Systems and methods for capturing, separating, and purifying biomolecules

Abstract

Systems and methods for capturing, separating and purifying biomolecules. The present invention relates to a system for capturing, separating and purifying target biomolecules (7) of DNA, RNA and / or protein types from a liquid sample, the system comprising a liquid (1) containing - a DNA anchoring molecule (3) covalently bound to a polymer chain (2) and having a free DNA binding site (3.1), - a library of DNA cross-linking agents containing a large number of different DNA cross-linking agent molecules (5) forming double-stranded overlapping domains (5.1) and reversibly binding to the free DNA binding site (3.1) to form a three-dimensional polymer network, and - an interchangeable capture molecule (4) reversibly binding to the free DNA binding site (3.1) and having at least one binding structure (4.1) binding at least one specific target biomolecule (7) of DNA, RNA and / or protein types from the liquid sample, wherein the target biomolecule (7) can be released by DNA denaturation or foothold-mediated strand displacement reaction (TMSD).

Description

[0001] This invention relates to a system and method for capturing, separating and purifying biomolecules in a sample containing a target biomolecule.

[0002] Capturing, isolating, and purifying target biomolecules (such as nucleic acids or proteins) from complex mixtures remains a fundamental process in many applications, including biotechnology manufacturing 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 laboratories for basic research applications such as genetic engineering. A variety of commercial methods exist for biomolecule isolation, which can be categorized by their selectivity for targets in terms of biomolecule type and sequence.

[0003] Traditional nonselective methods use chromatographic techniques to separate complex mixtures on a solid support based on molecular size, charge, or other characteristics. For nucleic acid separation, these supports are typically made of low-cost inorganic materials (such as silica) that indiscriminately bind nucleic acids present in the sample. Large classes of nucleic acids (e.g., large fragments versus small fragments, or DNA versus RNA) can be selected by adjusting conditions affecting biomolecule adsorption (e.g., solvent composition or temperature). However, such methods cannot capture in a sequence-selective or programmable manner and are often tailored to only one specific biomolecule type, imposing limitations on the sample.

[0004] So-called "sequence-selective" methods can separate specific biomolecules based on complementary sequence hybridization in the case of nucleic acids, or through structure recognition in the case of proteins. Magnetic microbeads have emerged as an attractive solution for sequence-selective binding via affinity ligand functionalization. For example, streptavidin-coated beads can capture biomolecules bound by biotinylated probes. These magnetic particles can be easily manipulated by applying a magnetic field, simplifying the preparation steps required for biomolecule capture. Other popular methods utilize enzymes to amplify or degrade nucleic acids based on sequence selectivity. Currently, the biggest drawback of sequence-selective methods is their high cost and time-consuming sample preparation.

[0005] In recent decades, a new type of material known as stimulus-responsive or "smart" polymers has enabled novel applications in the field of bioseparation. These polymers exhibit significant and nonlinear responses in their structure or properties to minute changes in environmental conditions such as temperature, ionic strength, pH, or electric or magnetic fields. Sequence selectivity can be achieved through affinity precipitation, in which a target-specific ligand is covalently coupled to the polymer. A complex of the target and the polymer-ligand conjugate is formed, triggering phase separation and causing the complex to precipitate in an insoluble state. The purified target can be recovered by eluting from the precipitate, or by redissolving the precipitate, dissociating the target, and reprecipitating the polymer-ligand conjugate, leaving the purified target in the supernatant.

[0006] Recently, a novel DNA-functionalized smart polymer called methanol-responsive polymer (MeRPy) has been developed, which can programmatically bind and purify single-stranded and double-stranded DNA targets via affinity precipitation by incorporating short DNA oligonucleotides into the polymer chain to "capture" sequences. After binding the sequence of interest in solution, the addition of methanol causes reversible precipitation of MeRPy, thus facilitating the purification of the captured target 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 cannot specifically capture RNA or proteins because 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 purpose of this invention is to overcome known drawbacks and provide the possibility of isolating different target biomolecules (especially DNA, RNA or proteins) from a sample.

[0008] This objective is achieved by a system having the features described in claim 1 and a method having the features described in claim 11. Further extensions of the system and variations of the method are indicated in the corresponding dependent patent claims.

[0009] The concept of this invention is based on a system utilizing high-molecular-weight linear DNA-functionalized polymers grafted with short DNA oligonucleotides (DNA anchoring molecules) that confer functionality to the system. These DNA anchoring molecules act as binding sites for oligonucleotide hybridization probes (referred to as "capture molecules") that simultaneously bind in a sequence-selective manner to target biomolecules containing fully or partially single-stranded DNA and / or RNA, and / or proteins. DNA cross-linking molecules act as stimuli, inducing phase separation of the polymer by binding the DNA anchoring molecules across individual polymer chains to form amorphous polymer aggregates several micrometers in size. Because target capture and cross-linking 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 DNA, RNA, and / or protein types from a liquid sample is provided. The system comprises a liquid containing a polymer chain, a DNA anchoring molecule, a DNA cross-linking agent molecule, and an interchangeable capture molecule. The DNA anchoring molecule is covalently bound to the polymer chain and contains a free DNA binding site. Each DNA cross-linking agent molecule has a single-stranded overlapping domain and a single-stranded connective domain. The overlapping domain on one DNA cross-linking agent molecule reversibly binds to a complementary overlapping domain on another DNA cross-linking agent molecule to form a pair. The connective domain at each end of each DNA cross-linking agent molecule pair reversibly binds to a free DNA binding site on the DNA anchoring molecule, thereby cross-linking the polymer chain to form a three-dimensional polymer network. The interchangeable capture molecule reversibly binds to the free DNA binding site on the DNA anchoring molecule via the single-stranded connective domain and has at least one additional binding structure that binds at least one specific biomolecule of DNA, RNA, and / or protein type from the liquid sample. The captured target biomolecules can be released through DNA denaturation (e.g., by heating to above the melting temperature or by increasing the pH to above 11) or by foothold-mediated strand displacement (TMSD).

[0011] For the purposes of this invention, the DNA anchoring molecule, the DNA cross-linking agent molecule, and the trapping molecule are DNA oligonucleotides, wherein the DNA cross-linking agent molecule and the trapping molecule each have a single-stranded linker domain for binding to the free DNA binding site of the DNA anchoring molecule. The DNA cross-linking agent molecule forms pairs through the binding of complementary overlapping domains and contains single-stranded linker domains at both ends that bind the DNA anchoring molecule, thereby achieving cross-linking between individual polymer chains.

[0012] This system is characterized by the fact that phase separation is achieved without changes in solvent composition, temperature, pH, and / or buffer composition. Specifically, methanol is not used. Methanol is not part of this system and is not used in its application. Instead, the use of DNA cross-linking agents provides a phase separation mechanism that does not induce nonspecific adsorption or denaturation of target biomolecules.

[0013] According to an embodiment of the system, DNA cross-linking agent molecules are designed to form matching pairs with each other through the complementarity of their overlapping domains. Two matched DNA cross-linking agent molecules bind to each other through their complementary overlapping domains to form a pair, and an adaptor domain at each end of the pair binds to two different DNA anchoring molecules.

[0014] The above embodiments of the system require sufficient compositional complexity in the sequences of the double-stranded overlapping domains of the DNA cross-linking agent molecule pairs to properly cross-link between different polymer chains. The system may comprise a DNA cross-linking agent library containing different DNA cross-linking agent molecules that can form pairs, wherein the differences between the different DNA cross-linking agent molecule pairs lie in different nucleotide sequences within the double-stranded overlapping domains. The overlapping domains within the DNA cross-linking agent library can be synthesized using different defined predetermined sequences optimized for selective pairwise binding. Alternatively, high compositional complexity can also be achieved by using a combinatorial cross-linking agent library (CCL), wherein the double-stranded overlapping domains of different DNA cross-linking agent molecule pairs in a given CCL have predetermined nucleotide sequences diversified by introducing an ambiguous base (N) at specific positions, where each N nucleotide can be any of the four typical bases: adenine (A), thymine (T), cytosine (C), or guanine (G). The number of different DNA cross-linking agent molecule pairs in the CCL is equal to 4. n ,in n It refers to the number of undefined N-bases in the overlapping domain. Therefore, as an example, CCL-64 refers to a domain containing... n = A library of cross-linking agents consisting of 3 undefined N-bases and therefore containing 64 different DNA cross-linking agent molecule pairs.

[0015] According to embodiments of the system, the overlapping domain sequence contains indeterminate N-bases at exactly one, two, or four predetermined sequence positions. According to the most preferred embodiment, the overlapping domain sequence contains indeterminate N-bases at exactly three predetermined sequence positions. Except at the indeterminate N-base positions, the double-stranded overlapping domains of different DNA cross-linking agent molecule pairs have the same nucleotide sequence.

[0016] The polymer can be produced by free radical polymerization of acrylamide and acrylic acid monomers, wherein DNA-anchored molecules in the form of acrylamide-tagged DNA oligonucleotides are incorporated into the growing chain during polymerization. High molecular weight linear polymer chains (up to 50 MDa) are produced by polymerization with a small amount of initiator (e.g., ammonium persulfate) under a nitrogen atmosphere. It is important to note that the polymer chains and their synthetic scheme are identical to those of MeRPy. Alternatively, other acrylamide and acrylate monomers can be used to synthesize the backbone. Other examples include hydroxyethyl methacrylate, N-isopropylacrylamide, N,N-dimethylacrylamide, 2-acrylamido-2-methylpropanesulfonic acid, hydroxyethyl acrylate, and 2-(methacryloyloxy)ethyldimethyl(3-sulfopropyl)ammonium hydroxide. According to examples, the concentration of the polymer in the liquid is less than 0.2% (w / v), preferably less than or equal to 0.1% (w / v), and most preferably in the range of 0.1% (w / v) to 0.01% (w / v).

[0017] It has been shown that particularly suitable polymer aggregates can be formed by combining a DNA cross-linking agent library with a specified concentration of polymer. Polymer aggregates formed in this manner can be advantageously separated by centrifugal precipitation and thus. Following separation, further steps can be taken to purify the separated target biomolecules.

[0018] According to a preferred embodiment, the proportion of DNA cross-linking agent molecules relative to the proportion of DNA anchoring molecules can be in the range of 70% to 90%. Most preferably, the proportion of DNA cross-linking agent molecules relative to the proportion of DNA anchoring molecules is exactly 80%.

[0019] According to a preferred embodiment of the system, the three-dimensional polymer network exists in the liquid as multiple individual polymer aggregates. Therefore, the system contains multiple polymer aggregates loaded with trapping molecules.

[0020] The polymer chains are cross-linked via hybridization between the DNA cross-linking agent library and the DNA anchoring molecules, which occurs during sample cycling using a predefined temperature profile. The capture molecules bind to the DNA anchoring molecules in the same manner. To provide a capture solution, DNA hybridization is performed between the DNA cross-linking agent library and the DNA anchoring molecules, between complementary overlapping domains of the DNA cross-linking agent molecules within the DNA cross-linking agent library, and between the capture molecules and the DNA anchoring molecules. This allows for temperature treatment before and / or after contact with the liquid sample containing the target biomolecules, depending on which target biomolecules are to be captured from the sample.

[0021] The binding structure of the capture molecule is provided to bind at least one target biomolecule of DNA, RNA, and / or protein type. Therefore, the binding structure of the capture molecule may include DNA, RNA, aptamers, antibodies, protein traps, and / or drug molecules. It is therefore possible that the capture molecule may have several binding structures for binding different target biomolecules of DNA, RNA, and / or protein type.

[0022] According to embodiments of the system, the binding structure of the captured molecule is attached to the foothold domain, enabling the release of the captured target biomolecule via TMSD. This is advantageous because the release does not require any enzymes or harsh chemical reagents.

[0023] This invention also relates to a method for capturing, separating, and purifying target biomolecules of DNA, RNA, and / or protein types from liquid samples using the system of this invention. According to this method, a capture solution is provided comprising polymer aggregates loaded with capture molecules, wherein the polymer chains are cross-linked in the liquid by binding a DNA cross-linking agent library composed of DNA cross-linking agent molecules to DNA anchoring molecules covalently bound to the polymer chains to form three-dimensional polymer aggregates. The DNA cross-linking agent library may contain more than 16 but fewer than 256 different pairs of DNA cross-linking agent molecules. The capture solution is combined with a liquid sample containing biomolecules in a container, under conditions allowing the target biomolecules of DNA, RNA, and / or protein types to bind to the binding structure of the capture molecules. Subsequently, the polymer aggregates are separated from the solution, such that the target biomolecules of DNA, RNA, and / or protein types remain bound to the polymer aggregates.

[0024] This method can be carried out in a one-pot process, where polymer chains of covalently bound DNA anchoring molecules, a DNA cross-linking agent library, capture molecules, and target biomolecules of DNA, RNA, and / or protein types are all in a liquid within a container.

[0025] The liquid used to provide the capture solution can be a Tris-EDTA buffer. Importantly, the liquid itself should not significantly affect the properties of the system components so as not to adversely affect the system's function. In particular, substances that would contribute to the denaturation of DNA anchoring molecules, DNA cross-linking agent libraries, or capture molecules should not be used.

[0026] The capture solution can be cyclically processed according to a predetermined temperature profile to allow DNA hybridization between the DNA cross-linking agent library and the DNA anchoring molecule, between complementary overlapping domains of the DNA cross-linking agent molecules within the DNA cross-linking agent library, and between the capture molecule and the DNA anchoring molecule. According to a first embodiment, a one-pot method can be used to temperature-treat the capture solution together with the target biomolecule. Therefore, the cyclic temperature treatment according to the predetermined temperature profile occurs after contact with a liquid sample containing the target biomolecule. To capture temperature-sensitive target biomolecules of DNA, RNA, and / or protein types, the temperature treatment of the capture solution can be cyclically performed according to the temperature profile before contact with the liquid containing the target biomolecule. According to another embodiment of the method, the temperature treatment can be cyclically performed according to the temperature profile before and after contact with target biomolecules of DNA, RNA, and / or protein types. In this embodiment of the method, the container containing only the capture solution is first temperature-treated. After contact with target biomolecules of DNA, RNA, and / or protein types, further temperature treatment of the container, wherein the container contains the capture solution and a liquid sample having target biomolecules of DNA, RNA, and / or protein types, can be performed.

[0027] Separation of the target biomolecule bound to the binding structure of the trapping molecule can be accomplished by decanting the liquid or by pipetting to remove the liquid. The polymer aggregates with the trapping molecule, and then the bound target biomolecule remains in the container. According to a preferred embodiment of the method, the container is centrifuged, thereby precipitating the polymer aggregates containing the trapping molecule and the bound target biomolecule. Thus, the polymer aggregates are centrifuged after contact with a liquid sample containing the target biomolecule, and the resulting supernatant is removed by decanting or pipetting.

[0028] When preparing the capture solution, care should be taken to ensure that the polymer concentration is kept below the critical gelation concentration of 0.2% (w / v). Furthermore, it has been shown that at low polymer concentrations, the formed polymer aggregates can be better precipitated by centrifugation, as high concentrations will result in voluminous precipitates. Therefore, it is desirable to use polymers with concentrations less than 0.2% (w / v), preferably less than or equal to 0.1% (w / v), and most preferably in the range of 0.1% (w / v) to 0.01% (w / v).

[0029] According to embodiments of the method, the binding structure of the trapping molecule has a foothold domain, and the bound target biomolecule can be released from the polymer aggregate via TMSD. Alternatively, the bound target biomolecule can be released from the binding structure of the trapping molecule by increasing the temperature or pH.

[0030] This invention describes a method for capturing, separating, and purifying various target biomolecules (such as DNA, RNA, and / or proteins) on DNA-functionalized stimuli-responsive polymers using a DNA hybridization-based crosslinking mechanism. The advantages of this invention can be summarized as follows: This system and method precipitate polymers through mild, controlled phase separation via crosslinking and aggregation, without requiring chemicals, denaturation temperature changes, or other environmental alterations. Therefore, this system can capture target biomolecules under mild physiological conditions.

[0031] - Controlled phase separation of polymer aggregates can be achieved using a combined crosslinking agent library (CCL) containing different DNA crosslinking agent molecules.

[0032] - This system allows for the capture of target biomolecules in a programmable, sequence-selective manner by allowing predetermined binding structures for the captured molecules.

[0033] - The interchangeability of captured molecules allows the system to target different types of biomolecules without changing the polymer or scheme.

[0034] - Because capture molecules can be designed to have several different binding structures while maintaining the same function, different target biomolecules can be captured in multiple ways by using different capture molecules simultaneously.

[0035] -Capture occurs under physiological conditions, allowing chemically sensitive molecules to be captured selectively.

[0036] Further details, features, and advantages of embodiments of the present invention will become apparent from the following description of examples of implementations with reference to the accompanying drawings. The following shows: Figure 1 : A schematic diagram of an embodiment of a collection system and method for any stewing process. Figure 2 Polymerization with acrylamide, acrylic acid, and acrylamide-tagged DNA oligonucleotides as DNA anchoring molecules. Figure 3 The effect of polymer concentration in the capture solution. Figure 4 : A schematic diagram of a combined crosslinking agent library (CCL), which contains varying numbers of indeterminate N-bases and therefore varying numbers of DNA crosslinking agent molecule pairs, used to crosslink polymer chains. Figure 5 The principle of programmably capturing biomolecules using capture molecules with different binding structures. Figure 6 Examples of how to release captured target biomolecules via TMSD Figure 7 Quantification of specific and non-specific capture efficiency of various target biomolecules using the system according to the present invention. Figure 8 a: The effect of optimizing the diversity of crosslinking agent libraries (CCLs) on the phase separation of polymers (confocal microscopy image). Figure 8 b: The effect of combined crosslinking agent libraries (CCLs) on the centrifugibility of polymer aggregates. Figure 9 The effect of polymer concentration on the centrifugibility of polymer aggregates. Figure 10 The effect of the amount of DNA cross-linking agent molecules (as a percentage of the total amount of DNA anchoring molecules) on capture efficiency. Figure 11 An example of the application of this system is the capture of fluorescent single-stranded DNA (ssDNA) oligonucleotides as target biomolecules. Figure 12 Examples of the system's application include the capture of SARS-CoV-2 viral N gene RNA as a target biomolecule, and... Figure 13 Example of the application of this system, in which human thrombin is captured as a target biomolecule.

[0037] Figure 3 , Figure 5 and Figure 6 The portion was created using Biorender.com.

[0038] Figure 1A schematic diagram illustrating an embodiment of a collection system and method for any stewing process is shown. The system includes a liquid 1 containing a polymer chain 2 to which a DNA anchoring molecule 3, in the form of a DNA oligonucleotide, is covalently bound, a trapping molecule 4, a DNA cross-linking agent molecule 5, and a target biomolecule 7, which may be DNA, RNA, and / or a protein. Reference numeral A shows an enlarged portion of the polymer chain 2 with the covalently bound DNA anchoring molecule 3. The DNA anchoring molecule 3 has a free binding site 3.1 to which the trapping molecule 4 binds in the enlarged portion A shown. The trapping molecule 4 has a binding structure 4.1 in the form of a DNA oligonucleotide to which the target biomolecule 7 binds. Reference numeral B shows an enlarged portion of two polymer chains 2 with the covalently bound DNA anchoring molecule 3. The DNA cross-linking agent molecule 5 binds in pairs via complementary overlapping domains 5.1 and binds to the free binding site 3.1 of the DNA anchoring molecule 3, causing the two polymer chains 2 to cross-link. Due to the cross-linking, a polymer aggregate 6 is formed, shown in the rectangular enlarged portion C. Several of these polymer aggregates 6 are present in the liquid 1 in the container 8.

[0039] Because the system operates on a DNA hybridization model, capture and cross-linking occur only in the presence of sufficient ion concentrations to shield against repulsive interactions between DNA strands. Specifically, for this example, all components were dissolved in an aqueous solution of 10 mM Tris (pH 8.0), 1 mM EDTA, and 150 mM NaCl. When a specific protein was used for capture, bovine serum albumin was added to a final concentration of 0.1% (w / v) to prevent nonspecific binding of the protein to the plastic apparatus.

[0040] The procedure can be demonstrated as follows.

[0041] For nucleic acid binding, all components 1, 2, 3, 4, and 5 of the system are simultaneously mixed with the target biomolecule 7 (e.g., the target nucleic acid) and bound to the target nucleic acid by slowly decreasing the temperature to form target-bound polymer aggregates 6. To capture proteins (as target biomolecule 7) or other temperature-sensitive molecules, polymer chains 2 are pre-loaded with target-specific capture molecules 4 and pre-crosslinked with DNA crosslinking agent molecules 5 to form polymer aggregates 6. The target biomolecule 7 is then captured on polymer aggregates 6 in a homogeneous solution at a preferred temperature. The crosslinked polymer aggregates 6 maintain high permeability, thereby enabling rapid binding kinetics and high binding capacity. After binding the target biomolecule 7, the mixture is centrifuged at 17,000 xg for 30 minutes to precipitate the polymer aggregates 6 at the bottom of container 8, and the target biomolecule 7 is captured in the resulting precipitate 9. The centrifugation step is performed by… Figure 1The arrows indicate this. Any non-target molecules and / or contaminants remaining in the supernatant can be removed by decantation or by pipetting. This procedure will increase the concentration of target biomolecules, but when preparing RNA-seq libraries, this method is also used to remove interfering molecules, such as highly abundant transcripts like ribosomal RNA, from the supernatant.

[0042] The following examples illustrate the process steps and parameters using implementation methods.

[0043] Step 1: Mix the following components to the specified concentration:

[0044] Step 2: Thermal cycling with the following conditions:

[0045] If nucleic acids are captured, target biomolecule 7 can be added before step 2, or if temperature-sensitive molecules are captured, target biomolecule 7 can be added after step 2. If captured after step 2, the target biomolecule is added and the mixture is gently shaken to bind for 30 minutes at the preferred temperature.

[0046] Step 3: Centrifuge at 17,000 xg for 30 minutes to precipitate the target-bound polymer aggregate 6.

[0047] Step 4 (optional): Target biomolecule 7 can be released from polymer aggregate 6 by adding a 1.5-fold molar excess of sequence-specific release chain 10 relative to capture molecule 4. Incubate with gentle shaking at a preferred temperature for at least two hours, followed by centrifugation at 17,000 x g for 30 minutes to precipitate polymer aggregate 6. The purified target biomolecule 7 can be recovered from the supernatant.

[0048] Figure 2 The polymerization of the polymer with acrylamide, acrylic acid, and an acrylamide-labeled DNA oligonucleotide as a DNA anchoring molecule is illustrated. Free radical polymerization yields a linear high-molecular-weight polymer chain 2. The acrylamide-labeled DNA oligonucleotide (DNA anchoring molecule 3) is incorporated into the growing chain during polymerization. High-molecular-weight linear polymer chain 2 (up to 50 MDa) is produced by polymerization with a small amount of initiator (e.g., ammonium persulfate) under a nitrogen atmosphere. The polymerization method is well known and will therefore not be described in detail.

[0049] Figure 3The effect of polymer concentration in the capture solution is illustrated schematically. Experimental work has shown that after reaching a critical gelation concentration of approximately 0.2% (w / v) of polymer, the polymer forms an elongated polymer network that cannot be easily compressed into a compact precipitate 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 micron-sized cross-linked polymer aggregates 6 that remain separated but still sufficiently dense to precipitate the target biomolecules and effectively capture them (see [link to relevant documentation]). Figure 9 ).

[0050] Figure 4 A schematic diagram of a combined crosslinker library (CCL) is shown, which contains DNA crosslinker molecules 5 with varying numbers of indeterminate N-bases in their overlapping domains, and thus each CCL contains a different number of DNA crosslinker molecule pairs used to crosslink polymer chains. The different DNA crosslinker molecules 5 in the CCL are used to increase intermolecular crosslinks between polymer chains. The CCL consists of many different DNA crosslinker molecules 5 containing the same adaptor domain and a unique overlapping domain 5.1, which will bind only to another DNA crosslinker molecule 5 with a complementary overlapping domain 5.1 to form a pair. Indeterminate N-bases (which can be adenine (A), thymine (T), guanine (G), or cytosine (C)) are added to a predetermined sequence of the overlapping domain 5.1 to increase the diversity of the overlapping domain 5.1 of the DNA crosslinker molecules 5. Increasing the diversity of the cross-linking agent library reduces the likelihood that DNA cross-linking agent molecule 5 will find its match on the same polymer chain, favoring intermolecular cross-linking between polymer chains rather than intramolecular cross-linking, thus ensuring proper network formation. Since each indeterminate N-base can be one of four possible bases, the final CCL complexity is 4. n ,in n This refers to the number of undefined N-bases in the overlapping sequence. This method also uses CCL to ensure proper network formation and polymer aggregate formation. It has been shown that the number of different crosslinker pairs is crucial for proper particle formation (see [link to CCL]). Figure 8 Only when cross-linking is performed using a CCL-64 library containing 64 different DNA cross-linking agent molecules can polymer aggregates that can be centrifuged for effective target capture be formed.

[0051] The concentration of the DNA cross-linking agent library is also important for proper polymer aggregate formation and target biomolecule capture. If the DNA cross-linking agent library concentration is too low, the polymer chains will not be effectively cross-linked. If the DNA cross-linking agent library concentration is too high, not enough DNA anchoring molecules remain to bind with the capture molecules, thus reducing target biomolecule capture. This range depends on the number of available DNA anchoring molecules in the polymer solution. It has been shown that the proportion of DNA cross-linking agent molecules should be between 70% and 90% of the proportion of DNA anchoring molecules. For the example shown, a DNA anchoring concentration of 10 µM and a DNA cross-linking agent library (CCL-64) concentration of 8 µM (80% of the total DNA anchoring molecules) were used because this ratio provides the highest capture efficiency. Figure 10 The amount of capture molecules that can be used, and therefore the amount of target biomolecules that can be captured, ultimately depends on the amount of DNA cross-linking agent molecules used. In one example, up to 2 µM of capture molecules (20% of the total DNA anchoring molecules) can be added before the DNA anchoring molecules become saturated. To avoid accidental saturation of the DNA anchoring molecules due to pipetting errors, up to 1 µM of capture molecules (10% of the total DNA anchoring molecules) is used. The capture molecules are always present in excess relative to the target biomolecules. In one example, the target biomolecules are captured at a target:capture molecule ratio of at least 1:3 and at most 1:13.2. However, in other embodiments, the capture molecules or the DNA cross-linking agent library, or both, are directly grafted onto the polymer backbone (covalently linked), eliminating the need for binding to the DNA anchoring molecules. In this case, the target biomolecules will bind directly to the capture molecules on the polymer, making the capture molecules (i.e., the binding structure) less susceptible to alteration and thus allowing for optimization for specific applications.

[0052] Figure 5 The principle of programmable capture molecules is illustrated. A key feature of the system according to the invention is its ability to capture different biomolecules on the same polymer backbone in a sequence-selective and programmable manner. The capture molecules provide this programmability because the target-binding sequence of the binding structure of the capture molecules can be varied according to the target and the application. Figure 5A schematic diagram of a polymer chain 2 with three DNA anchoring molecules 3 is shown, in which a capture molecule 4 binds to its free binding site 3.1 in each case. The capture molecule 4 at position D is designed to bind single-stranded DNA as the target biomolecule 7. The capture molecule 4 at position E is designed to bind RNA as the target biomolecule 7. Any partial or complete 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 somehow linked to a partial or complete single-stranded DNA or RNA molecule. Position F shows the capture molecule 4 in the form of a DNA aptamer, which is designed to bind a protein as the target biomolecule 7. Experimental data demonstrate the high specificity and efficiency of capture (average >80% specific capture, <3% non-specific capture) for all three biomolecule types (positions D, E, and F) (including single-stranded DNA, SARS-CoV-2 viral RNA, human ribosomal RNA, and human thrombin). Figure 7 , Figures 11-13 It is possible to provide several types of capture molecules for simultaneous use in the capture solution, so as to capture and thereby separate different target biomolecules in multiple ways.

[0053] Figure 6 An example of how to release captured target biomolecules via TMSD is illustrated. The left side shows a polymer chain 2 with a covalently bound DNA anchoring molecule 3, to which a capture molecule 4 hybridizes to its free binding site 3.1. The capture molecule 4 comprises a foothold domain 4.2 and a binding structure 4.1 in aptamer form to which the target biomolecule 7 (protein thrombin) binds. To enable the release of the captured target biomolecule 7, a release chain 10 is added, which binds the capture molecule 4 at the foothold domain 4.2, and the bound target biomolecule 7 is displaced into solution via TMSD. This allows the target to be recovered in its native state without contamination by any DNA molecules used in the system. DNA cross-linking agents (not shown) can also be dynamically altered by enzymatic degradation, TMSD, or temperature changes to switch the polymer between densely cross-linked polymer aggregates and free linear polymer chains. Other options exist for releasing target biomolecules. For example, the target-bound precipitate can be redispersed and heated to release all DNA chains. Then, a blocking strand of binding structure 4.1 of the capture molecule 4 can be added to prevent rehybridization between the target biomolecule and the capture molecule. The polymer or polymer particles are then circulated using a predetermined temperature profile and centrifuged to remove all contaminating or unwanted DNA cross-linking agent molecules and capture molecules, while leaving the purified target biomolecule in the supernatant.

[0054] Figure 7The figure illustrates the quantification of specific and non-specific capture efficiency of various biomolecules using the system according to the invention. The target biomolecules, from left to right, are ssDNA oligonucleotides, SARS-CoV-2 N gene RNA, human ribosomal RNA, and human thrombin. All tested biomolecules exhibited highly specific and efficient binding.

[0055] Figure 8 This paper illustrates the effect of optimizing the diversity of crosslinking agent libraries on the phase separation of polymers. The complexity of the combined crosslinking agent library (CCL) affects precipitation and capture efficiency. Figure 8 Image a shows a fluorescence micrograph of polymers crosslinked with different crosslinking agent libraries at 100x magnification. The extended polymer aggregates form only in the CCL-64 state (i.e., having a combined crosslinking agent library consisting of 64 different DNA crosslinking agent molecule pairs), the overlapping domains of which contain three undefined N-bases.

[0056] Figure 8 b illustrates the effect of a combined crosslinker library (CCL) on the centrifugibility of polymer aggregates ("xlinker" refers to a DNA crosslinker molecule). The CCL-64 library was used to optimally observe efficient precipitation of polymer aggregates and, consequently, efficient target capture.

[0057] Figure 9 The effect of polymer concentration on the centrifugibility of polymer aggregates is shown. Images of fluorescent single-stranded DNA oligonucleotides as target biomolecules binding to polymers at different polymer concentrations are presented. 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), large molecular hydrogels that cannot precipitate are formed.

[0058] Figure 10 The effect of the amount of DNA cross-linking agent molecules (as a percentage of the total amount of DNA-anchoring molecules) is shown. The binding of fluorescent single-stranded DNA oligonucleotides to the polymer at different concentrations of DNA cross-linking agent molecules is shown. A concentration of 80% DNA cross-linking agent molecules indicates the highest target capture efficiency.

[0059] Figure 11 An example of the system’s application is shown, in which ssDNA is captured as a target biomolecule. Figure 11 a shows that for the complete polymer system (“polymer”), appropriate target capture and precipitation are observed only in the presence of both the crosslinking agent (“xlinker”) and the capturing molecule (“CSL”), indicating high binding specificity and efficient separation due to crosslinking. Figure 11b (right) shows the quantification of capture efficiency under the conditions in 11a. The significant capture of the target biomolecules is demonstrated by the decrease in normalized fluorescence in the supernatant, and this decrease in fluorescence is observed only when all components of the system are present.

[0060] Figure 12 An example of the system’s application is shown, in which SARS-CoV-2 viral N gene RNA is captured as a target biomolecule. Figure 12 Figure a shows the RT-qPCR curves for the amplification of SARS-CoV-2 N gene RNA in the supernatant after capture on polymer aggregates. The increased cycle number of the polymer sample (“polymer”) with all components present compared to the control (without the capture molecule, labeled “polymer-CSL”, or RNA only) indicates that the N gene RNA was captured and consumed from the supernatant. Figure 12 b shows the quantification of SARS-CoV-2 N gene RNA concentration in the supernatant after capture. Significant consumption of SARS-CoV-2 N gene RNA was observed only for the polymer system according to the invention, in which all components are present.

[0061] Figure 13 Figure a illustrates a cleavage assay in which only thrombin in the supernatant freely cleaves a colorimetric substrate, which changes color from transparent to dark after cleavage. The darker the color, the more substrate is cleaved, and therefore the higher the concentration of thrombin. The SDS-PAGE gel below shows the presence of thrombin protein in the supernatant from the corresponding samples described above during the cleavage assay. Capture was observed in the polymer system (“polymer”) of the present invention only when all components were present, compared to the control (capture molecule labeled “CSL”, crosslinker labeled “xlinker”). Upon addition of the release chain, the thrombin protein is released back into the supernatant via TMSD. Figure 13 Figure b shows the diagram from Figure 13 Quantification of the amount of free thrombin in the supernatant of the corresponding sample in section a.

[0062] List of reference numerals 1 liquid 2 polymer chain / polyacrylamide chain 3 DNA anchoring molecules 3.1 Free DNA binding sites 4 Capture Molecules 4.1 Combined Structure 4.2 Foothold Structural Domain 5 DNA cross-linking agent molecules 5.1 Double-chain overlapping structural domain 6. Polymer aggregates / polyacrylamide aggregates 7 target biomolecules 8 containers 9 sediments 10 Release Chain.

Claims

1. A system for capturing and separating target biomolecules (7) of DNA, RNA, and / or protein types from a liquid sample, the system comprising a liquid (1) containing - DNA anchoring molecule (3), which is covalently bound to polymer chain (2) and has free DNA binding site (3.1) - DNA cross-linking agent molecules (5), which form double-stranded overlapping domains (5.1) and reversibly bind to the free DNA binding site (3.1) to form a three-dimensional polymer network, and Interchangeable capture 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 DNA, RNA, and / or protein type from a liquid sample, wherein such target biomolecule (7) can be released by DNA denaturation or by foothold-mediated strand displacement (TMSD).

2. The system of claim 1, wherein the system comprises a DNA cross-linking agent library containing different DNA cross-linking agent molecules (5), wherein the differences between the different DNA cross-linking agent molecules (5) are located in different nucleotide sequences of the double-stranded overlapping domain (5.1).

3. The system according to claim 2, characterized in that... The overlapping domain sequence (5.1) contains an indefinite N-base at exactly one, two or four predetermined sequence positions, preferably at exactly three predetermined sequence positions.

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

5. The system according to any one of claims 1 to 4, characterized in that... The proportion of DNA cross-linking agent molecules (5) relative to DNA anchoring molecules (3) is 70% to 90%, preferably exactly 80%.

6. The system according to any one of claims 1 to 5, characterized in that... The three-dimensional polymer network exists in the liquid in the form of multiple individual polymer aggregates (6).

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

8. The system according to any one of claims 1 to 7, characterized in that... The binding structures (4.1) of these trapping molecules (4) include DNA, RNA, aptamers, antibodies, protein traps, and / or drug molecules.

9. The system according to any one of claims 1 to 8, characterized in that... These capture molecules (4) have several different binding structures (4.1) that bind to different target biomolecules of DNA, RNA and / or protein types (7).

10. The system according to any one of claims 1 to 9, characterized in that... The binding structure (4.1) of these capturing molecules (4) is connected to the foothold domain (4.2).

11. A method for capturing, separating and purifying target biomolecules (7) of DNA, RNA and / or protein types in a liquid sample using the system according to claims 1 to 10, wherein a capture solution is provided comprising polymer aggregates (6) loaded with capture molecules (4), wherein polymer chains (2) are cross-linked in the liquid by binding a DNA cross-linking agent library consisting of DNA cross-linking agent molecules (5) to DNA anchoring molecules (3) covalently bound to these polymer chains (2) to form three-dimensional polymer aggregates (6), wherein these polymer aggregates (6) are loaded with capture molecules (4). Under conditions that allow target biomolecules (7) of DNA, RNA, and / or protein types to bind to the binding structures (4.1) of these capture molecules (4), the capture solution is brought into contact with a liquid sample containing the target biomolecules in a container (8), and Separate these polymer aggregates (6) so that the target biomolecules (7) of these DNA, RNA and / or protein types remain bound to these polymer aggregates (6).

12. The method according to claim 11, characterized in that... The capture solution is cyclically treated according to a predetermined temperature profile to allow DNA hybridization of the DNA crosslinking agent molecules (5), the capture molecules (4), and the DNA anchoring molecules (3), wherein the temperature treatment is performed before and / or after contact with the liquid sample containing the target biomolecule (7).

13. The method according to claim 11 or 12, characterized in that... These polymer aggregates (6) are centrifuged after contact with the liquid sample containing the target biomolecules (7), and the resulting supernatant is removed by decanting or pipetting.

14. The method according to any one of claims 11 to 13, characterized in that These bound target biomolecules (7) are released from these polymer aggregates (6) via temperature elevation or foothold-mediated chain substitution (TMSD).

15. The method according to any one of claims 11 to 14, characterized in that... These polymer aggregates (6) have a polymer concentration of less than 0.2% (w / v), preferably less than or equal to 0.1% (w / v), and most preferably a polymer concentration in the range of 0.1% (w / v) to 0.01% (w / v).

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