nucleic acid chain (126, 128) is complementary to a portion of the first nucleic acid chain (106) or a portion of the second nucleic acid chain (110), wherein a portion of the first nucleic acid chain (106) and a portion of the second nucleic acid chain (110) are complementary to each other.

10. The marker according to any one of the preceding claims, wherein the at least one first marking portion (108) and / or the at least one second marking portion (112) is optically detectable.

11. The marker according to any one of the preceding claims, wherein the at least one first marking portion (108) and / or the at least one second marking portion (112) have the same optical characteristics, or The at least one first marking portion (108) and / or the at least one second marking portion (112) have different optical characteristics.

12. The marker according to any one of the preceding claims, wherein the at least one first marker portion (108) and the at least one second marker portion (112) are configured for nonradiative energy transfer between them.

13. The marker according to any one of the preceding claims, wherein the at least one first marker portion (108) and the at least one second marker portion (112) are both attached to the first nucleic acid chain (106) or the second nucleic acid chain (110).

14. The marker according to any one of claims 1 to 12, wherein the at least one first marker portion (108) is attached to the first nucleic acid chain (106).

15. The marker according to any one of the preceding claims, wherein the first nucleic acid chain (106) extends along a first direction, and a plurality of the first marker portions (108) are disposed on the first nucleic acid chain (106) along the first direction, and / or The second nucleic acid chain (110) extends along a second direction, and a plurality of the second label portions (112) are arranged on the second nucleic acid chain (110) along the second direction.

16. The marker according to any one of the preceding claims, comprising a third marker portion, the third marker portion comprising a third nucleic acid strand (708), wherein the third nucleic acid strand (708) is configured to form a triplet with the first nucleic acid strand (106) and the second nucleic acid strand (110).

17. The marker according to any one of the preceding claims, wherein the spacing between each marker portion (108, 112) and any adjacent marker portion (108, 112) is equal.

18. A biomarker (100, 500, 600, 700, 1000) for analyzing biological samples having multiple target analytes (116, 120), comprising: The marker according to any one of the preceding claims comprises a first marker portion and a second marker portion. The first marker portion (102) includes a first affinity reagent (114) and the first marker portion, and The second marker portion (104) includes the second affinity reagent (118) and the second marker portion, and The first affinity reagent (114) and the second affinity reagent (118) are both configured to specifically bind to one of the target analytes (116, 120) of the biological sample.

19. A method for analyzing biological samples, comprising the following steps: Introducing at least one biomarker (100, 500, 600, 700, 1000) according to claim 18 into the biological sample, Optionally, a first optical readout of the biological sample having the aforementioned markers (100, 500, 600, 700, 1000) is generated. Remove blocking nucleic acid chains (126, 128) from the markers (100, 500, 600, 700, 1000), and Generate optical readout or another optical readout of the biological sample having the aforementioned markers (100, 500, 600, 700, 1000), and Optionally, the multiple change between the first and second optical readouts is calculated, or the first optical readout is compared with the other optical readout.

Images