Affinity reagents, markers and methods for analyzing biological samples

Abstract

The present invention provides an affinity reagent (102, 102a, 102b) for analyzing a biological sample. The affinity reagent (102, 102a, 102b) comprises a nucleic acid backbone (200, 300, 402). The nucleic acid backbone (200, 300, 402) is configured to specifically bind to a target analyte (106) through its complex structure (400c). Further, the nucleic acid backbone (200, 300, 402) comprises at least one pair of reactive moieties (202, 204, 302, 304, 308, 310) for cross-linking the nucleic acid backbone (200, 300, 402). In other aspects, the present application provides markers and methods for analyzing a biological sample.

Description

Technical Field

[0001] The present invention relates to an affinity reagent for analyzing biological samples. In other aspects, a marker comprising the affinity reagent and a method for analyzing biological samples using the marker are provided. Background Art

[0002] Nucleic acid-based affinity reagents offer advantages over antibodies in many respects. For example, aptamer discovery via SELEX (Selex aptamer expression) is simple and economical. Furthermore, information about nucleic acid-based affinity reagents can be stored digitally and used to produce the corresponding nucleic acid-based affinity reagents at low cost via oligonucleotide synthesis with excellent batch-to-batch variability. They generally exhibit high affinity and are easily functionalized and polymerized.

[0003] However, there is a key challenge that remains unresolved. The binding properties of nucleic acid-based affinity reagents depend on their 3D structure. However, due to the lack of side chain interactions, the stability of nucleic acid-based 3D structures is lower than that of protein-based 3D structures. For this reason, a specific aptamer can dynamically change between different 3D structures, only one or a few of which are active and specifically bind to the target molecule. Similarly, during storage, nucleic acid-based affinity reagents may partially unfold or misfold, and therefore may need to be refolded before use. This significantly reduces the ease of use and practical application of nucleic acid-based affinity reagents. Summary of the Invention

[0004] The object of the present invention is to provide a robust affinity reagent and marker and a method for analyzing a biological sample using the marker.

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

[0006] In a first aspect, an affinity reagent for analyzing a biological sample is provided. The affinity reagent comprises a nucleic acid backbone. The nucleic acid backbone is configured to specifically bind to a target analyte through its composite structure. Further, the nucleic acid backbone comprises at least one pair of reaction moieties for cross-linking the nucleic acid backbone. This can provide a robust and stable affinity reagent.

[0007] For example, the nucleic acid backbone can be a deoxyribonucleic acid molecule. In particular, the nucleic acid backbone can be a single, continuous deoxyribonucleic acid molecule. The nucleic acid backbone can have an unfolded primary structure, such as a linear sequence of nucleotides linked together by phosphodiester bonds.

[0008] In a preferred embodiment, the I nucleic acid backbone can comprise, in whole or in part, a heterologous nucleic acid backbone, i.e., the backbone can comprise phosphorothioate bonds, borophosphate bonds, amide bonds, or triazole bonds. Such backbones can impart higher stability and degradation resistance. Similarly, the nucleic acid backbone can comprise L-DNA and / or modified nucleobases, including nucleobases with side chain residues, such as, for example, the slow-off-rate modified aptamer SOMAmer (SOMAmer).

[0009] For example, the composite structure or conformation of the nucleic acid backbone of an affinity reagent can be a secondary, tertiary or quaternary structure of a nucleic acid backbone. Therefore, the nucleic acid backbone of an affinity reagent can preferably have a secondary, tertiary or quaternary structure. In particular, an affinity reagent is configured to specifically bind to a target analyte, such as a target analyte of a biological sample, due to the composite structure of the backbone of the affinity reagent. Specifically, the individual discontinuous nucleotides of the nucleic acid backbone bind to the target analyte, rather than the continuous nucleotides in the sequence of the nucleic acid hybridizing with the target analyte, especially the complementary sequence of the target analyte. This enables the binding of the affinity reagent to have high specificity, such as binding with a target analyte based on amino acids. In particular, the backbone of an affinity reagent, such as an aptamer, can bind to its target through a paratope-epitope interaction, rather than being bound to its target via hybridization of complementary nucleotide sequences. The binding of an affinity reagent to a target analyte can include intermolecular forces, such as hydrogen bonds, dipole-dipole interactions, ionic interactions, π-stacking, hydrophobic interactions and van der Waals forces.

[0010] The reactive part in reactive part can be arranged along the nucleotide linear sequence of nucleic acid backbone, and for example each reactive part all is attached to two Nucleotide in the linear sequence.Therefore, reactive part is arranged as non-adjacent to each other along the nucleic acid backbone sequence.Yet, in secondary, tertiary and / or quaternary structure of nucleic acid backbone, reactive part can change owing to corresponding change in the nucleic acid backbone conformation relative to each other's position.Preferably, when having unfolding, linear primary structure with nucleic acid backbone, compare, when nucleic acid backbone had composite structure, reactive part distance between each other was less.

[0011] In particular, the nucleic acid backbone can comprise the crosslinking between at least one reactive part. In particular, crosslinking and / or at least one reactive part are to be used to maintain the composite structure of the nucleic acid backbone. For this reason, reactive part and / or crosslinking are preferably crosslinking positions along the nucleic acid backbone, and they are non-adjacent along the nucleic acid backbone. For example, the reactive part in the reactive part is arranged as, as discussed above, along the linear sequence of the nucleic acid backbone, at a certain distance from each other. Therefore, intrachain crosslinking can be formed, especially the intrachain crosslinking of the reactive part in the reactive part. For example, intrachain crosslinking can crosslink two separate positions along the linear sequence of the nucleic acid molecule of the nucleic acid backbone.

[0012] In a preferred embodiment, the nucleic acid backbone can comprise a plurality of nucleic acid molecules that combine and fold together to form a composite structure. In this case, crosslinking can be formed between the reactive moieties in the reactive moiety pair, which are arranged on different nucleic acid molecules in the plurality of nucleic acid molecules. This can be referred to as interchain crosslinking.

[0013] Preferably, the reactive part in the reactive part pair is configured to react with each other. This can result in forming a covalent bond between the reactive part, and can produce a kind of robust affinity reagent with stable composite structure. In particular, the reactive part is configured to react specifically or directly with each other. This can form a covalent bond specifically between the reactive part. Therefore, the reactive part is preferably covalently attached to each other, that is, they react with each other to form a covalent bond, and the reaction is not combined with each other by hybridization or by the intermolecular forces such as hydrogen bond, dipole-dipole interaction and van der Waals force. The reaction of the reactive part can especially be copper-dependent azide alkyne cycloaddition (CuAAC), strain-promoted azide alkyne cycloaddition (SpAAC), Staudinger reaction (Staudinger reaction), Diels-Alder reaction (Diels-Alder), thiol-ene reaction or other any suitable reaction. The reactive moiety can be, among others, an azide, an alkyne, dibenzocyclooctyne (DBCO), a thiol, an alkene, a maleimide, N-hydroxysuccinimide, an amino group.

[0014] Preferably, the reactive part in the reactive part pair is configured to react under catalytic conditions, in particular only react under catalytic conditions. This can control the reaction of the reactive part in the reactive part pair. For example, the catalytic conditions can be the presence of a catalyst. In particular, the catalyst can be light (such as UV light), copper ions or molecules.

[0015] Preferably, the affinity reagent further comprises a linking element, and the reactive portion in the reactive portion pair is configured to react with the corresponding reactive portion of the linking element. This can flexibly cross-link the nucleic acid backbone. For example, when the nucleic acid backbone has a composite structure, the distance between the reactive portions in the reactive portion pair can be selected to form a linking element. The linking element can include polyethylene glycol (PEG) molecules, which have various lengths and can be selected. Further, the corresponding reactive portion of the linking element can be a click chemistry group or an NHS group. Therefore, the PEG molecule can be functionalized. Each of the reactive portions in the reactive portion pair preferably forms a covalent bond with a corresponding reactive portion of the linking element.

[0016] Preferably, the reactive moieties in the reactive moiety pair are modified nucleotides, particularly functionalized nucleotides. This allows for efficient integration of the reactive moieties in the nucleic acid backbone. For example, the reactive moieties may be non-natural or artificial nucleotides, preferably not (particularly nucleobases) that bind to the natural nucleotides adenine, thymine, guanine and cytosine via base pairing.

[0017] Preferably, the reactive moiety in the reactive moiety pair comprises a click chemistry group. This can effectively generate crosslinks. For example, the reactive moiety can be azide / alkyne group for CuAAC, or DBCO / azide group for SpAAC. For example, in the case of CuAAC, the catalytic condition can be the presence of a copper catalyst.

[0018] Preferably, the reactive moieties in the reactive moiety pair are photoreactive. This allows for efficient crosslinking. For example, the reactive moieties can be photoreactive nucleobases. In particular, each of the reactive moieties is a thymine nucleotide, which reacts under UV light to form a thymine dimer.

[0019] Preferably, the nucleic acid backbone comprises 10 to 150 nucleotides. This allows for a particularly compact affinity reagent that can effectively penetrate biological samples, such as tissue sections.

[0020] Preferably, the nucleic acid backbone is an aptamer. This allows for a compact and robust affinity reagent. Further, this allows for efficient generation of nucleic acid backbones that specifically bind to a specific target analyte, for example, via SELEX methods.

[0021] Preferably, the reactive moieties in the reactive moiety pairs are separated from one another along the nucleic acid backbone. This can provide a robust nucleic acid backbone with stable crosslinks. In particular, the reactive moieties are separated from one another along the primary structure or sequence of the nucleic acid backbone. Thus, the reactive moieties are not adjacent or proximal to one another along the sequence of the nucleic acid backbone.

[0022] Preferably, when the affinity reagent has its composite structure, the reactive part in the reactive part pair is close to each other. This can provide a kind of robust nucleic acid backbone with stable crosslinking. Therefore, when the nucleic acid backbone is in its composite structure, the reactive part is arranged in a position close to each other in space along the nucleic acid backbone. Especially, the reactive part is close to each other in space so that the reactive parts can react with each other.

[0023] Preferably, the affinity reagent comprises a second reactive moiety pair, wherein the reactive moiety in the second pair is different from the reactive moiety in at least one reactive moiety pair. This can generate a robust affinity reagent. In particular, the reactive moiety in the second pair is different from the reactive moiety in at least one reactive moiety pair in that they do not react with each other. Therefore, only the reactive moieties in the second pair react with each other, and the reactive moieties in at least one reactive moiety pair react with each other.

[0024] In a preferred embodiment, the affinity reagent may comprise at least one additional reactive moiety pair, wherein the additional reactive moiety pair is different from the first reactive moiety pair and the second reactive moiety pair.

[0025] On the other hand, a marker for analyzing a biological sample is provided. The marker comprises the affinity reagent described in detail above and a label (label), the label comprising at least one labeling portion. The labeling portion can be attached to the affinity reagent. Preferably, the labeling portion is optically detectable, for example, a fluorophore. The affinity reagent can provide a robust marker, for example, it can be coupled with a thermosensitive labeling portion. In particular, the need for nucleic acid-based affinity reagents to be refolded by heating after storage may have a negative impact on the thermosensitive labeling portion.

[0026] In another aspect, a method for analyzing a biological sample is provided. The method comprises the steps of introducing at least one marker (eg, as described above) into the biological sample, and generating a readout of the biological sample using the marker.

[0027] For example, each element of at least one marker can be introduced separately into a biological sample. Thus, the affinity reagent and the label in the marker can be introduced separately to generate the marker in the biological sample. The readout is preferably an optical readout, for example, an optical readout generated with the aid of a microscope.

[0028] Preferably, prior to introducing the at least one marker, the affinity reagent for the marker is generated by cross-linking the reactive moiety pairs of the marker. Thus, cross-linking of the reactive moiety pairs of the affinity reagent is achieved. This may include applying catalytic conditions.

[0029] Typically, multiple markers can be introduced into a biological sample that are specific for the corresponding target analyte in order to simultaneously identify a large number of (different or identical) target analytes. Preferably, the target analyte is identified and / or localized within the biological sample based on a marker, particularly a labeling moiety, in the marker that is associated with the target analyte during optical reading.

[0030] The markers and methods have the same advantages as the affinity reagents.Furthermore, the features of the affinity reagents described in this document, in particular the features in the dependent claims of the affinity reagents, may be employed to supplement the markers and methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The following describes specific embodiments with reference to the accompanying drawings, in which:

[0032] Figure 1 is a schematic diagram of a marker comprising an affinity agent in a folded and unfolded conformation, and

[0033] Figure 2 is a schematic diagram of a marker having an affinity reagent comprising a reactive moiety,

[0034] Figure 3 is a schematic diagram of a marker having an affinity reagent comprising a linking element, and

[0035] Figure 4 is a schematic diagram of the steps for cross-linking the nucleic acid backbone of an affinity reagent. DETAILED DESCRIPTION

[0036] Figure 1 is a schematic diagram of a marker 100 comprising an affinity reagent 102 in a folded and unfolded conformation. The marker 100 further comprises a label having a labeling portion 104. When the affinity reagent 102 has a folded conformation, the affinity reagent 102, and therefore the marker 100, can specifically bind to a target analyte 106 in a biological sample. Figure 1 The left view shows a folded conformation of affinity agent 102. For example, affinity agent 102 can be a nucleic acid-based aptamer.

[0037] The labeling portion 104 may be an optically detectable portion, such as a fluorophore. Thus, the marker 100 may be detected with the aid of a microscope. Alternatively, the marker 100 may comprise a plurality of labeling portions.

[0038] Under unfolding conditions, such as at high temperature, the affinity reagent 102 can unfold into Figure 1 The partially unfolded conformation shown in the upper right panel, or unfolded into Figure 1The fully unfolded conformation is shown in the lower right view. After the unfolding condition is removed, the affinity reagent 102 can return to the folded conformation. Only in the folded conformation of the affinity reagent 102 can the affinity reagent 102 specifically bind to the target analyte 106.

[0039] In order to maintain the affinity reagent 102 of the marker 100 in a folded conformation in which the affinity reagent 102 specifically binds to the target analyte 106, the affinity reagent 102 can include at least one reactive moiety pair. These reactive moieties can cross-link the affinity reagent 102 so as to maintain the composite structure of the affinity reagent 102. These aspects are further described below.

[0040] Figure 2 is a schematic diagram of a marker 100a having an affinity reagent 102a comprising a nucleic acid backbone 200 and reactive moieties 202 and 204 of a reactive moiety pair. The nucleic acid backbone 200 of the affinity reagent 102a is configured to specifically bind to a particular target analyte, such as a target analyte 106, e.g., a protein from a biological sample. In particular, the nucleic acid backbone 200 can be formed or folded into a complex structure or conformation. The complex structure of the backbone 200 of the affinity reagent 102a can be a secondary, tertiary, and / or quaternary structure of the backbone 200. Thus, in particular, the backbone 200 of the affinity reagent 102a has a secondary, tertiary, or quaternary structure. Due to the complex structure of the backbone 200 of the affinity reagent 102a, the affinity reagent 102a specifically binds to the target analyte 106. Specifically, individual, discrete nucleotides within the nucleic acid of the backbone 200 bind to the target analyte 106. This binding can include intermolecular forces such as hydrogen bonding, dipole-dipole interactions, and van der Waals forces. This is in contrast to nucleic acid hybridization, which is characterized by the hybridization of a contiguous nucleotide sequence in the nucleic acid to the target analyte, particularly to a complementary nucleotide sequence of the target analyte.

[0041] Based on the composite structure of the scaffold 200 , the scaffold 200 of the affinity reagent 102 a can bind to the target analyte 106 with high affinity and specificity.

[0042] The nucleic acid of backbone 200 may comprise multiple individual nucleotide sequences or chains. These individual sequences may bind to each other and form a quaternary structure.

[0043] In order to make Figure 2 For clarity, the nucleic acid backbone 200 is shown as an unfolded linear structure. When the nucleic acid backbone 200 of the affinity reagent 102a of the marker 100a has a composite structure, the reactive portions 202 and 204 are in close proximity to form a covalent bond. Figure 2The covalent bond is represented by reference numeral 206. The covalent bond between the reaction parts 202, 204 can cross-link the nucleic acid backbone 200 of the affinity reagent 102a to maintain the composite structure of the nucleic acid backbone 200.

[0044] The reactive moieties 202, 204 are modified nucleotides of the nucleic acid backbone 200. For example, the reactive moieties 202, 204 can comprise click chemistry groups attached to the nucleobases guanine or cytosine, respectively, such as via an azide / alkyne group for CuAAC or via a DBCO / azide group for SpAAC.

[0045] Figure 3 is a schematic diagram of a marker 100b having an affinity reagent 102b comprising a nucleic acid backbone 300, reactive moieties 302, 304 of a reactive moiety pair, and a linking element 306. The nucleic acid backbone 300 of the affinity reagent 102b is configured to specifically bind to a particular target analyte, such as target analyte 106.

[0046] As explained above for nucleic acid backbone 200, nucleic acid backbone 300 can be formed or folded into a composite structure or conformation. Figure 3 As can be seen clearly, the nucleic acid backbone 300 is shown as an unfolded linear structure. When the nucleic acid backbone 300 of the affinity reagent 102b of the marker 100b has a composite structure, the reactive portions 302 and 304 are in close proximity, and the connecting element 306, in particular the corresponding reactive portions 308 and 310 of the connecting element 306, can react with the reactive portions 302 and 304 of the nucleic acid backbone 300 to crosslink the nucleic acid backbone 300. This maintains the composite structure of the nucleic acid backbone 300. The reactive portions 302, 304, 308, and 310 can react with each other to form covalent click chemistry groups between the reactive portions 302 and 308 and between the reactive portions 304 and 310.

[0047] In particular, when the nucleic acid backbone 300 has a composite structure that is too large to allow the reaction portions 302, 304 to react directly with each other, the connecting element 306 can connect the reaction portions 302, 304 at a certain distance from each other. When the nucleic acid backbone 300 has a composite structure, the length of the connecting element 306 can be selected based on the distance between the reaction portions 302, 304.

[0048] Figure 4 Schematic diagram of the steps of cross-linking the nucleic acid backbone 402 of the affinity reagent in its complex structure in the marker 400. The marker 400 further comprises a labeling moiety 404. The nucleic acid backbone 402 comprises a reactive moiety pair 406 that cross-links the nucleic acid backbone 402.

[0049] First, by applying unfolding conditions (such as high temperature), the partially folded conformation 400a of the marker 400, especially the nucleic acid backbone 402, can be completely unfolded. This generates a linear conformation 400b of the unfolding of the marker 400. The partially folded conformation 400a can alternatively be a misfolded conformation that cannot specifically bind to a specific target analyte. Subsequently, by applying folding conditions, such as low temperature or physiological temperature, the unfolded conformation 400b can be refolded into the natural composite structure 400c of the marker 400. The composite structure 400c can specifically bind to a specific target analyte with high affinity. However, the composite structure 400c is still easy to unfold into partially or completely unfolded conformations 400a, 400b. This can be caused by applying unfolding conditions or randomly due to environmental influences or storage conditions.

[0050] In order to maintain the composite structure 400c of the affinity reagent of the marker 400, the nucleic acid backbone 402 can be cross-linked. The reactive moiety pairs 406 are configured to react with each other under catalytic conditions (e.g., UV light 408). After folding into the composite structure 400c, UV light 408 can be applied to react the reactive moiety pairs 406 and form cross-links. This can maintain the composite structure of the affinity reagent of the marker 400 and avoid undesirable unfolding of the affinity reagent of the marker 400.

[0051] In an alternative embodiment, the composite structure 400c can be refolded in the absence of the tag moiety 404 attached to the nucleic acid backbone 402, and the reactive moiety pair 406 can be cross-linked. Only in a subsequent step can the tag moiety 404 be attached to the nucleic acid backbone 402. This avoids damage to the tag moiety, such as by catalytic conditions or unfolding conditions.

[0052] Markers 100, 100a, 100b, 400 can be used in methods for analyzing biological samples. Markers 100, 100a, 100b, 400, and in particular corresponding affinity reagents, are specific for a target analyte in the biological sample. When markers 100, 100a, 100b, 400 are introduced into the biological sample, the markers 100, 100a, 100b, 400 specifically bind to the target analyte. Subsequently, the markers 100, 100a, 100b, 400 can be used to generate a readout of the biological sample, in particular an optical readout, for example, using a microscope. In this readout, the corresponding labeled portion of the markers 100, 100a, 100b, 400 can be detected, and the presence or location of the target analyte in the biological sample can be determined. Preferably, the markers 100, 100a, 100b, 400 are generated before or during their introduction into the biological sample, in particular as described above.

[0053] In all figures, the same reference numerals are used to denote elements having the same or similar functions. 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 " / ".

[0054] Although some aspects have been described in the context of an apparatus, it is clear that these aspects also represent a description of a corresponding method, where a block or device corresponds to a method step or a feature of a method step. Similarly, aspects described in the context of a method step also represent a description of a corresponding block or item or feature of a corresponding apparatus.

[0055] Reference Signs List

[0056] 100, 100a, 100b, 400 markers

[0057] 102, 102a, 102b affinity reagents

[0058] 104, 404 mark part

[0059] 106 target analytes

[0060] 200, 300, 402 nucleic acid backbone

[0061] 202, 204, 302, 304, 308, 310 reaction parts

[0062] 206 Covalent Bond

[0063] 306 connecting element

[0064] 400a Partially unfolded conformation

[0065] 400b unfolded conformation

[0066] 400c composite structure

[0067] 406 Reaction Partial

[0068] 408 Catalytic Conditions

Claims

1. An affinity reagent (102, 102a, 102b) for analyzing a biological sample, comprising: Nucleic acid backbone (200, 300, 402), wherein the nucleic acid backbone (200, 300, 402) is configured to specifically bind to a target analyte (106) through its composite structure (400c), and wherein the nucleic acid backbone (200, 300, 402) comprises at least one reactive moiety pair (202, 204, 302, 304, 308, 310) for cross-linking the nucleic acid backbone (200, 300, 402).

2. The affinity reagent of claim 1, wherein the reactive moieties (202, 204, 302, 304, 308, 310) of the reactive moiety pair are configured to react with each other.

3. The affinity reagent of any of the preceding claims, wherein the reactive moieties (202, 204, 302, 304, 308, 310) of the reactive moiety pair are configured to react under catalytic conditions (408).

4. An affinity reagent according to any of the preceding claims, further comprising a connecting element (306), wherein the reaction parts (202, 204, 302, 304, 308, 310) in the reaction part pair are configured to react with the corresponding reaction parts (202, 204, 302, 304, 308, 310) of the connecting element (306).

5. The affinity reagent according to any one of the preceding claims, wherein the reactive moieties (202, 204, 302, 304, 308, 310) of the reactive moiety pair are modified nucleotides.

6. The affinity reagent of any one of the preceding claims, wherein the reactive moiety (202, 204, 302, 304, 308, 310) of the reactive moiety pair comprises a click chemistry group.

7. The affinity reagent of any one of the preceding claims, wherein the reactive moieties (202, 204, 302, 304, 308, 310) of the reactive moiety pair are photoreactive.

8. The affinity reagent according to any one of the preceding claims, wherein the nucleic acid backbone (200, 300, 402) comprises 10 to 100 nucleotides.

9. The affinity reagent according to any one of the preceding claims, wherein the nucleic acid backbone (200, 300, 402) is an aptamer.

10. The affinity reagent of any one of the preceding claims, wherein the reactive moieties (202, 204, 302, 304, 308, 310) of the reactive moiety pairs are separated from each other along the nucleic acid backbone (200, 300, 402).

11. An affinity reagent according to any one of the preceding claims, wherein the reactive moieties (202, 204, 302, 304, 308, 310) of the reactive moiety pairs are in proximity when the affinity reagent has its composite structure (400c).

12. An affinity reagent according to any one of the preceding claims, comprising a second pair of reactive moieties, wherein the reactive moieties in the second pair are different from the reactive moieties in the at least one reactive moiety pair.

13. A marker (100, 100a, 100b, 400) for analyzing a biological sample, comprising: An affinity reagent (102, 102a, 102b) according to any one of the preceding claims, and A label comprising at least one labeling moiety (104, 404).

14. A method for analyzing a biological sample, comprising the steps of: introducing at least one marker (100, 100a, 100b, 400) according to claim 13 into the biological sample, and A readout of the biological sample is generated using the markers (100, 100a, 100b, 400).

15. The method according to claim 14, wherein said affinity reagent for said marker is generated by cross-linking of reactive moiety pairs of said marker (100, 100a, 100b, 400) prior to introduction of said at least one marker.

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