System and method for capturing, separating, and purifying biomolecules

Abstract

System and method for capturing, separating, and purifying biomolecules The invention relates to a system for capturing, separating, and purifying target biomolecules (7) of the type DNA, RNA, and / or protein in a liquid sample, comprising a liquid (1) containing - DNA anchor molecules (3) that are covalently bound to polymer chains (2) and have free DNA binding sites (3.1) - DNA crosslinker libraries containing large numbers of distinct DNA crosslinker molecules (5), which form double-stranded overlap domains (5.1) and reversibly bind to the free DNA binding sites (3.1) to form a three-dimensional polymer network, and - interchangeable catcher molecules (4) that reversibly bind to the free DNA binding site (3.1) and have at least one binding structure (4.1) that binds at least one specific target biomolecule (7) of the type DNA, RNA and / or protein from a liquid sample, wherein the target biomolecules (7) are releasable either by DNA denaturation or a toehold-mediated strand displacement reaction (TMSD).

Description

[0001] System and method for capturing, separating, and purifying biomolecules

[0002] The invention relates to a system and a method for capturing, separating, and purifying biomolecules in a sample containing target biomolecules.

[0003] The capture, separation, and purification of target biomolecules such as nucleic acids or proteins from complex mixtures remains a fundamental process in many applications including biotechnological production and diagnostics. For example, nucleic acids from viral or bacterial pathogens present in patient samples can be captured and sequenced for disease identification, or tumor biomarkers can be isolated from blood samples for personalized cancer treatment. Nucleic acids are routinely purified in molecular biology labs for basic research applications such as genetic engineering. A variety of commercial methods exist for biomolecule separation that can be categorized by their selectivity for the target in terms of the type and sequence of the biomolecule.

[0004] Traditional non-selective methods use chromatographic techniques to separate complex mixtures based on molecular size, charge, or other features over solidphase supports. For nucleic acid isolation, these supports are often made of low- cost inorganic materials such as silica that non-discrim inately bind nucleic acids present in the sample. Broad categories of nucleic acid can be selected for (e.g. large versus small fragments, or DNA versus RNA) by adjusting conditions (e.g. solvent composition or temperature) that affect biomolecule adsorption. However, such methods cannot capture in a sequence-selective or programmable manner, and are often tailored to only one specific biomolecule type with restrictive sample requirements. So-called “sequence-selective” methods can isolate specific biomolecules based on complementary sequence hybridization in the case of nucleic acids, or through structural recognition in the case of proteins. Magnetic microbeads have emerged as an attractive solution for sequence-selective binding through functionalization with affinity ligands. For example, streptavidin-coated beads can capture biomolecules bound by biotinylated probes. These magnetic particles can be readily manipulated by the application of a magnetic field, simplifying the preparative steps required for biomolecule capture. Other popular approaches utilize enzymes to either selectively amplify or degrade nucleic acids based on sequence. Currently, the greatest drawbacks to sequence-selective methods are their high cost and time-consuming sample preparation.

[0005] Over recent decades, a new material known as stimuli-responsive or “smart” polymers has enabled novel applications in the field of bioseparation. Such polymers exhibit significant and nonlinear responses in their structure or properties due to small changes in environmental conditions such as temperature, ionic strength, pH, or electric or magnetic fields. Sequenceselectivity can be achieved through affinity precipitation, in which a ligand, specific for a target, is covalently coupled to the polymer. A complex of the target and polymer-ligand conjugate is formed and phase separation is triggered, precipitating the complex in an insoluble state. The target can be recovered by elution from the precipitate, or through re-dissolving the precipitate, dissociating the target, and reprecipitating the polymer-ligand conjugate, leaving the purified target in the supernatant.

[0006] Recently, a new type of DNA-functionalized smart polymer termed methanolresponsive polymer (MeRPy) was developed that can programmably bind and purify single- and double-stranded DNA targets via affinity precipitation through the incorporation of short DNA oligonucleotide “catcher” sequences on the polymer chain. After binding a sequence of interest in solution, the addition of methanol leads to the reversible precipitation of MeRPy and thus the facile purification of captured targets from complex mixtures. MeRPy exhibits high stability, low-cost, high binding capacity, and rapid capture and release of DNA targets. However, in its current form, MeRPy is unable to specifically capture RNA or proteins, since precipitation in the presence of methanol can lead to undesirable non-specific binding (in the case of RNA) and denaturation (in the case of proteins).

[0007] The object of the present invention is to overcome the known disadvantages and to provide possibilities for the separation of diverse target biomolecules, in particular DNA, RNA, or proteins, from a sample.

[0008] The object is solved by a system with the features according to patent claim 1 and a method with the features according to patent claim 11 . Further extensions of the system and variants of the method are indicated in the respective dependent patent claims.

[0009] The concept of the invention is based on a system which utilizes a high molecular weight linear DNA-functionalized polymer which is grafted with short DNA oligonucleotides (DNA anchor molecules) that impart functionality to the system. These DNA anchor molecules serve as binding sites for oligonucleotide hybridization probes (referred to as “catcher molecules”) that simultaneously bind to a target biomolecule, containing either fully or partially single-stranded DNA and / or RNA, and / or protein in a sequence-selective manner. DNA crosslinker molecules act as the stimulus to induce phase separation of the polymer by binding DNA anchor molecules across separate polymer chains to form amorphous polymer aggregates with the size of several microns. Because target capture and crosslinks are formed through non-covalent and reversible DNA hybridization, the system is modular, dynamic, and programmable.

[0010] According to the present invention, a system for capturing, separating, and purifying target biomolecules of the type DNA, RNA, and / or protein in a liquid sample is provided. The system comprises a liquid containing polymer chains, DNA anchor molecules, DNA crosslinker molecules, and interchangeable catcher molecules. The DNA anchor molecules are covalently bound to the polymer chains and contain a free DNA binding site. The DNA crosslinker molecules each have a single-stranded overlap domain and a single-stranded adapter domain. The overlap domain on one DNA crosslinker molecule binds reversibly to a complementary overlap domain on another DNA crosslinker molecule to form a pair. The adapter domains on each end of the DNA crosslinker molecule pair each bind reversibly to the free DNA binding site on a DNA anchor molecule, crosslinking polymer chains to form a three-dimensional polymer network. The interchangeable catcher molecules bind reversibly to the free DNA binding site on the DNA anchor molecules through a single-stranded adapter domain, and have at least one additional binding structure which binds at least one specific biomolecule of the type DNA, RNA, and / or protein from a liquid sample. The captured target biomolecules can be released either by DNA denaturation (e.g. by heating above the melting temperature or by increasing the pH above 11 ) or through toehold-mediated strand displacement (TMSD).

[0011] For the sake of invention, the DNA anchor molecules, the DNA crosslinker molecules, and the catcher molecules are DNA oligonucleotides, with the DNA crosslinker molecules and the catcher molecules each having a single-stranded adapter domain in order to bind to the free DNA binding site of the DNA anchor molecules. The DNA crosslinker molecules form pairs through the binding of complementary overlap domains, and contain single-stranded adapter domains at both ends that bind the DNA anchor molecules, so that crosslinking between the individual polymer chains is achieved.

[0012] The system is characterized by the fact that phase separation is achieved without changes in solvent composition, temperature, pH, and / or buffer components. In particular, no methanol is used. Methanol is not part of the system and is not used in the application of the system. Instead, the use of DNA crosslinker molecules provides a mechanism for phase separation that does not induce non-specific adsorption or denaturation of target biomolecules. According to an embodiment of the system, the DNA crosslinker molecules are designed to form matching pairs with each other through the complementarity of their overlap domain. Two matching DNA crosslinker molecules bind to each other through their complementary overlap domain, forming a pair, and the adapter domains on each end of the pair bind to two different DNA anchor molecules.

[0013] With regard to the above embodiment of the system, a sufficient compositional complexity in the sequences of the double-stranded overlap domains of the DNA crosslinker molecule pairs is required to properly crosslink between different polymer chains. The system may comprise a DNA crosslinker library containing different DNA crosslinker molecules that can form pairs, wherein the differences between different DNA crosslinker molecule pairs are located in different nucleotide sequences of the double-stranded overlap domain. The overlap domains within the DNA crosslinker library could be synthesized with different explicit predetermined sequences that are optimized for selective pairwise binding. Alternatively, a high compositional complexity can also be achieved through the use of combinatorial crosslinker libraries (CCLs) in which the doublestranded overlap domain of the different DNA crosslinker molecule pairs in a given CCL has a predetermined nucleotide sequence that is diversified through the introduction of ambiguous bases (N) at specific positions, where each N nucleotide can be either of the four canonical bases adenine (A), thymine (T), cytosine (C), or guanine (G). The number of different DNA crosslinker molecule pairs in the CCL equals 4nwhere n is the number of ambiguous N-bases in the overlap domain. Thus, as an example, CCL-64 refers to a combinatorial crosslinker library containing n = 3 ambiguous N-bases and thus 64 different DNA crosslinker molecule pairs.

[0014] According to an embodiment of the system, the overlap domain sequence contains an ambiguous N-base at exactly one, two, or four predetermined sequence positions. According to a most preferred embodiment, the overlap domain sequence contains an ambiguous N-base at exactly three predetermined sequence positions. Except at the ambiguous N-base positions, the doublestranded overlap domains of different DNA crosslinker molecule pairs have identical nucleotide sequences.

[0015] The polymer can be produced from the free radical polymerization of acrylamide and acrylic acid monomers, wherein DNA anchor molecules in form of acrylamide-tagged DNA oligonucleotides are incorporated into the growing chain during polymerization. By polymerizing under nitrogen atmosphere with a small amount of initiator (e.g. ammonium persulfate), high molecular weight linear polymer chains (up to 50 MDa) are produced. It is important to note that the polymer chains and the protocol to synthesize it are identical to the synthesis of MeRPy. Alternatively, other acrylamide and acrylate monomers can be used for the synthesis of the backbone. Other examples include hydroxyethyl methacrylate, N-isopropylacrylamide, N,N-dimethylacrylamide, 2-acrylamido-2- methylpropanesulfonic acid, hydroxyethyl acrylate, and 2-(methacryloyloxy)ethyl dimethyl (3-sulfopropyl) ammonium hydroxide. According to an embodiment, a concentration of the polymer in the liquid is less than 0.2% (w / v), preferably less than or equal to 0.1 % (w / v), most preferably in a range between 0.1 % (w / v) and 0.01 % (w / v).

[0016] It has been shown that particularly suitable polymer aggregates can be formed at the specified concentrations of the polymer in combination with the DNA crosslinker library. The polymer aggregates formed in this way can be advantageously pelleted by centrifugation and thus separated. After separation, further steps can be carried out to purify the separated target biomolecules.

[0017] According to a preferred embodiment, the proportion of DNA crosslinker molecules relative to the proportion of DNA anchor molecules can be in a range of 70% to 90%. Most preferably the proportion of DNA crosslinker molecules relative to the proportion of DNA anchor molecules is exactly 80%. According to a preferred embodiment of the system, the three-dimensional polymer network is present in the liquid in the form of a multiplicity of separate polymer aggregates. The system thus contains a plurality of polymer aggregates loaded with the catcher molecules.

[0018] The polymer chains are crosslinked by hybridization of the DNA crosslinker library to the DNA anchor molecules, which occurs when the sample is treated with a predefined temperature profile cycle. The catcher molecules are bound to the DNA anchor molecules in the same way. To provide a capture solution, a DNA hybridization between the DNA crosslinker library and the DNA anchor molecules, between the complementary overlap domains of the DNA crosslinker molecules within the DNA crosslinker library, and between the catcher molecules and the DNA anchor molecules is carried out, whereby the temperature treatment can be carried out before and / or after contact with the liquid sample containing target biomolecules, depending on which target biomolecules are to be captured from a sample.

[0019] A binding structure of the catcher molecules is provided to bind at least one target biomolecule of the type DNA, RNA, and / or protein. Accordingly, the binding structure of the catcher molecules may comprise DNA, RNA, aptamers, antibodies, protein traps, and / or drug molecules. It is therefore possible that the catcher molecules may have several binding structures for binding different target biomolecules of the type DNA, RNA, and / or protein.

[0020] According to an embodiment of the system, the binding structure of the catcher molecules is connected to a toehold domain to enable the release of a captured target biomolecule via TMSD. This is advantageous because the release does not require any enzymes or harsh chemical reagents.

[0021] The invention also relates to a method for capturing, separating, and purifying target biomolecules of the type DNA, RNA, and / or protein in a liquid sample using the inventive system. According to the method, a capture solution comprising polymer aggregates loaded with catcher molecules is provided, wherein the polymer chains are crosslinked in a liquid by binding a DNA crosslinker library consisting of DNA crosslinker molecules to DNA anchor molecules covalently bound to the polymer chains, to form three-dimensional polymer aggregates. There may be more than 16 but less than 256 different DNA crosslinker molecule pairs within a DNA crosslinker library. The capture solution is combined with a biomolecule-containing liquid sample in a vessel under conditions that allow target biomolecules of the type of DNA, RNA, and / or protein to bind to a binding structure of the catcher molecules. Subsequently, polymer aggregates are separated from the solution, such that the target biomolecules of the type DNA, RNA, and / or protein remain bound to the polymer aggregates.

[0022] The method can be carried out in a one-pot process, whereby the polymer chains with the covalently bound DNA anchor molecules, the DNA crosslinker library, the catcher molecules, and the target biomolecules of the type DNA, RNA, and / or protein are all in a liquid in a vessel.

[0023] The liquid used to provide the capture solution may be a Tris-EDTA buffer. It is important that the liquid as such does not significantly affect the nature of the system’s components, so as not to adversely affect the function of the system. In particular, no substances are used that would contribute to the denaturing of the DNA anchor molecules, the DNA crosslinker library, or the catcher molecules.

[0024] The capture solution can be treated according to a predetermined temperature profile cycle to allow DNA hybridization between the DNA crosslinker library and the DNA anchor molecules, between the complementary overlap domains of the DNA crosslinker molecules within the DNA crosslinker library, and between the catcher molecules and the DNA anchor molecules. According to the first embodiment, the temperature treatment of the capture solution can be carried out together with the target biomolecules using the one-pot process. Thus, the temperature treatment according to the predetermined temperature profile cycle occurs after contact with the liquid sample containing the target biomolecules. To capture temperature-sensitive target biomolecules of the DNA, RNA, and / or protein type, the temperature treatment of the capture solution can be carried out according to the temperature profile cycle before contact with the liquid containing the target biomolecules. According to a further embodiment of the method, the temperature treatment can be carried out according to the temperature profile cycle before and after contact with the target biomolecules of the type DNA, RNA, and / or protein. In this embodiment of the method, a temperature treatment of the vessel, in which only the capture solution is contained, is carried out first. After contact with the target biomolecules of the DNA, RNA, and / or protein type, a further temperature treatment of the vessel, in which the capture solution and the liquid sample with the target biomolecules of the DNA, RNA, and / or protein type are contained, can then take place.

[0025] The separation of the target biomolecules bound to the binding structure of the catcher molecules can be done by decanting the liquid or by removing the liquid by pipetting. The polymer aggregates with the catcher molecules and the bound target biomolecules then remain in the vessel. According to a preferred embodiment of the method, the vessel is centrifuged, whereby the polymer aggregates with the catcher molecules and the bound target biomolecules pellet. Accordingly, the polymer aggregates are centrifuged after being brought into contact with the liquid sample containing target biomolecules and the supernatant formed is decanted or removed by pipetting.

[0026] When preparing the capture solution, care should be taken to ensure that the concentration of the polymer remains below the critical gelation concentration of the polymer of 0.2% (w / v). Furthermore, it has been shown that the formed polymer aggregates can be pelleted through centrifugation better at a low concentration of the polymer, as high concentrations would lead to a very voluminous sediment. It is therefore intended to employ the polymer at a concentration of less than 0.2% (w / v), preferably less than or equal to 0.1 % (w / v), most preferably with a polymer concentration in the range between 0.1 % (w / v) and 0.01 % (w / v).

[0027] According to an embodiment of the method, wherein the binding structure of the catcher molecules has a toehold domain, the bound target biomolecules can be released from the polymer aggregates by TMSD. Alternatively, it is possible to release the bound target biomolecules from the binding structure of the catcher molecules by increasing the temperature or pH.

[0028] This invention describes a method for capturing, separating, and purifying various target biomolecules, such as DNA, RNA, and / or proteins, on a DNA- functionalized stimuli-responsive polymer using a crosslinking mechanism based on DNA hybridization. The advantages of the invention can be summarized as follows:

[0029] - The system and method utilize a gentle, controlled phase separation through crosslinking and aggregation to precipitate the polymer without the need for chemicals, denaturing temperature changes, or other environmental changes. The system thus enables the capturing of target biomolecules under gentle, physiological conditions.

[0030] - The use of combinatorial crosslinker libraries (CCLs) containing different DNA crosslinker molecules enables controlled phase separation of polymer aggregates.

[0031] - The system allows capture of target biomolecules in a programmable, sequence-selective manner by allowing the binding structure of catcher molecules to be predetermined.

[0032] - Interchangeability of catcher molecules allows the system to target different types of target biomolecules without changing the polymer or protocol.

[0033] - Different target biomolecules can be captured in a multiplexed manner with the simultaneous use of different catcher molecules, since a catcher molecule can be designed to have several different binding structures while remaining functionally identical.

[0034] - The capture occurs under physiological conditions, so that chemically sensitive molecules can be captured in a selective manner.

[0035] Further details, features, and advantages of the embodiments of the invention will become apparent from the following description of examples of implementation with reference to the associated figures. The following show:

[0036] Fig. 1 : a schematic representation of an embodiment of the collection system and the method of any stewing process,

[0037] Fig. 2: the polymerization of the polymer with acrylamide, acrylic acid, and acrylamide-labeled DNA oligonucleotides as DNA anchor molecules,

[0038] Fig. 3: the influence of the concentration of the polymer in the capture solution, Fig. 4: a schematic representation of the combinatorial crosslinker libraries (CCL) containing different numbers of ambiguous N-bases and thus different numbers of DNA crosslinker molecule pairs, which are used to crosslink the polymer chains,

[0039] Fig. 5: the principle of programmable capture of biomolecules using catcher molecules with different binding structures,

[0040] Fig. 6: an example of how to release captured target biomolecules through TMSD,

[0041] Fig. 7: the quantification of the specific and non-specific capture efficiency for various target biomolecules using the system according to the invention,

[0042] Fig. 8a: the influence of optimizing the diversity of the combinatorial crosslinker library (CCL) on phase separation of the polymer (confocal microscopy images),

[0043] Fig. 8b: the influence of the combinatorial crosslinker library (CCL) on the centrifugability of the polymer aggregates,

[0044] Fig. 9: the influence of polymer concentration on the centrifugability of the polymer aggregates,

[0045] Fig. 10: the influence of the amount of DNA crosslinker molecules, as a percentage of the total amount of DNA anchor molecules, on capture efficiency,

[0046] Fig. 11 : an example of an application of the system, wherein a fluorescent singlestranded DNA (ssDNA) oligonucleotide was captured as a target biomolecule,

[0047] Fig. 12: an example of an application of the system, wherein SARS-CoV-2 viral N-gene RNAwas captured as a target biomolecule, and Fig. 13: an example of an application of the system, wherein human thrombin was captured as a target biomolecule.

[0048] Parts of figures 3, 5, and 6 were created with Biorender.com.

[0049] Figure 1 shows a schematic representation of an embodiment of the collection system and the method of any stewing process. The system comprises a liquid 1 containing polymer chains 2, to which DNA anchor molecules 3 in the form of DNA oligonucleotides are covalently bound, catcher molecules 4, DNA crosslinker molecules 5, and target biomolecules 7, which can be DNA, RNA, and / or proteins. The reference sign A shows an enlarged section of a polymer chain 2 with the covalently bound DNA anchor molecule 3. The DNA anchor molecule 3 has a free binding site 3.1 , to which a catcher molecule 4 is bound in the shown enlarged section A. The catcher molecule 4 has a binding structure 4.1 in the form of a DNA oligonucleotide to which a target biomolecule 7 is bound. Reference B shows an enlarged section of two polymer chains 2 with covalently bound DNA anchor molecules 3. DNA crosslinker molecules 5 are bound in a pair through a complementary overlap domain 5.1 and are bound to the free binding sites 3.1 of the DNA anchor molecules 3, so that the two polymer chains 2 are crosslinked. Due to the crosslinking, a polymer aggregate 6 is formed, which is shown in the rectangular enlargement section C. Several of these polymer aggregates 6 are present in the liquid 1 in the vessel 8.

[0050] Since the mode of action of the system is DNA hybridization, capture and crosslinking occur only in the presence of a sufficient concentration of ions to screen the repulsive interactions between the DNA strands. In particular, for the present example all components are dissolved in an aqueous solution of 10 mM Tris, pH 8.0, 1 mM EDTA, and 150 mM NaCI. When proteins are specified for capture, a final concentration of 0.1 % (w / v) bovine serum albumin is added to prevent non-specific binding of the protein to plasticware.

[0051] The procedure can be carried out as follows by way of example. For the binding of nucleic acids, all components 1 , 2, 3, 4, 5 of the system are mixed simultaneously with the target biomolecules 7, for example target nucleic acids, and bound together to the target nucleic acids by the slow reduction of the temperature to form target-bound polymer aggregates 6. For capturing proteins as target biomolecule 7 or other temperature-sensitive molecules, polymer chains 2 are pre-loaded with target-specific catcher molecules 4 and precrosslinked with DNA crosslinker molecules 5 into polymer aggregates 6. The target biomolecules 7 are then captured at the preferred temperature in homogeneous solution on the polymer aggregates 6. The crosslinked polymer aggregates 6 remain highly permeable, enabling fast binding kinetics and high binding capacities. After binding of the target biomolecule 7, the mixture is centrifuged at 17,000 xg for 30 minutes to pellet the polymer aggregates 6 at the bottom of the vessel 8 and trap the target biomolecule 7 in the resulting pellet 9. The centrifugation step is indicated by the arrow in Figure 1. Any non-target molecules and / or contaminants remaining in the supernatant can be removed by decanting or by removal of the supernatant by pipetting. This procedure would increase the concentration of the target biomolecule 7, but the method is also used to remove interfering molecules from the supernatant, e.g. high-abundance transcripts such as ribosomal RNAwhen preparing RNA-seq libraries.

[0052] The following example illustrates the process steps and parameters using an implementation example.

[0053] Step 1 : Mix the following components to the specified concentrations: Step 2: Thermocycle with the following conditions:

[0054] The target biomolecule 7 can be added before step 2 if capturing nucleic acids, or after step 2 if capturing temperature-sensitive molecules. If capturing after step 2, add target biomolecule and bind for 30 minutes with light shaking at preferred temperature.

[0055] Step 3: Centrifuge for 30 minutes at 17,000 xg to pellet the target-bound polymer aggregates 6.

[0056] Step 4 (optional): The target biomolecule 7 can be released from the polymer aggregates 6 through addition of a sequence-specific release strand 10 at 1.5x molar excess over the catcher molecules 4. Incubate for at least two hours at the preferred temperature with light shaking, followed by centrifugation for 30 minutes at 17,000 xg to pellet the polymer aggregates 6. The purified target biomolecule 7 can be recovered from the supernatant.

[0057] Figure 2 shows the polymerization of the polymer with acrylamide, acrylic acid, and acrylamide-tagged DNA oligonucleotides as DNA anchor molecules. Free radical polymerization generates linear high molecular weight polymer chains 2. The acrylamide-tagged DNA oligonucleotides (DNA anchor molecules 3) are incorporated into the growing chain during polymerization. By polymerizing under nitrogen atmosphere with a small amount of initiator (e.g. ammonium persulfate), high molecular weight linear polymer chains 2 (up to 50 MDa) are produced. The polymerization process is sufficiently well known and will therefore not be covered in any more detail. Figure 3 schematically illustrates the influence of the concentration of the polymer in the capture solution. The experimental work has shown that after reaching the critical gelation concentration of the polymer of ~0.2% (w / v), the polymer forms an extended polymer network that cannot be easily compressed into a compact pellet 9 by centrifugation. For this reason, the method according to the invention uses a much lower polymer concentration, for example 0.05% (w / v), to produce micrometer-sized crosslinked polymer aggregates 6 that remain separate but are still dense enough to pellet and efficiently capture the target biomolecules (see Figure 9).

[0058] Figure 4 shows a schematic representation of the combinatorial crosslinker libraries (CCL) containing DNA crosslinker molecules 5 with different numbers of ambiguous N-bases in their overlap domain, and thus each CCL contains different numbers of DNA crosslinker molecule pairs, which are used to crosslink the polymer chains. Different DNA crosslinker molecules 5 in a CCL are used to increase the intermolecular crosslinks between polymer chains. CCLs consist of many different DNA crosslinker molecules 5 that contain an identical adapter domain and a unique overlap domain 5.1 that will only bind another DNA crosslinker molecule 5 with a complementary overlap domain 5.1 to form a pair. Ambiguous N-bases, which can be either adenine (A), thymine (T), guanine (G), or cytosine (C), are added to the predetermined sequence of the overlap domain 5.1 to increase the diversity of the overlap domain 5.1 of the DNA crosslinker molecules 5. Increasing diversity in the crosslinker libraries decreases the likelihood that a DNA crosslinker molecule 5 will find its match on the same polymer chain, favoring intermolecular crosslinking between polymer chains over intramolecular crosslinking, thereby ensuring proper network formation. Since each ambiguous N-base can be one of four possible bases, the complexity of the final CCL is 4n, where n is the number of ambiguous N-bases in the overlap sequence. This method also uses CCLs to ensure proper network formation and the formation of polymer aggregates. The number of different crosslinker pairs has been shown to be critical for proper particle formation (see Figure 8). Only when the CCL-64 library of 64 different DNA crosslinker molecules 5 is used for crosslinking can polymer aggregates be formed that can be centrifuged for efficient target capture.

[0059] The concentration of the DNA crosslinker library is also important for proper formation of the polymer aggregates and target biomolecule capture. If the DNA crosslinker library concentration is too low, the polymer chains will not crosslink as efficiently. If the DNA crosslinker library concentration is too high, there are not enough DNA anchor molecules left to bind with the catcher molecule, so target biomolecule capture is reduced. This range depends on the number of available DNA anchor molecules in a solution of the polymer. It has been shown that the proportion of DNA crosslinker molecules should be between 70% and 90% of the proportion of DNA anchor molecules. For the examples shown, a DNA anchor concentration of 10 pM and a DNA crosslinker library (CCL-64) concentration of 8 pM (80% of total DNA anchor molecules) was used, as this ratio provides the highest capture efficiency (Figure 10). The amount of catcher molecules that can be used, and thus the amount of target biomolecules that can be captured, ultimately depends on the amount of DNA crosslinker molecules used. In the examples, up to 2 pM of the catcher molecules (20% of the total DNA anchor molecules) can be added before the DNA anchor molecules are saturated. To avoid accidental saturation of the DNA anchor molecules due to pipetting errors, up to 1 pM of the catcher molecules (10% of the total DNA anchor molecules) are used. Catcher molecules are always present in excess over the target biomolecule. In the examples, target biomolecules were captured at a target: catcher molecule ratio of at least 1 :3 and at most 1 :13.2. However, in other embodiments, either the catcher molecules or the DNA crosslinker library, or both, are grafted directly onto the polymer backbone (covalently linked), eliminating the need for binding to DNA anchor molecules. In this case, the target biomolecule would bind to the catcher molecule directly on the polymer, so that the catcher molecule, i.e. the binding structure, could not be easily changed and would thus be optimized for specific applications. Figure 5 shows the principle of programmable catcher molecules. A key feature of the system according to the invention is the ability to capture different biomolecules on the same polymer backbone in a sequence-selective and programmable manner. The catcher molecules provide this programmability because the target binding sequence of the catcher molecule’s binding structure can be varied depending on the target and application. Figure 5 shows a schematic illustration of a polymer chain 2 with three DNA anchor molecules 3, to whose free binding site 3.1 a catcher molecule 4 is bound in each case. The catcher molecule 4 at position D is designed to bind a single-stranded DNA as target biomolecule 7. The catcher molecule 4 at position E is designed to bind RNA as a target biomolecule 7. Any partially or fully single-stranded DNA or RNA sequence of interest can be used as a target. Proteins can also be captured if they can be bound by a suitable DNA aptamer or are linked in some way to a partially or fully single-stranded DNA or RNA molecule. Position F shows a catcher molecule 4 in the form of a DNA aptamer that is designed to bind a protein as a target biomolecule 7. The experimental data show highly specific and efficient capture (>80% specific capture, <3% non-specific capture, on average) of all three of these biomolecule types (positions D, E, and F), including singlestranded DNA, SARS-CoV-2 viral RNA, human ribosomal RNA, and human thrombin (Figures 7, 11 -13). It is possible to provide for the simultaneous use of several types of catcher molecules in a capture solution in order to capture different target biomolecules in a multiplexed manner and thereby separate them.

[0060] Figure 6 shows an example of how to release captured target biomolecules through TMSD. The left side shows a polymer chain 2 with a covalently bound DNA anchor molecule 3, to whose free binding site 3.1 a catcher molecule 4 is hybridized. The catcher molecule 4 comprises a toehold domain 4.2 and a binding structure 4.1 in the form of an aptamer, to which the target biomolecule 7, the protein thrombin, is bound. To enable the release of the captured target biomolecule 7, a release strand 10 is added, which binds the catcher molecule 4 at the toehold domain 4.2 and displaces the bound target biomolecule 7 into solution by TMSD. This allows the recovery of targets in a native state without contamination by any of the DNA molecules used in the system. The DNA crosslinker molecules (not shown) can also be dynamically altered to switch the polymer between dense crosslinked polymer aggregates and free linear polymer chains, either by enzymatic degradation, TMSD, or temperature changes. There are also other options for releasing the target biomolecules. For example, the target-bound pellet can be redispersed and heated to release all DNA strands. Blocking strands that bind the binding structure 4.1 of the catcher molecules 4 can then be added to prevent re-hybridization between the target biomolecules and the catcher molecules. The polymer or polymer particles are then treated with the predefined temperature profile cycle and pelleted by centrifugation, removing all contaminating or unwanted DNA crosslinker molecules and catcher molecules, while leaving the purified target biomolecule in the supernatant.

[0061] Figure 7 shows the quantification of the specific and non-specific capture efficiency for various biomolecules using the system according to the invention in a diagram. The target biomolecules are, from left to right, ssDNA oligonucleotide, SARS-CoV-2 N-gene RNA, human ribosomal RNA, and human thrombin. All biomolecules tested exhibit highly specific and efficient binding.

[0062] Figure 8a shows the influence of optimizing the diversity of the crosslinker library on phase separation of the polymer. The complexity of the combinatorial crosslinker library (CCL) affects pelleting and capture efficiency. Figure 8a shows fluorescence micrographs of polymers crosslinked with different crosslinker libraries at 100x magnification. Extended polymer aggregates form only in the CCL-64 state, i.e., with a combinatorial crosslinker library consisting of 64 different DNA crosslinker molecule pairs whose overlap domains contain three ambiguous N-bases.

[0063] Figure 8b shows the influence of the combinatorial crosslinker library (CCL) on the centrifugability of the polymer aggregates (“xlinkers” refers to the DNA crosslinker molecules). Efficient pelleting of the polymer aggregates and thus efficient capture of the target is best observed with the CCL-64 library. Figure 9 shows the influence of polymer concentration on the centrifugability of the polymer aggregates. Shown are images of the binding of a fluorescent singlestranded DNA oligonucleotide as a target biomolecule to the polymer at different concentrations of the polymer. The highest binding efficiency is achieved at a polymer concentration of 0.05% (w / v). At concentrations above the critical gelation concentration of 0.2% (w / v), macro-hydrogels form that cannot be pelleted.

[0064] Figure 10 shows the influence of the amount of DNA crosslinker molecules as a percentage of the total amount of DNA anchor molecules. The binding of a fluorescent single-stranded DNA oligonucleotide to the polymer is shown at different DNA crosslinker molecule concentrations. A DNA crosslinker molecule concentration of 80% shows the highest target capture efficiency.

[0065] Figure 11 shows an example of an application of the system, wherein ssDNAwas captured as a target biomolecule. Figure 11a shows that proper target capture and pelleting is observed only in the presence of both crosslinkers (“xlinkers”) and catcher molecules (“CSL”) for the complete polymer system (“polymer”), indicating high binding specificity and efficient separation as a result of crosslinking. Figure 11 b (right) shows the quantification of the capture efficiency under the conditions in 11 a. Significant capture of the target biomolecule is evidenced by a decrease in normalized fluorescence in the supernatant, and such a decrease in fluorescence is only observed when all components of the system are present.

[0066] Figure 12 shows an example of an application of the system, wherein SARS- CoV-2 viral N-gene RNAwas captured as a target biomolecule. Figure 12a shows RT-qPCR curves of SARS-CoV-2 N-gene RNA amplification in the supernatant after capture on the polymer aggregates. An increased number of cycles for the polymer sample with all components present (“Polymer”) compared to the controls (without catcher molecules labeled as “Polymer-CSL”, or RNA only) indicates the capture and depletion of N-gene RNA from the supernatant. Figure 12b shows the quantification of the concentration of SARS-CoV-2 N-gene RNA in the supernatant after capture. Significant depletion of SARS-CoV-2 N-gene RNA is observed only for the polymer system according to the invention with all components present.

[0067] Figure 13a shows a cleavage assay in which only the thrombin in the supernatant is free to cleave a colorimetric substrate which changes color from clear to dark following cleavage. The darker the color, the more substrate is cleaved and thus the higher the concentration of thrombin. The SDS-PAGE gel below shows the thrombin protein present in the supernatant from the corresponding samples above in the cleavage assay. Capture is observed only in the invention’s polymer system when all components are present (“Polymer”) compared to the controls (catcher molecules are labeled as “CSL”, crosslinkers are labeled as “xlinkers”). After addition of the release strand, the thrombin protein is released back into the supernatant by TMSD. The figure of Figure 13b shows the quantification of the amount of free thrombin in the supernatant from the corresponding samples in Figure 13a.

[0068] Bezugszeichenliste

[0069] 1 Liquid

[0070] 2 Polymer chain I Polyacrylamide chain

[0071] 3 DNA anchor molecule

[0072] 3.1 Free DNA binding site

[0073] 4 Catcher molecule

[0074] 4.1 Binding structure

[0075] 4.2 Toehold domain

[0076] 5 DNA crosslinker molecule

[0077] 5.1 Double-stranded overlap domain

[0078] 6 Polymer aggregate I Polyacrylamide aggregate

[0079] 7 Target biomolecules

[0080] 8 Vessel

[0081] 9 Pellet

[0082] 10 Release strand

Claims

Patent claims1. System for capturing and separating target biomolecules (7) of the type DNA, RNA, and / or protein in a liquid sample, comprising a liquid (1 ) containing- DNA anchor molecules (3) that are covalently bound to polymer chains (2) and have a free DNA binding site (3.1 )- DNA crosslinker molecules (5) that form a double-stranded overlap domain (5.1 ) and reversibly bind to the free DNA binding site (3.1 ) to form a three-dimensional polymer network, and interchangeable catcher molecules (4) which bind reversibly to the free DNA binding site (3.1 ) and have at least one binding structure (4.1 ) which binds at least one specific target biomolecule (7) of the type DNA, RNA, and / or protein from a liquid sample, wherein the target biomolecules (7) are releasable either by DNA denaturation or through toehold-mediated strand displacement (TMSD).

2. System according to claim 1 , wherein the system comprises a DNA crosslinker library containing different DNA crosslinker molecules (5), wherein the differences between different DNA crosslinker molecules (5) are located in different nucleotide sequences of the double-stranded overlap domain (5.1 ).

3. System according to claim 2, characterized in that the overlap domain sequence (5.1 ) contains an ambiguous N-base at exactly one, two or four, preferably at exactly three predetermined sequence positions.

4. System according to one of claims 1 to 3, characterized in that a concentration of the polymer is less than 0.2% (w / v), preferably less than or equal to 0.1 % (w / v), particularly preferably in a range between 0.1 % (w / v) and 0.01 % (w / v).

5. System according to one of claims 1 to 4, characterized in that theproportion of DNA crosslinker molecules (5) relative to the proportion of DNA anchor molecules (3) is 70% to 90%, preferably exactly 80%.

6. System according to one of claims 1 to 5, characterized in that the three- dimensional polymer network is present in the liquid in the form of a multiplicity of separate polymer aggregates (6).

7. System according to one of claims 1 to 6, characterized in that the polymer is synthesized from acrylamide derivatives and / or acrylic acid derivatives.

8. System according to one of claims 1 to 7, characterized in that the binding structure (4.1 ) of the catcher molecules (4) comprises DNA, RNA, aptamers, antibodies, protein traps, and / or drug molecules.

9. System according to one of claims 1 to 8, characterized in that the catcher molecules (4) have several different binding structures (4.1 ) which bind different target biomolecules (7) of the type DNA, RNA, and / or protein.

10. System according to one of claims 1 to 9, characterized in that the binding structure (4.1 ) of the catcher molecules (4) is connected to a toehold domain (4.2).11 . Method for capturing, separating, and purifying target biomolecules (7) of the type DNA, RNA, and / or protein in a liquid sample using a system according to claims 1 to 10, wherein a capture solution comprising polymer aggregates (6) loaded with catcher molecules (4) is provided, wherein polymer chains (2) are crosslinked in a liquid by binding a DNA crosslinker library consisting of DNA crosslinker molecules (5) to DNA anchor molecules (3) covalently bound to the polymer chains (2), to form three-dimensional polymer aggregates (6), wherein the polymer aggregates (6) are loaded with catcher molecules (4), the capture solution is contacted with a target biomolecule-containing liquid sample in a vessel (8) under conditions that allow targetbiomolecules (7) of the type of DNA, RNA, and / or protein to bind to a binding structure (4.1 ) of the catcher molecules (4), and the polymer aggregates (6) are separated such that the target biomolecules (7) of the type DNA, RNA, and / or protein remain bound to the polymer aggregates (6).

12. Method according to claim 11 , characterized in that the capture solution is treated according to a predetermined temperature profile cycle to allow DNA hybridization of the DNA crosslinker molecules (5), the catcher molecules (4), and the DNA anchor molecules (3), wherein the temperature treatment is carried out before and / or after contact with the liquid sample containing target biomolecules (7).

13. Method according to claim 11 or 12, characterized in that the polymer aggregates (6) are centrifuged after being brought into contact with the liquid sample containing target biomolecules (7) and the supernatant formed is decanted or removed by pipetting.

14. Method according to one of claims 11 to 13, characterized in that the bound target biomolecules (7) are released from the polymer aggregates (6) by temperature increase or toehold-mediated strand displacement (TMSD).

15. Method according to one of claims 11 to 14, characterized in that the polymer aggregates (6) are provided with a polymer concentration of less than 0.2% (w / v), preferably less than or equal to 0.1 % (w / v), most preferably with a polymer concentration in a range between 0.1 % (w / v) and 0.01 % (w / v).

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