METHOD FOR IDENTIFYING HIGHLY ACCENT COMPLEXES CONTAINING TWO LIGANDS AND A RECEPTOR, DEVICE FOR PERFORMING THE METHOD AND SELF-ASSEMBLING CHEMICAL LIBRARY FOR USE IN THE METHOD

Description

[0001] A method for identifying high-affinity complexes of two ligands and a receptor is proposed. The method uses ligands from a library, each containing a single-stranded DNA or RNA. Since the DNA or RNA of one part of the ligands is complementary to the DNA or RNA of another part of the ligands, both parts of the ligands can form binary complexes with each other. In one embodiment of the invention, the length of the DNA or RNA can be 3 to 10 bases. According to the invention, hybridization and dissociation of the single-stranded DNAs or RNAs is provided in a dynamic manner, which is achieved even at room temperature for the embodiment of the invention, but requires a cycle of heating and cooling for a non-inventive embodiment.Furthermore, a microfluidic device for carrying out the method according to the invention and a dynamic, self-assembling chemical library containing binary complexes of the respective ligands are proposed.

[0002] A major challenge in using DNA-encoded chemical libraries and DNA / RNA aptamer libraries to discover effective binders and inhibitors against diverse target proteins of biomedical interest is improving the signal-to-noise ratio in selection experiments to distinguish molecules with high binding affinity from weak or non-binding compounds.

[0003] To date, the state of the art has mostly involved parallel selection experiments with different selection stringencies to optimize the selection conditions for a known target protein. For example, solid supports were loaded with different amounts of the target protein. Furthermore, various washing procedures were established, using buffers with different ionic strengths and detergents.

[0004] To conduct parallel selection experiments, many additional experiments must be performed for a target protein. This is both time-consuming and costly. Furthermore, it is very difficult to design parallel selection experiments with different stringencies when the binding affinity distributions of the entire library for the target protein are unknown. To select high-affinity binders, specific selection conditions must be applied. Therefore, it is a disadvantage that a stringent, parallel selection experiment can only be designed after it is known that high-affinity binding molecules are present in the library.

[0005] On the other hand, characterizing components of a first-time selection experiment (de novo selection experiment) is also very time-consuming and expensive. It is also difficult to design a parallel selection experiment without the knowledge and information resulting from a previous primary selection.

[0006] US 2010 / 159455 A1 discloses a method for the biological detection of receptors and proteins and the creation of profiles of receptors and proteins.

[0007] WO 97 / 43232 A1 discloses a substance library, a method for producing the substance library, a method for producing supramolecular complexes using the substance library, a use of the produced supramolecular complexes and a use of the substance library.

[0008] The object of the present invention is therefore to provide a method that enables the identification of high-affinity ternary complexes of two ligands and one receptor in a simple, economical, and highly sensitive manner. Furthermore, a device for carrying out the method and a self-assembling chemical library for use in the method should be provided.

[0009] The object is achieved by the method according to claim 1, the dynamic, self-assembling chemical library according to claim 10 and the use of the library according to claim 15.

[0010] A method for the sensitive identification of high-affinity complexes of two ligands and a receptor is provided, which comprises the following steps: a) Interaction of a plurality of different complexes of ligands from a chemical library with at least one receptor in a solution, wherein the ligands of the library comprise single-stranded DNA or RNA with a base length of more than 10 bases, which is chemically covalently bound to the ligands, and wherein more than 10 bases of the single-stranded DNA or RNA from a first part of the ligands are complementary to bases of the single-stranded DNA or RNA from a second part of the ligands, and wherein the ligands are complexed to form ligand complexes via hybridization of the DNA or RNA; b) Incubation of the solution for a certain period of time, whereby complexes of ligand complexes and the receptor are formed; c) Dissociation of the ligand complexes to form free ligands; d) Re-hybridization of the free ligands to form further ligand complexes;e) Incubating the solution for a certain period of time, whereby further complexes are formed between the further ligand complexes and the receptor; f) identifying the resulting complexes between the ligand complexes and the receptor.

[0011] The method not according to the invention is characterized in that incubation is carried out (step b), step d) and / or step e)) at a temperature at which the first part of the ligands and the second part of the ligands in the solution are hybridized to form ligand complexes.

[0012] In the presence of a receptor that exhibits high affinity for a specific combination of two ligands (a specific binary ligand complex of ligand 1 and ligand 2 via dsDNA or dsRNA), a high-affinity ternary ligand-receptor complex forms between the two ligands and the receptor. High-affinity ligand pairs are thus depleted from the pool of binary ligand pairs and enriched in the form of a ternary ligand-receptor complex.

[0013] The disadvantage of prior art methods is that a certain percentage of high-affinity ligands hybridize with low-affinity or no-affinity ligands and are thus "trapped" in low-affinity ligand complexes. This problem is solved by the method not according to the invention. Through a process step in which ligand pairs are specifically dissociated, the high-affinity ligands previously "trapped" in the ligand complex are "freed" again to hybridize with other free high-affinity ligands to form new ligand complexes in a further hybridization step of the process. The newly formed ligand complexes are, in turn, available for the formation of ligand-receptor complexes.As a result, the high-affinity ligands can be specifically converted – ideally quantitatively – into ligand-receptor complexes, resulting in a larger quantity of ligand-receptor complexes and thus increasing the sensitivity of the method not according to the invention compared to prior art methods (improved signal-to-noise ratio). Indeed, with a number of N theoretically possible high-affinity ligand pairs in a library, the frequency of formation of the high-affinity ligand pair can ideally be increased by a factor of N (quantitative formation of high-affinity complexes, no high-affinity ligands bound to low-affinity ligands and thus "trapped").

[0014] In the process not according to the invention, the ligand complexes can be dissociated, for example, by increasing the temperature of the solution, in particular to a temperature of 35 °C to 95 °C, preferably 50 °C to 95 °C, particularly preferably 70 °C to 95 °C, in particular 80 °C to 95 °C.

[0015] In the non-inventive method, the dissociation of the ligand complexes is preferably carried out in a portion of the solution that is spatially separated from the receptor, preferably in such a way that the receptor is not functionally impaired by the conditions leading to dissociation. The advantage of this embodiment of the non-inventive method is that causing the dissociation does not have a negative effect on the (biological) function of the receptor. In particular, this protects receptors from thermal and / or pH-induced denaturation (e.g., receptors that contain or consist of proteins).

[0016] In the process not according to the invention, the free ligands can be (re)hybridized by lowering the temperature of the solution, in particular by lowering it to a temperature of 1 °C to 30 °C, preferably 5 °C to 28 °C, particularly preferably 10 °C to 25 °C, in particular 15 to 20 °C.

[0017] Steps c) to e) of the non-inventive method can be repeated at least once or twice, preferably at least 3 or 4 times, particularly preferably at least 5 or 6 times, especially at least 7 or 8 times. The more often the steps are repeated, the more high-affinity ligand complexes are gradually converted into the ligand-receptor complex (increase in sensitivity).

[0018] In a preferred embodiment of the method not according to the invention, at least 20 bases, preferably at least 40 bases, particularly preferably at least 100 bases, in particular at least 200 bases, of the single-stranded DNA or RNA of a first part of the ligands are complementary to the single-stranded DNA or RNA of a second part of the ligands.

[0019] Furthermore, an inventive solution to the inventive problem is provided which does not require heating and cooling steps.

[0020] In this regard, the invention provides a method for the sensitive identification of high-affinity complexes of two ligands and a receptor, comprising the following steps: i) interacting a plurality of different complexes of ligands from a chemical library with at least one receptor in a solution, wherein the ligands of the library comprise single-stranded DNA or RNA with a base length of more than 2 bases, which is chemically covalently bound to the ligands and wherein only 2 to 10 bases of the single-stranded DNA or RNA from a first portion of the ligands are complementary to bases of the single-stranded DNA or RNA from a second portion of the ligands; ii) incubating the solution for a certain period of time, whereby complexes of ligand complexes and the receptor are formed; and iii) identifying the resulting complexes of ligand complexes and the receptor.

[0021] The method is characterized in that the solution is incubated at a room temperature at which an equilibrium of hybridization to form ligand complexes and dissociation to form free ligands is established between the first part of the ligands and the second part of the ligands (room temperature, i.e., 15°C to 30°C). This results in dynamic hybridization and dissociation. Furthermore, the method is characterized in that in step i), the ligands are complexed to form ligand complexes via hybridization of the DNA or RNA, wherein the single-stranded DNA or RNA in the first part of the ligands and the single-stranded DNA or RNA in the second part of the ligands contain a base sequence that encodes the chemically covalently bound ligand.

[0022] The difference from the first-mentioned, non-inventive method is that, due to the smaller number of complementary bases between the two parts of the ligands, association (via hybridization) and dissociation of the ligands already occurs at room temperature (dynamic equilibrium). In the presence of a receptor, high-affinity ligand pairs are "removed" from the dynamic equilibrium and transferred into the ligand-receptor complex. High-affinity ligand pairs are thus depleted from the general ligand pool of the chemical library, and the high-affinity, ternary ligand-receptor complex is enriched. Dynamic equilibrium therefore allows all high-affinity ligand pairs to be transferred (ideally quantitatively) into the receptor complex even without heating and cooling, which increases the concentration of ligand-receptor complexes and thus improves the sensitivity of the detection method.The disadvantage, however, is an overall somewhat lower affinity of the ligand pairs to the receptor compared to the ligand pairs from the first-mentioned, non-inventive method, since the stability of the ligand pairs is somewhat lower due to the smaller number of complementary bases and thus the ternary ligand-receptor complex is also less populated in equilibrium.

[0023] In this variant of the invention, only 3 to 9 bases, preferably only 4 to 8 bases, particularly preferably only 5 to 7 bases, of the single-stranded DNA or RNA of a first part of the ligands may be complementary to the single-stranded DNA or RNA of a second part of the ligands.

[0024] In both methods, the length of the DNA or RNA of the first part of the ligands and / or second part of the ligands may be at least 20 bases, preferably at least 40 bases, particularly preferably at least 100 bases, in particular at least 200 bases.

[0025] The first and / or second portion of the ligands may comprise more than 20%, preferably more than 30%, particularly preferably more than 40%, and especially 50%, of the total amount of ligands. The first portion of the ligands preferably comprises L different ligands, and the second portion of the ligands preferably comprises M different ligands, so that L M different binary ligand complexes are formed. For a library of L M binary ligand complexes, a particular high-affinity ligand complex can be enriched with an increased efficiency of at least 1.5 to N-fold, preferably 2 to N (1 / 2)-fold, especially 3 to 10-fold.

[0026] In a preferred embodiment, the solution in at least one of steps b), e), and ii) is incubated at a temperature of 1°C to 50°C, preferably 5°C to 37°C, particularly preferably 10°C to 25°C, especially 15 to 20°C. These temperature ranges are advantageous because many receptor types (especially protein receptors) are stable and have not lost their ability to bind ligands.

[0027] The solution can be incubated in at least one of steps b), e), and ii) for a period of 0.1 to 48 hours, preferably 0.2 to 24 hours, particularly preferably 0.5 to 12 hours, especially 1 to 6 hours. Longer incubation times have proven advantageous because the binding of the ligand pairs to the receptor may be thermodynamically favored but may still exhibit slow kinetics.

[0028] The receptor used in the method according to the invention can be immobilized, preferably on a substrate selected from the group consisting of glass, ceramic, biopolymer, Sepharose, synthetic polymer, and hydrogel. In particular, the solution containing ligands can be circulated past the immobilized receptor. This embodiment is advantageous if, in the method, the dissociation of the ligand complexes is carried out in a part of the solution that is spatially separated from the receptor, since in this case, dissociation and hybridization of the ligands can occur spatially separately but nevertheless simultaneously (e.g., dissociation far from the receptor, hybridization near the receptor).

[0029] The receptor preferably contains or consists of a protein, a DNA, an RNA, a cell and / or an organic molecule with a molecular mass < 200 kDa, preferably ≤ 100 kDa, particularly preferably ≤ 10 kDa, in particular ≤ 3 kDa.

[0030] The ligands used can contain or consist of a molecule selected from the group consisting of protein, peptide, lipid, carbohydrate, dsDNA, ssDNA, dsRNA and ssRNA, aptamer, organic molecule with a molecular mass ≤ 200 kDa, preferably <_ 100 kDa, particularly preferably <_ 10 kDa, in particular < 3 kDa.

[0031] In a preferred embodiment, the complexes of ligand complexes and the receptor are identified using an analytical method. The method is preferably selected from the group consisting of mass spectrometry, HPLC, gas chromatography, IR spectroscopy, and DNA sequencing, preferably DNA sequencing of a DNA or RNA barcode.

[0032] According to the invention, the single-stranded DNA or RNA in the first and second part of the ligands contains a base sequence that codes for the chemically covalently bound ligand (base sequence as barcode), wherein this base sequence is preferably hybridized to a complementary base sequence of another single-stranded DNA or RNA. The advantage of the barcode lies in the simple identification of complex-bound ligands via DNA or RNA sequencing. It is particularly advantageous if the base sequence of the ligands contains a complementary single-stranded DNA or RNA hybridized, which can also serve as a barcode. If both ligands of the ligand-receptor complex (ligand 1 and 2) contain a single-stranded DNA or RNA, this can, for example,by adding a ligase, resulting in a single-stranded DNA or RNA that codes for the specific combination of ligand 1 and 2. In this case, the (ligated) barcode codes not only for a single ligand, but for two ligands that together form a high-affinity ligand complex.

[0033] The single-stranded i) DNA may contain adenine, thymine, guanine and / or cytosine; and / or ii) RNA may contain adenine, uracil, guanine, cytosine and / or inosine; contain.

[0034] In an advantageous embodiment, the method is characterized in that the single-stranded DNA or RNA of the ligands of the library is hybridized in regions to a complementary base sequence of another single-stranded DNA or RNA, wherein 2 to 10 bases of the another single-stranded DNA or RNA of a first part of the ligands are complementary to bases of the single-stranded DNA or RNA of a second part of the ligands and bind to the complementary bases during the method (preferably forming a Y-shape, in particular as in Fig. 6 shown), wherein during the process a ligase is added which chemically covalently ligates the further single-stranded DNA or RNA from the first part of the ligands with the further single-stranded DNA or RNA from the second part of the ligands.

[0035] Ligation of the two additional DNAs or RNAs thus only occurs if hybridization of the respective bases occurs, i.e., if the overall complex is sufficiently stable. However, the advantage of this embodiment is that once complex formation has occurred, it is "stored" by the chemically covalent ligation of the two additional DNAs or RNAs. In other words, the ligated additional DNAs or RNAs accumulate during the process if the basis for their ligation was a stable formation of the overall complex. This approach can significantly increase the sensitivity of the process, especially since the accumulated, ligated DNA or RNA can be further amplified using known methods (PCT, RT-PCR). Consequently, even very low ligand concentrations can be used in this way, and high-affinity ligands can be identified even at very low receptor concentrations.

[0036] In the above-mentioned embodiment, in the case of a single-stranded DNA or RNA, a first part of the ligands, i) which has the ligand bound at its 5'-end, which has bases complementary to the bases of the single-stranded DNA or RNA of a second part of the ligands at its 5'-end and the 2 to 10 bases of the further single-stranded DNA or RNA complementary to bases of the single-stranded DNA or RNA of a second part of the ligands are arranged at the 3'-end of the further single-stranded DNA or RNA; or ii) which has the ligand bound at its 3'-end, which has bases complementary to the bases of the single-stranded DNA or RNA of a second part of the ligands at its 3'-end and the 2 to 10 bases of the further single-stranded DNA or RNA complementary to bases of the single-stranded DNA or RNA of a second part of the ligands are located at the 5'-end of the further single-stranded DNA or RNA.

[0037] This specific arrangement of the respective complementary bases promotes the formation of a Y-structure, which in turn increases the efficiency of the ligation of the two other single-stranded DNAs or RNAs by the ligase.

[0038] Furthermore, a microfluidic device for carrying out the method according to the invention is provided. The device comprises: a) a container for holding immobilized receptor, the container having an access with a valve which serves for the injection of ligands from a library and for the isolation of ligand-receptor complexes, and the container having an inlet and an outlet to a fluidic line; b) a fluidic line which is connected to an inlet and an outlet of the container and which has an outlet with a valve at one point; c) a heating device which is arranged in a first region of the fluidic line; and d) a cooling region which is located in a second region of the fluidic line which is different from the first.

[0039] In the microfluidic device, the heating of ligands in solution and the selection of ligands bound to immobilized receptor can take place locally separately, thus preventing thermal damage to the receptor (e.g., protein denaturation). The container can retain immobilized receptor and prevent it from entering the fluidic lines. Preferably, the container thus has filters that prevent immobilized receptor from passing into the fluidic line.

[0040] In a preferred embodiment, the cooling region of the device has a cooling device and / or is arranged in the region of the outlet with a valve.

[0041] The device may comprise a pump arranged to pump a liquid through the fluidic line. In particular, the container of the device may comprise filters that prevent immobilized receptor from passing into the fluidic line.

[0042] The microfluidic device can be characterized in that the container and / or the fluidic line contains a dynamic, self-assembling chemical library according to the invention (see below).

[0043] A chemical library is further provided which contains ligands comprising single-stranded DNA or RNA with a base length of more than 2 bases, which are chemically covalently bound to a ligand, wherein 2 to 10 bases of the single-stranded DNA or RNA of a first portion of the ligands are complementary to bases of the single-stranded DNA or RNA of a second portion of the ligands. The chemical library is characterized in that it contains binary complexes of the respective ligands, wherein the single-stranded DNA or RNA in the first portion of the ligands and the single-stranded DNA or RNA in the second portion of the ligands contain a base sequence encoding the chemically covalently bound ligand.

[0044] In this case, only 3 to 9 bases, preferably only 4 to 8 bases, particularly preferably only 5 to 7 bases, of the single-stranded DNA or RNA of a first part of the ligands can be complementary to the single-stranded DNA or RNA of a second part of the ligands.

[0045] The length of the DNA or RNA of the first part of the ligands and / or second part of the ligands may be at least 20 bases, preferably at least 40 bases, particularly preferably at least 100 bases, in particular at least 200 bases.

[0046] In a preferred embodiment, the first and / or second part of the ligands makes up more than 20%, preferably more than 30%, particularly preferably more than 40%, especially 50%, of the total amount of ligands in the library. The first part of the ligands L preferably comprises L different ligands, and the second part of the ligands preferably comprises L different ligands, so that the library comprises L 2< different ligand complexes.

[0047] The ligands of the library can contain or consist of a molecule selected from the group consisting of protein, peptide, lipid, carbohydrate, dsDNA, ssDNA, dsRNA and ssRNA, aptamer, organic molecule with a molecular mass ≤ 200 kDa, preferably ≤ 100 kDa, particularly preferably ≤ 10 kDa, in particular ≤ 3 kDa.

[0048] The single-stranded i) DNA may contain adenine, thymine, guanine and / or cytosine; and / or ii) RNA may contain adenine, uracil, guanine, cytosine and / or inosine; contain.

[0049] The library can be characterized in that the single-stranded DNA or RNA of the ligands of the library is hybridized in each case in regions to a complementary base sequence of another single-stranded DNA or RNA, wherein 2 to 10 bases of a first further single-stranded DNA or RNA of a first part of the ligands are complementary to bases of the second single-stranded DNA or RNA of a second part of the ligands.

[0050] The single-stranded DNA or RNA from a first part of the ligands, i) which has the ligand at its 5'-end, may have the bases complementary to the bases of the single-stranded DNA or RNA of a second part of the ligands at its 5'-end and the 2 to 10 bases of the further single-stranded DNA or RNA complementary to bases of the second single-stranded DNA or RNA of a second part of the ligands at the 3'-end of the further single-stranded DNA or RNA; or ii) which has the ligand at its 3'-end, may have the bases complementary to the bases of the single-stranded DNA or RNA of a second part of the ligand at its 3'-end and the 2 to 10 bases of the further single-stranded DNA or RNA which are complementary to bases of the single-stranded DNA (9) or RNA of a second part of the ligands are arranged at the 5'-end of the further single-stranded DNA or RNA.

[0051] Finally, the use of the chemical library according to the invention for the selective and sensitive identification of high-affinity ternary ligand-receptor complexes is proposed.

[0052] The subject matter of the invention will be explained in more detail with reference to the following figures, without wishing to restrict it to the specific embodiments shown here.

[0053] Of the following embodiments, the Figure 1 , 3 and 4 not embodiments within the meaning of the invention, but are helpful to understand certain aspects of the invention.

[0054] Figure 1shows a plurality of different ligands 2, 3, 4, 5, 6, 7 of a chemical library and at least one receptor 1. The ligands 2, 3, 4, 5, 6, 7 are each chemically covalently bound to a single-stranded DNA 8, 9, wherein the single-stranded DNA 8, 9 has a base length of more than 10 bases. The single-stranded DNA 8, 9 encodes the respective ligand to which it is bound via its base sequence. In this non-inventive example, 18 bases of the single-stranded DNA 8 from a first part of the ligands 2, 6, 7 are complementary to bases of the single-stranded DNA 9 from a second part of the ligands 3, 4, 5 (see dashed line between the single-stranded DNAs 8, 9). Here, only a specific ligand pair (formed by the ligands of reference particles 2 and 3) binds with high affinity to receptor 1. Other ligand pairs (formed by the ligands of reference particles 4, 5, 6, 7) bind weakly or not at all to receptor 1.The high number of complementary bases between the single-stranded DNAs 8, 9 of two ligands 2, 3, 4, 5, 6, 7 stabilizes the ligand complexes, resulting in high sensitivity when used in a detection method. To achieve dynamic hybridization and dissociation, constant heating (dissociation) and cooling (reassociation or re-hybridization), i.e., an energy supply, is necessary.

[0055] Figure 2 shows a Figure 1 corresponding figure with the difference that in this embodiment only 6 bases of the single-stranded DNA from a first part of the ligands are complementary to bases of the single-stranded DNA or RNA from a second part of the ligands (otherwise the molecules and designations are Figure 1identical). The lower number of complementary bases results in the advantage that the association (hybridization) of two ligands is highly dynamic even at room temperature (15°C to 30°C), i.e., a permanent hybridization and dissociation takes place. The high-affinity binding to receptor present in certain ligand pairs inhibits the dissociation of these ligand pairs, whereby complexes of receptor and high-affinity ligand pairs are populated for a longer time, "accumulate" over time, and ultimately are more strongly populated in equilibrium. The advantage of this embodiment is that the dynamics of association and hybridization take place at room temperature, i.e., in contrast to the non-inventive embodiment of the Figure 1 no thermal energy needs to be supplied. The disadvantage of this embodiment is a lower sensitivity than in the non-inventive embodiment in Figure 1, because the smaller number of base pairs (6 instead of 18) makes the ligand complex and thus the complex of the ligands with the receptor more unstable, which makes it less populated in equilibrium than in the embodiment in Figure 1 .

[0056] Figure 3 illustrates how the high-affinity ligand pairs "accumulate" as a complex with the receptor over time (see first arrow above). In addition to the Figure 1 and Figure 2 The ligand pairs have a complementary single-stranded DNA 10, 11 to the single-stranded DNA covalently bound to the ligand (otherwise the molecules and names are Figure 1identical). This additional single-stranded DNA 10, 11 can be generated, for example, using suitable primers and a PCR reaction. The lower arrow 12 symbolizes a ligation reaction (e.g., by adding a ligase enzyme), which results in the two additional single-stranded DNAs 10, 11 being chemically covalently ligated to one another. The advantage of this is that the ligated DNA fragment codes for a specific ligand pair via its base sequence, which can thus be easily identified via DNA sequencing.

[0057] Figure 4describes a microfluidic device. In a container 14 there is immobilized receptor, which has been filled into the container via an opening 15 with a valve. The container 14 is connected on one side and the other to a line through which a liquid can be passed. The line is located in a zone that can be heated. Heating can be achieved using an IR radiator as the heat source 13. The device further has a cooling zone 17. This zone optionally contains a cooling device. In addition, the line, here in the cooling area 17, has an outlet 16 with a valve from which liquid containing (free) ligands can be withdrawn and fed for analysis. In addition, after a certain incubation time, immobilized receptor loaded with high-affinity ligands can be withdrawn via the opening 15.

[0058] Figure 5shows the result of an experiment demonstrating the effectiveness of the method according to the invention. Iminobiotin was used as the ligand, which was chemically covalently bound to ssDNA (= Im-ssDNA). The Im-ssDNA was equally divided into a first part of Im-ssDNA1 and a second part of Im-ssDNA2, with Im-ssDNA1 and Im-ssDNA2 having a different number of complementary bases depending on the experiment (e.g., 6 complementary bases for "6-mer"). Im-ssDNA1 was chemically covalently coupled to the fluorescent dye Cy5 to enable, on the one hand, the detection and quantification of free Im-ssDNA1 or the free binary complex of Im-ssDNA1 and Im-ssDNA2 without receptor, and, on the other hand, the detection of the ternary Im-ssDNA1·Im-ssDNA2·receptor complex. Immobilized streptavidin was used as receptor and an aqueous buffer with pH 9.2 was used as solution.The figure shows the ratio of the amount of ligand-receptor complex to ligand not bound to the receptor ("bound / unbound" on the y-axis) under competitive conditions, i.e. in the presence of iminobiotin-free ssDNA (ssDNA) as an example of a non-affinity ligand (see "single-armed", "6-mer", "8-mer" and "21-mer" on the x-axis) or non-competitive conditions, i.e. without the presence of competitive ssDNA ("21mer, no comp." on the x-axis).

[0059] Except for the non-competitive experiment ("21-mer, no comp." on the x-axis), the iminobiotin-free ssDNA was present in a 300-fold excess in the solution. In the experiment labeled "single-arm" (also a 6-mer), only Im-ssDNA1 was present, i.e., no Im-ssDNA2, so no binary ligand complexes could form. The "single-arm" experiment thus shows the binding ratio for a monomeric iminobiotin to streptavidin. A direct comparison with the "6-mer" experiment clearly shows that, under the competitive conditions tested, the binding equilibrium for dimeric iminobiotin (= binary ligand complex) is significantly shifted toward the ligand-receptor complex. This effect is even more pronounced due to the higher stabilization through the hybridization of 8 base pairs in the "8-mer" experiment. However, if the number of complementary bases increases further (e.g.,here 21 complementary bases in the "21-mer" experiment), the amount of ligand-receptor complex obtained falls to a value that approximately corresponds to the value of the "one-armed" experiment.

[0060] This observation can be explained by the fact that the Cy5-labeled iminobiotin-bound ssDNA (Im-ssDNA1) is "trapped" in low-affinity binary complexes with iminobiotin-free ssDNA (ssDNA) and can therefore no longer bind to complementary Cy5-free, but iminobiotin-containing ssDNA (Im-ssDNA2). This effect is therefore only observed in the "21mer" experiment, since the formed Im-ssDNA1·ssDNA complex is so stable that practically no further dissociation of this complex occurs at room temperature (without further energy input). With a number of complementary bases of 6 and 8 bases ("6-mer" and "8-mer" respectively), the resulting complex is highly stable.However, this effect does not occur in the 8-mer (or "8-mer"), since the formation of the lm-ssDNA1·ssDNA complex is not static but dynamic. Thus, with this smaller number of complementary bases, an iminobiotin-bound ssDNA (Im-ssDNA1) that is "blocked" by ssDNA is released and can react with another iminobiotin-bound ssDNA (Im-ssDNA2) to form a high-affinity binary complex. Consequently, with a number of base pairs of 6 and 8 at room temperature, an "accumulation" of complexes consisting of receptor (here streptavidin) and high-affinity ligand pairs (here a pair of two iminobiotin molecules) is achieved, making this method superior to a static method in terms of sensitivity. The sensitivity can be increased by using a number of 21 base pairs and alternating heating (dissociation) and cooling (hybridization), ie energy is supplied.

[0061] Figure 6shows a Y-shape that the overall complex assumes in a preferred embodiment of the invention. The first ligand 2, which is chemically covalently bound to the first single-stranded DNA 8, has a section 18 that codes for the first ligand 2. The second ligand 3, which is chemically covalently bound to the second single-stranded DNA 9, has a section 19 that codes for the second ligand 3. Here, six bases of the first single-stranded DNA 8 are complementary to six bases of the second single-stranded DNA 9 and can form base pairs 20 during the process (see the lines between the single-stranded DNAs 8, 9). Furthermore, in this embodiment, the first single-stranded DNA 8 is partially hybridized (here: at the 3' end) with a first additional single-stranded DNA 10.The first additional single-stranded DNA 10 is characterized by containing an "overhang" of two to twelve bases (here: five bases, located at its 3'-end) that are complementary to two to twelve bases of the second single-stranded DNA 9 (here: five bases) and that can form base pairs 20 with the complementary bases during the process (see the lines between the additional first single-stranded DNA 10 and the second single-stranded DNA 9).

[0062] If the second ligand 3 is partially hybridized via the second single-stranded DNA 9 with a suitable second additional DNA 11, the formation of the overall complex (here: in Y-shape) causes the first additional DNA 10 to be brought together with the second additional DNA 11 in such a way that they can be chemically covalently ligated by adding a ligase enzyme. If a ligase enzyme is added to the process, this ensures that ligation products of the two single-stranded additional DNAs 10, 11, which encode high-affinity ligand complexes, accumulate during the process. This increases the sensitivity of their detection. Since the accumulation product is DNA, it can be further amplified (e.g., by PCR), thereby further increasing the sensitivity of the process.In addition, sequencing of the ligated DNA allows rapid identification of the two ligands 2, 3, since the ligated DNA contains segments 18, 19, which code for both ligands 2, 3.

Claims

1. A method for sensitive identification of high-affinity complexes made of two ligands (2, 3, 4, 5, 6, 7) and one receptor (1), comprising the steps i) interaction of a large number of different complexes made of ligands (2, 3, 4, 5, 6, 7) of a chemical library with at least one receptor (1) in a solution, the ligands (2, 3, 4, 5, 6, 7) of the library having a single-strand DNA (8, 9) or RNA with a base length of more than 2 bases, which is bonded chemically covalently to the ligands (2, 3, 4, 5, 6, 7), and only 2 to 10 bases of the single-strand DNA (8) or RNA of a first part of the ligands (2, 6, 7) being complementary to bases of the single-strand DNA (9) or RNA of a second part of the ligands (3, 4, 5); ii) incubation of the solution for a specific period of time, complexes made of ligand complexes and the receptor (1) being produced; iii) identification of the resulting complexes made of ligand complexes and the receptor (1), characterized in that the solution is incubated at a room temperature at which, between the first part of the ligands (2, 6, 7) and the second part of the ligands (3, 4, 5), an equilibrium of hybridisation to form ligand complexes and dissociation to form free ligands (2, 3, 4, 5, 6, 7) arises, wherein, in step i), the ligands are complexed to form ligand complexes via hybridisation of the DNA (8, 9) or RNA, wherein the single-strand DNA (8) or RNA in the first part of the ligands (2, 6, 7) and the single-strand DNA (9) or RNA in the second part of the ligands (3, 4, 5) comprises a base sequence which codes for the chemically covalently-bonded ligand (2, 3, 4, 5, 6, 7).

2. The method according to the preceding claim, characterized in that only 3 to 9 bases, preferably only 5 to 9 bases, particularly preferably only 6 to 8 bases, of the single-strand DNA (8) or RNA of a first part of the ligands (2, 6, 7) are complementary to the single-strand DNA (9) or RNA of a second part of the ligands (3, 4, 5).

3. The method according to one of the preceding claims, characterized in that, in step ii), the solution is a) incubated at a temperature of 1°C to 50°C, preferably 5 °C to 37 °C, particularly preferably 10 °C to 25 °C, especially 15 °C to 20 °C; and / or b) incubated for a period of time of 0.1 to 48 hours, preferably 0.2 to 24 hours, particularly preferably 0.5 to 12 hours, especially 1 to 6 hours.

4. The method according to one of the preceding claims, characterized in that the receptor i) is immobilized, preferably on a substrate selected from the group consisting of glass, ceramic, biopolymer, sepharose, synthetic polymer and hydrogel; and / or ii) comprises or consists of a protein, a DNA, an RNA, a cell and / or an organic molecule with a molecular mass ≤ 200 kDa, preferably ≤ 100 kDa, particularly preferably ≤ 10 kDa, especially ≤ 3 kDa.

5. The method according to one of the preceding claims, characterized in that the ligands (2, 3, 4, 5, 6, 7) comprise or consist of a molecule selected from the group consisting of protein, peptide, lipid, carbohydrate, dsDNA, ssDNA, dsRNA and ssRNA, aptamer, organic molecule with a molecular mass ≤ 200 kDa, preferably ≤ 100 kDa, particularly preferably ≤ 10 kDa, especially ≤ 3 kDa.

6. The method according to one of the preceding claims, characterized in that the complexes made of ligand complexes and the receptor (1) are identified via an analytical method selected from the group consisting of mass spectrometry, HPLC, gas chromatography, IR spectroscopy and DNA sequencing, in particular via DNA sequencing of a DNA or RNA barcode.

7. The method according to one of the preceding claims, characterized in that the base sequence which codes for the chemically covalently bound ligand is hybridised at least partially to a complementary base sequence of a further single-stranded DNA or RNA.

8. The method according to one of the preceding claims, characterized in that the single-strand DNA (8, 9) or RNA of the ligands (2, 3, 4, 5, 6, 7) of the library is hybridised in regions to form a complementary base sequence of a further single-strand DNA (10, 11) or RNA, 2 to 10 bases of the further single-strand DNA (10) or RNA of a first part of the ligands (2, 6, 7) being complementary to bases of the single-strand DNA (9) or RNA of a second part of the ligands (3, 4, 5) and binding to the complementary bases during the method, a Y-form being formed, and a ligase being added during the method, which ligates chemically covalently the further single-strand DNA or RNA of the first part of the ligands (2.

6. 7) to the further single-strand DNA (11) or RNA of the second part of the ligands (3, 4, 5).

9. The method according to claim 8, characterized in that, in the case of a single-strand DNA (8) or RNA of a first part of the ligands (2, 6, 7), i) which has the ligand (2, 6, 7) at the 5' end thereof, has the bases which are complementary to the bases of the single-strand DNA (9) or RNA of a second part of the ligands (3, 4, 5), at the 5' end thereof, and the 2 to 10 bases of the further single-strand DNA (10) or RNA, which are complementary to bases of the single-strand DNA (9) or RNA of a second part of the ligands (3, 4, 5), are disposed at the 3' end of the further single-strand DNA (10) or RNA; or ii) which has the ligand (2, 6, 7) at the 3' end thereof, has the bases, which are complementary to the bases of the single-strand DNA (9) or RNA of a second part of the ligands (3, 4, 5), at the 3' end thereof, and the 2 to 10 bases of the further single-strand DNA (10) or RNA, which are complementary to bases of the single-strand DNA (9) or RNA of a second part of the ligands (3, 4, 5), are disposed at the 5' end of the further single-strand DNA (10) or RNA.

10. A chemical library, comprising ligands (2, 3, 4, 5, 6, 7) which have bonded chemically covalently a single-strand DNA (8, 9) or RNA with a base length of more than 2 bases, wherein only 2 to 10 bases of the single-strand DNA (8) or RNA of a first part of the ligands (2, 6, 7) are complementary to bases of the single-strand DNA (9) or RNA of a second part of the ligands (3, 4, 5), characterized in that the chemical library comprises binary complexes of the respective ligands (2, 3, 4, 5, 6, 7), the single-stranded DNA (8) or RNA in the first part of the ligands (2, 6, 7) and the single-stranded DNA (9) or RNA in the second part of the ligands (3, 4, 5) comprising a base sequence which codes for the chemically covalently bound ligand (2, 3, 4, 5, 6, 7)..

11. The library according to claim 10, characterized in that only 3 to 9 bases, preferably only 5 to 9 bases, particularly preferably only 6 to 8 bases, of the single-strand DNA (8) or RNA of a first part of the ligands (2, 6, 7) are complementary to the single-strand DNA (9) or RNA of a second part of the ligands (3, 4, 5).

12. The library according any one of claims 10 or 11, characterized in that the ligands (2, 3, 4, 5, 6, 7) comprise or consist of a molecule selected from the group consisting of protein, peptide, lipid, carbohydrate, dsDNA, ssDNA, dsRNA and ssRNA, aptamer, organic molecule with a molecular mass ≤ 200 kDa, preferably ≤ 100 kDa, particularly preferably ≤ 10 kDa, especially ≤ 3 kDa.

13. The library according to any one of claims 10 to 12, characterized in that the single-strand DNA (8, 9) or RNA of the ligands of the library is hybridised respectively in regions to a complementary base sequence of a further single-strand DNA (10, 11) or RNA, 2 to 10 bases of a first further single-strand DNA (10) or RNA of a first part of the ligands (2, 6, 7) being complementary to bases of the second single-strand DNA (9) or RNA of a second part of the ligands (3, 4, 5).

14. The library according to claim 13, characterized in that the single-strand DNA (8) or RNA of a first part of the ligands (2, 6, 7), i) which has the ligand (2, 6, 7) at the 5' end thereof, has the bases, which are complementary to the bases of the single-strand DNA (9) or RNA of a second part of the ligands (3, 4, 5), at the 5' end thereof, and the 2 to 10 bases of the further single-strand DNA (10) or RNA, which are complementary to bases of the second single-strand DNA (9) or RNA of a second part of the ligands (3, 4, 5), are disposed at the 3' end of the further single-strand DNA (10) or RNA; or ii) which has the ligand (2, 6, 7) at the 3' end thereof, has the bases, which are complementary to the bases of the single-strand DNA (9) or RNA of a second part of the ligands (3, 4, 5), at the 3' end thereof, and the 2 to 10 bases of the further single-strand DNA (10) or RNA, which are complementary to bases of the single-strand DNA (9) or RNA of a second part of the ligands (3, 4, 5), are disposed at the 5' end of the further single-strand DNA (10) or RNA.

15. A use of the library according to any one of claims 10 to 14 for selective and sensitive identification of high-affinity ternary ligand-receptor complexes.