Solid supports, articles, and methods comprising cyclic amine ligands suitable for polynucleic acid processing.

Abstract

A method for processing polynucleic acids is described, comprising: a) providing a solid support containing ligands, wherein at least a portion of the ligands contain a cyclic amine group having more than six ring members; exposing the solid support to polynucleic acid molecules in a buffer having a pH less than 5.5 to bind at least a portion of the polynucleic acid molecules to the ligands; exposing the solid support having the bound polynucleic acid molecules to a pH greater than 6 to release a portion of the bound polynucleic acid molecules from the ligands of the solid support, optionally retaining a portion of the polynucleic acid molecules bound to the solid support; and utilizing the released portion of the bound polynucleic acid molecules and / or the retained portion of the polynucleic acid molecules bound to the solid support, or a suspension thereof. A solid support containing ligands bound to a solid support (e.g., magnetic beads), wherein at least a portion of the ligands contain a cyclic amine group having more than six ring members, and a kit containing such a solid support are also described.

Description

Technical Field

[0001] Summary of the Invention In one embodiment, a method for processing a polynucleic acid, comprising: a) providing a solid support comprising a ligand, wherein at least a portion of the ligand comprises a cyclic amine group comprising ring members exceeding 6 members; b) exposing the solid support to a polynucleic acid molecule in a buffer having a pH of less than 5.5 to bind at least a portion of the polynucleic acid molecule to the ligand; c) exposing the solid support having the bound polynucleic acid molecule to a pH of more than 6 to release a portion of the bound polynucleic acid molecule from the ligand of the solid support, and optionally retaining a portion of the polynucleic acid molecule bound to the solid support; d) optionally, washing the solid support comprising the bound and / or retained polynucleic acid molecule; e) optionally, preparing a suspension from the solid support comprising the retained bound polynucleic acid molecule; f) using the released portion of the bound polynucleic acid molecule of c), and / or the retained portion of the polynucleic acid molecule bound to the solid support or a suspension thereof. A method is described.

[0002] In another embodiment, a solid support (e.g., magnetic beads) comprising a ligand bound to the solid support, wherein at least a portion of the ligand comprises a cyclic amine group comprising ring members exceeding 6 members, is described.

[0003] In another embodiment, a kit comprising a solid support (e.g., magnetic beads) and a buffer having a pH of less than 5.5 is described.

Mode for Carrying Out the Invention

[0004] The methods and articles described herein include a solid support. The surface of the solid support includes ligands. The ligands can reversibly bind to or otherwise interact with polynucleic acids, including, for example, ionic / electrostatic interactions, hydrogen bonding interactions, hydrophobic interactions, and combinations thereof.

[0005] Various solid supports are described in the literature. Solid supports are typically composed of organic polymers (e.g., plastics), inorganic materials, or a combination of both. Examples of suitable solid support materials include metal oxides such as Al2O3, TiO2, ZrO2, and Ta2O3; and silica materials such as SiO2 and polysilicic acid. Solid supports may be magnetic materials such as iron, cobalt, or nickel and their oxides, alloys, ceramics, or amalgams. Suitable organic polymers include polystyrene, poly(meth)acrylic polymers including poly(meth)acrylate and poly(meth)acrylamide, polyamides such as polyurethane and nylon; polyolefins such as polyethylene, polypropylene, and polybutadiene, and copolymers thereof. Other solid support materials include polysaccharides, especially hydrogels, such as agarose, cellulose, dextran, SEPHADEX®, SEPHACRYL®, and chitosan. Examples of inorganic supports include glass or metal surfaces such as gold. In some embodiments, the ligands described herein are bonded (e.g., covalently) to the solid support material. In some embodiments, the solid support is not gold nanoparticles.

[0006] Various particles (e.g., magnetic and non-magnetic) can be used as solid supports. In some embodiments, the particles have an average particle size of at least 0.5 or 1 micron. In some embodiments, the particles typically have an average particle size of 500, 250, 100, 75, 50, 25, 15, 10, or 5 microns or less. The particles are typically spherical, but particles of other shapes can also be used.

[0007] In some embodiments, the solid support is a plurality of particles, including magnetic beads, particularly paramagnetic beads. In a typical embodiment, the ligand (e.g., ionizable) is formed by bonding (e.g., covalently) a cyclic amine compound to (para)magnetic particles (also called beads) having functional groups on their surface, as described herein.

[0008] Typical reaction schemes are as follows:

[0009] [ka] (In the formula, X is a functional group on the support surface that reacts with the cyclic amine compound, and Y is the reaction product of X and the cyclic amine compound.)

[0010] It is understood that an organic linking group (e.g., alkylene) may be present between a solid support (e.g., magnetic particles) and a functional group X. In some embodiments, a cyclic amine ligand is bonded to a solid support (e.g., magnetic particles) using an organic linking group lacking a sulfur moiety.

[0011] In some embodiments, solid supports containing carboxylic acid groups or salts thereof (e.g., carboxylate groups) on their surface are commercially available. Several commercially available beads are described in the following examples. Gold nanoparticles (10 nm carboxylic acid-functionalized polyethylene glycol 3000 g / mol) are commercially available from MilliporeSigma (product number 765457). Other commercially available products include carboxyl multiwell plates (Corning® PureCoat® Carboxyl Plates), carboxyl-modified polystyrene (Polybead® Carboxylate Microspheres), carboxy-terminated biosensor surfaces (Octet® Amine Reactive 2nd-Generation Biosensors), carboxylic acid silica gel (SiliaBond Carboxylate (WCX), product number R70030B), and carboxylate polystyrene monodisperse microspheres (commercially available from Polysciences as Polybead® Carboxylate Sampler Kits).

[0012] As described for other amine compounds in U.S. Patent Application Publication No. 2019 / 0071662, a carboxylic acid group or a salt thereof on the surface of a solid support (e.g., particles) covalently bonds with the amine group of a cyclic amine compound to form an amide linkage group. An exemplary reaction scheme is as follows:

[0013] [ka]

[0014] The amide group can also be a reaction product of an amine and an ester. In yet another embodiment, the ligand having the amide group may be a reaction product of an amine and an acid halide.

[0015] Although the solid support particles described above are shown as having a single ligand, it is understood that the solid support (e.g., each particle) may contain multiple ligands. For example, the solid support particles used in the examples are presumed to have about 0.6 mmol of carboxylic acid or carboxylate groups per gram of particle. Therefore, if all such carboxylic acid or carboxylate groups react with amine compounds as described herein, the number of ligands will be approximately equal to the number of carboxylic acid or carboxylate groups. If the particles have an average diameter of 1 μm, then the particles will have about 1.8 × 10⁻⁴ picomoles of ligands / μm 2 It may contain up to [a certain amount]. If the particles contain a second ligand or an unreacted carboxylic acid / carboxylate group, the particles may contain a smaller amount of ligand per unit surface area of ​​the particles.

[0016] Cyclic amine compounds can also react with other functional groups on the surface of a support (e.g., magnetic beads).

[0017] In some embodiments, the functional group on the surface of the support is a p-toluenesulfonate (i.e., tosylate) group. Magnetic beads having surface p-toluenesulfonate (i.e., tosylate) groups are available from Thermo Fisher Scientific Inc. under the trade name "DYNABEADS MYONE TOSYLACTIVATED" and product number 65502.

[0018] In some embodiments, the functional groups on the surface of the support are epoxy groups. Magnetic beads having surface epoxy groups are available from Thermo Fisher Scientific Inc. under the trade name "DYNABEADS M-270 EPOXY" and product number 14301. Amine groups react to open the epoxy group ring, thereby allowing a cyclic amine group to be covalently bonded to the support (e.g., magnetic beads).

[0019] In some embodiments, the functional group on the surface of the support is a (meth)acryloxy group. The amine group can react with the (meth)acryloxy group, thereby covalently bonding the cyclic amine group to the support (e.g., magnetic beads). In one synthetic approach, a support (e.g., magnetic beads) containing a silanol group reacts via condensation with a (meth)acryloxy silane compound (such as acryloxypropyltrimethoxysilane) to form a (meth)acryloxy terminal group. The linking group (L) between the support (e.g., magnetic beads) and the (meth)acryloxy terminal group contains the condensation reaction product of silanol and alkoxysilane. Magnetic beads having surface silanol groups are available from Thermo Fisher Scientific Inc, under the trade name "DYNABEADS MYONE SILANE", product number 37002D.

[0020] Other supports containing functional groups capable of reacting with the cyclic amine compound are commercially available or described in the literature.

[0021] In some embodiments, the cyclic amine compound that reacts with the functional group on the surface of the solid support (e.g., nanoparticles) does not contain a thio or thiolate functional group.

[0022] The functional groups on the surface of a solid support (e.g., particles) are reacted with a cyclic amine compound containing ring members exceeding 6 members. The resulting ligand is a cyclic amine group containing ring members exceeding 6 members. In some embodiments, the cyclic group of the ligand bound to the support may contain one fewer amine group than the cyclic amine compound. The cyclic amine compound and the cyclic amine group contain at least 7, 8, 9, 10, 12, 14, 15, 16, or 18 members. The total number of ring members of the heterocycle is typically 24 or less. In some embodiments, the cyclic amine compound contains at least 2 nitrogen atoms. In some embodiments, the cyclic amine contains at least 2, 3, or 4 amine groups. The total number of amine groups is typically 4, 5, 6, 7, or 8 or less. At least one of the amine groups reacts with the functional group of the solid support. In some embodiments, the cyclic amine compound / group contains at least 2 heteroatoms selected from nitrogen and oxygen. In some embodiments, the cyclic amine compound / group contains at least 2, 3, or 4 oxygen atoms (e.g., ether groups). The total number of oxygen atoms (e.g., ether groups) is typically 4 or less. Some representative cyclic amine compounds are shown below.

[0023]

Chemical formula

[0024] Various other cyclic amine compounds having ring members exceeding 6 members are described in the literature. See, for example, International Publication No. 92 / 05804, which is incorporated herein by reference.

[0025] The cyclic amine compound and the cyclic amine group of the ligand may optionally further contain substituents under the condition that their presence does not substantially impair the binding and / or release of the polynucleic acid molecule at different pHs.

[0026] Substituents typically contain 1 to 20 carbon atoms. Representative organic groups include alkyl, substituted alkyl, aryl, substituted aryl, and combinations thereof. Organic groups may be linear or branched, and may optionally include aliphatic or aromatic cyclic groups. Representative substituents include hydroxy, alkoxy, halo, ether, thioether, phenyl, benzyl, pyridinyl, nitro, cyano, sulfonyl, ester, and combinations thereof.

[0027] The cyclic amine groups of cyclic amine compounds and ligands typically do not contain immobilized metal ions, as is the case with metal chelates.

[0028] In some embodiments, the cyclic amine group lacks a carboxylic acid or carboxylate functional group.

[0029] In a typical embodiment, a single cyclic amine group having more than six members is bonded to a solid support (e.g., magnetic particles). Therefore, a cyclic amine group having more than six members is not a polymerization unit of a polymer such as a polyamide, poly(organic)phosphazene, or polyacrylic acid (e.g., superabsorbent) polymer.

[0030] In some embodiments, the surface of a solid support (e.g., particles) is subjected to passivation before reaction with a cyclic amine compound. Passivation involves reacting surface functional groups present on a solid support that can be positively ionized in an aqueous buffer with a chemical that prevents such ionization. In some embodiments, the solid support is passivated with acetic anhydride.

[0031] While not intended to be theoretically binding, it is hypothesized that amine, oxygen, and / or amide linking groups may be involved in nucleic acid binding. Referring to Table 2 of the following examples, when cyclic amine compounds having more than six ring members were reacted with their functional groups (e.g., carboxylic acid groups or salts) on the surface of a solid support (e.g., particles), the resulting solid support (e.g., particles) exhibited a greater zeta potential at pH 4.5 than at pH 8.5. This change in zeta potential indicates a change in potential near the electrical double layer, which includes the negative carboxylic acid / carboxylate surface charge of any unreacted particles and (e.g., positive) counterions in the solution associated with the particle surface. Such a change in potential appears to contribute to the ligand's ability to bind to and release DNA, which may be beneficial for subsequent processing, including DNA size selection, amplification, or modification.

[0032] The zeta potential of a solid support (e.g., particles) containing the described ligands can be measured according to the test methods in the examples. The solid support (e.g., particles) containing the ligands has a negative or positive zeta potential in the presence of a low pH buffer. In some embodiments, the pH of the low pH buffer is at least 3.5, 4, or 4.5. The solid support (e.g., particles) containing the ligands has a lower zeta potential in the presence of a high pH buffer than in the presence of a low pH buffer. In some embodiments, the pH of the high pH buffer is at least 5.5, 6, or 6.5. The absolute difference between the zeta potential in the presence of a low pH buffer and the zeta potential in the presence of a high pH buffer is typically at least 10, 20, 30, 40, or 50 mV. In one embodiment, the absolute difference between the zeta potential at pH 4.5 and the zeta potential at pH 8.5 is at least 10, 20, 30, 40, or 50 mV. The absolute value of the pH difference between a low-pH buffer and a high-pH buffer is typically at least 2, 3, or 4. In a typical embodiment, a solid support (e.g., particles) containing ligands has a lower zeta potential at pH 8.5 than at pH 4.5. In some embodiments, buffers used to characterize the zeta potential of the solid support (e.g., particles) containing ligands described are trishydroxymethylaminomethane (TRIS) and sodium acetate buffers.

[0033] In other embodiments, the ligand further comprises an ionizable amine group. For example, when a cyclic amine compound containing at least two amine groups reacts with a carboxylic acid group or a salt thereof on the surface of a solid support (e.g., particles), one of the amine groups forms an amide bond, and the other amine group of the ligand can be reversibly ionized. The above-mentioned cyclic amine compounds and ligands formed from such compounds each include a secondary amine or a tertiary amine (if substituents are present). The function of amine groups as ionizable groups has been documented in the literature.

[0034] In some embodiments, a solid support (e.g., a particle) containing a functional group reacts with a single cyclic amine compound having more than six ring members, as described above, to form a ligand. In other embodiments, the solid support (e.g., a particle) reacts with at least two different amine compounds to form first and second ligands.

[0035] In some embodiments, both the first and second ligands are cyclic amine compounds having more than six ring members, as described herein.

[0036] In other embodiments, the second ligand is a cyclic amine compound having six or fewer members in the heterocycle. In some embodiments, the cyclic amine compound further comprises an ether group, such as morpholine. Referring to Table 9, supports further comprising morpholine as the second ligand had higher elution concentrations of smaller cell-free DNA having a size in the range of approximately 75 to 500 base pairs compared to the first ligand alone. In some embodiments, the elution concentrations were greater than 65, 70, 75, 80, 85, 90, 95, or 100% of base pairs having a size of 75, 334, or 501 base pairs.

[0037] In other embodiments, the second different ligand is a piperazine or a derivative thereof, as described in International Applications PCT / IB2022 / 060641 and PCT / IB2022 / 060640, which are incorporated herein by reference. Representative compounds include, for example, N-methylpiperazine, N-phenylpiperazine, N-(2-hydroxyethyl)piperazine, N-(4-methoxyphenyl)piperazine, N-(4-trifluoromethylphenyl)piperazine, and 1-(4-bromophenyl)piperazine. In yet another embodiment, the second ligand is a fluorinated amine (e.g., a monoamine or diamine). Various other ligands for processing polynucleic acids are described in the art.

[0038] In some embodiments, the second amine compound may not function to bind to the polynucleic acid, but rather to simply reduce the concentration of the first ligand.

[0039] The molar ratio of the first amine compound to the second amine compound is typically in the range of 1:10 to 10:1. Similarly, the molar ratio of the first ligand to the second ligand is in the range of 1:10 to 10:1. In some embodiments, the molar ratio of the first amine compound or the first ligand to the second amine compound or ligand is at least 2:10, 3:10, 4:10, 5:10, 6:10, 7:10, 8:10, or 9:10. In some embodiments, the molar ratio of the first amine compound or the first ligand to the second amine compound or ligand is 9:10, 8:10, 7:10, 6:10, 5:10, 4:10, 3:10, or 2:10 or less.

[0040] Solid supports (e.g., particles) and kits containing the described ligands can be used in a variety of polynucleotide processing techniques, including, for example, size separation, purification, quantification, amplification, tagged fragmentation, digestion, and library preparation (e.g., for nucleic acid sequencing).

[0041] A method for processing polynucleic acids typically includes a) providing a solid support containing ligands, wherein at least a portion of the ligands contain a cyclic amine group having more than six ring members, as described herein. The method also includes b) The method further includes exposing a solid support to polynucleic acid molecules at a first pH (e.g., in a buffer) to bind at least a portion of the polynucleic acid molecules to ligands. Typically, the method further includes exposing the solid support having the bound polynucleic acid molecules to a second different pH to release a portion of the bound polynucleic acid molecules from the ligands of the solid support. In some embodiments, the method includes retaining a portion of the bound polynucleic acid molecules on the solid support. In some embodiments, the method includes washing the solid support containing the bound and / or retained bound polynucleic acid molecules. In some embodiments, the method includes preparing a suspension from the solid support containing the retained bound polynucleic acid molecules. The method includes utilizing the released portion of the bound polynucleic acid molecules (e.g., eluate) and / or the retained portion of the polynucleic acid molecules bound to the solid support or the suspension thereof.

[0042] Regarding step b), the binding of polynucleic acids generally occurs in the presence of a low-pH buffer. In some embodiments, the pH of the low-pH buffer is at least 3.5, 4, 4.5, 5, or 5.5. In some embodiments, the pH of the low-pH buffer is 5.5, 5, 4.5, 4, or 3.5 or less. In some embodiments, the solid support is exposed to polynucleic acid molecules in a buffer having a low pH for 10 minutes at ambient temperature (e.g., 25°C).

[0043] Regarding step c), the release of polynucleic acid generally occurs in the presence of a high-pH buffer. In some embodiments, the pH of the high-pH buffer is at least 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, or 11. In some embodiments, the pH of the high-pH buffer is 11, 10.5, 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, or 6 or less. In some embodiments, the solid support is exposed to polynucleic acid molecules in a high-pH buffer for 10 minutes at ambient temperature (e.g., 25°C).

[0044] Various low-pH and high-pH biological buffers are known for use with this method and kit.

[0045] Some suitable buffers include, for example, citrate buffers (sodium citrate and citrate monohydrate), acetate buffers, and TE buffers, as further described in the Examples; phosphate-buffered saline (PBS); N-2-acetamido-2-aminoethanesulfonic acid (ACES); N-2-acetamido-2-iminodiacetic acid (ADA); aminomethylpropanediol (AMP); 3-1,1-dimethyl-2-hydroxyethylamino-2-hydroxypropanesulfonic acid (AMPSO); N,N-bis-2-hydroxyethyl-2-aminoethanesulfonic acid ( BES); N,N-bis-2-hydroxyethylglycine (BICINE); bis-2-hydroxyethyliminotrishydroxymethylmethane (BIS-TRIS); 1,3-bistrishydroxymethylmethylaminopropane (BIS-TRISpropane); 4-cyclohexylamino-1-butanesulfonic acid (CABS); 3-cyclohexylamino-1-propanesulfonic acid (CAPS); 3-cyclohexylamino-2-hydroxy-1-propanesulfonic acid (CAPSO); 2-N-cyclohexylaminoethanesulfonic acid (CHES); 3-N, N-bis-2-hydroxyethylamino-2-hydroxypropanesulfonic acid (DIPSO); N-2-hydroxyethylpiperazine-N-3-propanesulfonic acid (EPPS or HEPPS); N-2-hydroxyethylpiperazine-N-4-butanesulfonic acid (HEPBS); N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES); N-2-hydroxyethylpiperazine-N-2-propanesulfonic acid (HEPPSO); 2-N-morpholinoethanesulfonic acid (MES); 4-N-morpholinobutanesulfonic acid (MOBS) 3-N-morpholinopropanesulfonic acid (MOPS); 3-N-morpholino-2-hydroxypropanesulfonic acid (MOPSO); piperazine-NN-bis-2-ethanesulfonic acid (PIPES); piperazine-NN-bis-2-hydroxypropanesulfonic acid (POPSO); N-trishydroxymethyl-methyl-4-aminobutanesulfonic acid (TABS); N-trishydroxymethyl-methyl-3-aminopropanesulfonic acid (TAPS); 3-N-trishydroxymethyl-methylamino-2-hydroxypropanesulfonic acid (TAPSO);Examples include N-trishydroxymethyl-methyl-2-aminoethanesulfonic acid (TES); N-trishydroxymethylmethylglycine (TRICINE); trishydroxymethylaminomethane (TRIS); histidine and polyhistidine; imidazole and its derivatives; triethanolamine dimers, oligomers and polymers; and di / tri / oligoamino acids, such as Gly-Gly; as well as Ser-Ser, Gly-Gly-Gly, and Ser-Gly.

[0046] In some embodiments, the low-pH buffer is a citrate buffer (sodium citrate and citrate monohydrate) or an acetate buffer. In some embodiments, the high-pH buffer is PBS or TRIS.

[0047] This method may optionally include one or more washing steps. In one embodiment, the method includes washing the solid support containing the bound polynucleic acid molecules after step b). This washing step typically utilizes a low pH buffer. In some embodiments, this washing step utilizes the same buffer as in step b). In another embodiment, the method includes washing the solid support containing the retained polynucleic acid molecules after step c). This washing step typically utilizes a high pH. Exemplary suspension buffers (also referred to as storage buffers) include PBS and TE buffers. In some embodiments, the suspension buffer has a pH of about 8. Neutral pH buffers or water may also be used.

[0048] Buffers generally have an ionic salt concentration of less than about 1 M. In some embodiments, the salt concentration of the buffer during binding is less than 500 mM, 250 mM, 100 mM, 50 mM, 25 mM, or 10 mM. (For example, (poly)nucleic acid capture) A generally preferred salt for use in buffers is sodium chloride. In one embodiment, the buffer has a pH greater than 6 and a salt concentration of less than 1 M, 500 mM, 250 mM, 100 mM, 50 mM, 25 mM, or 10 mM.

[0049] In some embodiments, at least some of the method steps may be carried out using a kit. The kit typically comprises a solid support containing ligands and a low pH buffer (e.g., having a pH less than 5.5), as described above. In some embodiments, the kit further comprises a high pH buffer, as described above, which is suitable for releasing a portion of the polynucleic acid molecule. In some embodiments, the kit further comprises a washing buffer and / or suspension buffer, as described above.

[0050] Various polynucleic acids can be processed using solid supports, methods, and kits comprising ligands described herein. In some embodiments, the polynucleic acid comprises at least 100, 200, 300, 400, or 500 base pairs. In some embodiments, the polynucleic acid comprises at least 1000, 1500, 2000 (e.g., 2027, 2322), 2500, 3000, 3500, 4000 (e.g., 4361), 4500, or 5000 base pairs. In some embodiments, the polynucleic acid comprises at least 5500, 6000, 6500 (e.g., 6557), 7000, 7500, 8000, 8500, 9000 (e.g., 9461), or 10,000 base pairs. In some embodiments, the polynucleic acid comprises at least 150,000;20,000 (e.g., 23130), 25,000;30,000;35,000;40,000;45,000, 50,000 (e.g., 48502) base pairs or more. In some embodiments, the polynucleic acid comprises a size distribution having minimum and maximum values ​​defined by the intervals of the number of base pairs described just.

[0051] In some embodiments, the following performance criteria described herein were obtained with respect to DNA standards or mixtures of standards, as will be described in more detail in the following examples. Typical commercially available DNA standards include, for example, λDNA (i.e., double-stranded DNA isolated from bacteriophage lambda, with a length of 48,502 base pairs), λDNA-HindIII digest (i.e., DNA isolated from bacteriophage lambda, digested with the restriction endonuclease HindIII to produce eight DNA fragments ranging in size from 125 bp to 23,130 bp), and cell-free DNA ("cfDNA") (double-stranded DNA fragments of 75 bp, 167 bp, 334 bp, and 501 bp).

[0052] Unless otherwise specified, DNA standard processing is performed with a polynucleic acid (e.g., DNA):solid support (e.g., particles) weight ratio of 1:250 w / w. Other ratios are also preferred. For example, the polynucleic acid (e.g., DNA):solid support (e.g., particles) weight ratio may range from 1:10 to 1:2500. In some embodiments, the polynucleic acid (e.g., DNA):solid support (e.g., particles) weight ratio is at least 1:25, 1:50, 1:100, 1:150, or 1:200. In some embodiments, the polynucleic acid (e.g., DNA):solid support (e.g., particles) weight ratio is 1:2500; 1:2000; 1:1500, 1:1000, or 1:500 or less.

[0053] In some embodiments, the size and distribution of polynucleic acids are known, as in the case of standards. In other embodiments, the size and distribution of polynucleic acids can be determined using methods known in the art, such as pulsed-field gel electrophoresis or Qubit fluoroscopy.

[0054] Unless otherwise specified, the following terms are defined as follows: The term "supernatant" refers to the solution remaining after polynucleotide molecules (e.g., DNA) have bound to a solid support (e.g., particles). Therefore, characterization of the supernatant is related to the unbound polynucleotide molecules (e.g., DNA). The percentage of bound polynucleotide molecules (e.g., DNA) can be calculated according to the formula 100 × (initial DNA ng - supernatant DNA ng) / initial DNA ng.

[0055] "Elution" refers to a solution of polynucleic acid molecules (e.g., DNA) that were initially bound to the beads but are released when exposed to a buffer with a higher pH at room temperature for 10 minutes.

[0056] A “solid support (e.g., particles) suspension” refers to providing a solid support (e.g., particles) in an aqueous liquid after separation of an eluate containing released polynucleic acid molecules (e.g., DNA). The aqueous liquid may be characterized as a carrier liquid that carries the solid support (e.g., particles) with bound polynucleic acid to subsequent processing and analysis steps. A portion of the polynucleic acid bound to the solid support (e.g., particles) may be released into the aqueous liquid of the suspension. However, in typical embodiments, the amount of polynucleic acid bound to the solid support (e.g., particles) is significantly greater than the amount released into the aqueous liquid of the suspension. For example, the amount of polynucleic acid released into the aqueous liquid of the suspension may be less than 10, 5, or 1% by weight compared to the total amount of polynucleic acid in the suspension (i.e., the sum of polynucleic acid bound to the solid support and released into the aqueous liquid of the suspension).

[0057] In some embodiments, the described ligands in solid supports (e.g., particles), methods, and kits may be used to process (e.g., bind) polynucleic acids (e.g., DNA) in the size range of 100 to 50,000 base pairs, such as λDNA and λDNA-HindIII digestion mixes or cell-free DNA in an 80:20 v:v ratio as described above.

[0058] In some embodiments, the DNA in the supernatant and eluate was quantified. The bound DNA was calculated as described above.

[0059] Referring to Tables 3 and 4, in some embodiments, the unbound polynucleotide molecules in the supernatant constitute less than 80, 70, 60, 50, 40, 30, or 20% of the total initial polynucleotide mass. In some embodiments, the retained bound polynucleotide molecules constitute more than 50, 60, 70, 80, or 90% of the total initial polynucleotide mass. In some embodiments, the mass of released polynucleotide molecules is more than 10, 20, 30, 40, 50, 60, 70, or 80% of the total initial polynucleotide mass. In some embodiments, the solid support contains a greater amount of retained bound polynucleotide molecules than released polynucleotide molecules. In contrast, when the solid support (e.g., particles) contains a carboxylic acid / carboxylate (i.e., CE1) in the absence of the described ligands, the amount of bound polynucleotide was 4%, and no polynucleotide was released. Furthermore, when the solid support (e.g., particles) contained a morpholine ligand (i.e., CE3), the amount of bound polynucleic acid was 27%, and 12% of the polynucleic acid was released.

[0060] Tables 11 and 12 show that large amounts of polynucleic acid can bind to and release from cyclic amines lacking one or more ether moieties.

[0061] In other embodiments, after steps a-c), the method further includes quantifying the size and distribution of polynucleic acid (e.g., DNA) fragment sizes using methods known in the art, such as pulsed-field gel electrophoresis. Referring to Table 8, the initial DNA (e.g., the standard) contains certain DNA fragments at specific concentrations in the size range of 125 to 48,505 base pairs. In particular, a higher amount of DNA of a particular size fragment in the eluate compared to the initial DNA indicates enrichment of that DNA fragment size. Specifically, the amounts of DNA in the eluate were enriched for fragment sizes of 125, 564, 2027, 2322, 4361, 6557, and 9416, or in other words, for fragments in the size range of 125 to approximately 10,000.

[0062] These results indicate that the process of binding and releasing polynucleotide molecules is suitable for size selection or size separation of polynucleotides, particularly high molecular weight fragments. The ability to perform size selection by eluting a biased distribution of molecular weight fragments, while both populations (bound nucleic acids and eluted nucleic acids) remain available, was unexpected but beneficial because all nucleic acids, whether eluted or not, are now accessible for use. This allows for size selection by direct use of both populations without buffer exchange or desalting. Both populations can be used in the same way or subjected to different process steps. This also maximizes the amount of usable and accessible nucleic acid after size selection, as both populations are available.

[0063] Referring to Table 5, large amounts of bound and released polynucleic acids (e.g., DNA) can be achieved with polynucleic acids (e.g., DNA) in the size range of 75 to 500 base pairs (e.g., 75, 167, 334, or 501 base pairs). In some embodiments, the amount of bound polynucleic acid (e.g., DNA) was at least 35, 40, 50, 60, 70, 80, 90, or 100%. In some embodiments, the amount released was at least 40, 50, 60, 70, 80, or 90%.

[0064] Referring to Table 10, a large number of 167 bp and 334 bp fragments can be bound and released. After elution, the amount remaining on the beads was less than 10%.

[0065] In some embodiments, solid supports (e.g., particles), methods, and kits containing the described ligands may be used to amplify polynucleic acids. Numerous techniques are available for amplifying nucleic acids. These techniques include polymerase chain reaction (PCR), ligase chain reaction (LCR), autologous persistent sequence replication (3SR), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), transcription-mediated isothermal CR cycling probe techniques, cascade rolling circle amplification (CRCA), nickel endonuclease amplification (NEAR), transcription-mediated amplification (TMA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), CRISPR-Cas-based amplification, and in vitro transcription (IVT).

[0066] PCR (polymerase chain reaction) is a method for amplifying a target DNA sequence using a thermostable DNA polymerase and two nucleotide primers (one complementary to the (+) strand at one end of the sequence to be amplified, and the other complementary to the (-) strand at the other end). The newly synthesized DNA strand can then serve as a further template for the same primer sequence, so a series of rounds of primer annealing, strand elongation, and dissociation results in rapid and highly specific amplification of the desired sequence.

[0067] Rolling circle amplification (RCA) is an amplification process driven by DNA polymerase that can replicate in either linear or geometric dynamics under isothermal (single temperature) conditions. Geometric amplification occurs via DNA strand substitution and superbranching in the presence of two suitably designed primers, with a rate of 10 in 1 hour. 12 More than one copy of the DNA template is generated.

[0068] Referring to Tables 6 and 7, lower Ct values ​​indicate greater (e.g., PCR) amplification. In particular, a difference of 6 in Ct value is generally equivalent to a 100-fold difference in the amount of target polynucleic acid. Thus, even a slight decrease in Ct value is significant. In some embodiments, the decrease in Ct value is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 compared to a buffer lacking a functionalized support (e.g., beads) with a cyclic amine ligand. The decrease in Ct value is evident in the eluate and bead suspension at 1:10 and 1:100 dilutions. Therefore, solid supports (e.g., particles), methods, and kits containing the described ligands are advantageous for amplifying higher molecular weight fragments, such as 23kb HindIII digest fragments of λDNA, and for amplifying lower molecular weight fragments, such as 4kb-HindIII digest fragments of λDNA.

[0069] The solid supports (e.g., particles), methods, and kits containing the ligands described may be used for library preparation for nucleic acid sequencing. A sequencing library is a collection of DNA fragments modified using nucleic acid adapters into a format compatible with the sequencing technology and instrument in use. Each sequencing instrument has its own library preparation workflow for attaching the necessary barcodes and adapters and modifying the fragments to enable sequencing. Libraries can be prepared for high-throughput sequencing instruments that rely on methods such as synthetic sequencing, ligation sequencing, binding sequencing, pyrosequencing, or impedance-based sequencing, or for real-time long-read instruments that rely on methods such as synthetic sequencing in zero-mode waveguides or nanopore sequencing. In some embodiments, the sequencing involves tagged fragmentation and enzymatic reactions for fragmenting the DNA. In some embodiments, the enzyme may be bound to beads, such as those exemplified by the bead-linked transposomes in the "Illumina DNA Prep" reference guide.

[0070] Utilizing released and / or retained and / or suspended polynucleic acid molecules may include processing or analyzing the polynucleic acid, for example, size separation, purification, quantification, detection or modification (e.g., via exposure to at least one enzyme), amplification, transcription, tagged fragmentation, digestion, ligation, or library preparation (e.g., for nucleic acid sequencing).

[0071] Considering the favorable test results obtained by testing DNA standards, it is inferred that the described ligands, methods, and kits, including solid supports (e.g., particles), may be used for polynucleic acids extracted from any suitable living source (including human, animal, microbial, plant, or viral sources) (including cells, saliva, fresh tissue, or other materials containing DNA), whether initially whole-destroyed or otherwise whole- or partially destroyed. In some embodiments, the described ligands, including solid supports (e.g., particles), methods, and kits, may be used to test target DNA from microbial or food samples, etc.

[0072] A variety of polynucleic acid molecules can be processed, including those obtained from bacteria, microorganisms, fungi, plants, or animals. Biological samples containing polynucleic acids may be naturally occurring samples or samples or libraries that are intentionally designed or synthesized. In one embodiment, the sample contains a population of cells or cell fragments, including but not limited to cell membrane components, exosomes, and intracellular components. The cells may be a homogeneous population of cells, such as a specific type of isolated cell, or a mixture of different cell types from a biological fluid or tissue of a human, mammal, or other species. Biological samples may be simple, for example, containing isolated DNA or derived from a homogeneous cell culture or tissue source, or complex, such as a sample of a tumor, blood, or whole organ. Biological samples may be derived from any suitable source, such as a healthy tissue or cell source, a diseased tissue or cell source, a cell culture or cell line, a cell extract or lysate, or a biopsy.

[0073] Other biological samples containing polynucleic acids for use include blood samples (including serum, plasma, whole blood, and peripheral blood), saliva, urine, vaginal or cervical secretions, amniotic fluid, placental fluid, cerebrospinal fluid, or serous or mucosal secretions (e.g., cheek, vagina, or rectum). Further samples include biological samples or cell lysates derived from blood or biopsy tissues (i.e., mixtures derived from tissues and / or cells). Other suitable tissues include hair, fingernails, etc. Additional samples may include libraries of antibody mimics such as antibodies, antibody fragments, and aphibodies. Other samples may be collections of synthesized or engineered chemical molecules, proteins, antibodies, or any other polyanions described herein. [Examples]

[0074] Unless otherwise specified, all parts, percentages, ratios, etc., in the examples and the rest of the specification are by weight. Unless otherwise indicated, all other reagents may be obtained from or available from fine chemical vendors such as MilliporeSigma in Burlington, Massachusetts, or synthesized by known methods. Table 1 (below) lists the materials used in the examples and their sources.

[0075] [Table 1-1]

[0076] [Table 1-2]

[0077] Synthetic cDNA standard (cfDNA) Synthetic cfDNA standards consisting of short double-stranded DNA fragments (75 bp, 167 bp, 334 bp, and 501 bp) and qPCR primer / probe sets were used for the simultaneous detection and quantification of individual low-copy-number fragments. Each fragment originated from random DNA sequences of appropriate lengths, shared the same primer binding site to facilitate amplification of approximately 75 bp, and contained a unique fragment-specific probe sequence. Each probe possessed a different fluorophore to facilitate the quantification of these standards in multiplexed reactions, either as a single DNA input or as a spike-in to a composite sample such as human-like plasma medium or bovine plasma. These standards were used experimentally to determine the recovery efficiency of short DNA fragments using the desired functional beads.

[0078] Test method DNA binding to beads Functionalized magnetic beads (25 μL) were added to the bottom of an unbound 96-well plate (Greiner Bio-One, Frickenhausen, Germany). Binding buffer (75 μL) and 10 mM citrate buffer at pH 4 were added to the wells and mixed thoroughly. The plate was placed on a plate magnet (Invitrogen, Waltham, MA) for 2 minutes. The liquid was removed, and then DNA (specific DNA shown in each example) and 10 mM citrate buffer at pH 4 were added to the wells. The beads were resuspended by pipetting and then allowed to stand at room temperature (RT) for 10 minutes. After placing on the plate magnet, the residual liquid was collected as the supernatant (also referred to as "super" in the table below). Washing buffer (10 mM citrate buffer at pH 4 (100 μL)) was added to the wells, and the beads were resuspended in the liquid by pipetting. After placing the plate on a magnet, the residual liquid was removed. Elution buffer (100 μL) and 10 mM Tris / 10 mM NaCl pH 8.5 were added, and the beads were resuspended by pipette aspiration. After standing at room temperature for 10 minutes, the beads were separated from the liquid using a plate magnet, and the liquid was collected as the eluate. The separated beads were resuspended as a bead suspension in 100 μL of storage solution (TE buffer). If necessary, the sample was stored at -20°C until further use.

[0079] DNA quantification method (QUBIT) The Qubit assay kit (Qubit dsDNA HS kit, Invitrogen) was used as instructed. All assay components were equilibrated to room temperature. The "working solution" stock solution was prepared by mixing 20 μL of "reagent" with 3980 μL of "buffer," and vortexing slowly to ensure complete mixing. For each sample, 10 μL was mixed with 190 μL of working solution in a Qubit tube and vortexed. Standards were prepared by mixing two 10 μL standards in 190 μL of working solution in a Qubit tube and then vortexing. The tubes were then read individually with a Qubit fluorometer, which reports the concentration of the analyte in the sample. Based on this concentration and the final volume of the sample, the amount of bound and recovered DNA was calculated and reported as a percentage. If the DNA concentration is too low to be detected by the Qubit fluorometer, the DNA% is reported as 0.

[0080] Binding and elution method 1: HindIII digestion and 20% lambda "high MW" DNA: Materials used: DNA mixture: 80% HindIII digest + 20% lambda "high MW" DNA at 100 ng / μl Buffering agent 1 (rinsing, binding, washing): 10 mM citrate pH 4.0 Buffer 2 (elution): 10mM Tris-HCl / 10mM NaCl pH 8.5

[0081] procedure: Immediately after obtaining a homogeneous suspension by vortexing and pipetting, 12.5 μL of bead suspension was transferred to a 1.5 mL tube pre-rinsed with 10 mM citrate buffer pH 4.0 (buffer 1). 87.5 μL of buffer 1 was added and mixed by pipetting to homogenize the mixture. The beads were precipitated by magnetic precipitation for 2 minutes, the liquid was removed, and replaced with 95 μL of buffer 1 and 5 μL of 80% HindIII digest + 20% lambda "high MW" DNA (100 ng / μL). The sample was thoroughly mixed with a pipette and allowed to stand for 10 minutes. The beads were precipitated by magnetic precipitation for 2 minutes, and the liquid was collected as the supernatant. The beads were washed with 100 μL of buffer 1 by mixing with a pipette, followed by magnetic precipitation for 2 minutes. The washing solution was collected, replaced with 100 μL of buffer 2, mixed with a pipette, and allowed to stand for 10 minutes. The beads were precipitated by magnetic field for 2 minutes, and the liquid was collected as the eluate. The beads were suspended for storage by adding 100 μL of TE buffer and then pipetted to suspend the mixture. Control samples were prepared by directly adding 5 μL of DNA to 10 mM citrate buffer pH 4 and to 10 mM Tris-HCl / NaCl pH 8.5. The recovered DNA was quantified using the Qubit method.

[0082] Binding and elution method 2: Artificial human plasma-like medium (HPLM) Samples were prepared in human plasma-like medium by adding the above cfDNA standards at biologically relevant concentrations (approximately 5 ng / ml and 1 × 10¹⁰ copies / ml for each fragment). Binding and elution tests were performed as follows: For each sample to be tested, 790 μL of HPLM and 10 μL of DNA standard were mixed. Then, 100 microliters (100 μL) of 1% Triton® X-100 was added and the tube was vortexed at high speed for 5 seconds. 100 microliters (100 μL) of 1 M sodium acetate pH 4.5 was added and the tube was vortexed at high speed for 5 seconds. The samples were kept on ice. K22 beads (10 mg / ml) were equilibrated with 10 mM sodium acetate pH 4.5 (1:100 v / v beads:sample ratio). 10 mM sodium acetate pH 4.5 (100 μL) was pipetteed into a 1.5 ml microcentrifuge tube. The bead stocks were resuspended by vortexing each for 10 seconds five times. 10 microliters (10 μL) of the bead stock was pipetteed into 10 mM sodium acetate pH 4.5, the tube was vortexed at high speed for 10 seconds, and then allowed to stand at room temperature for 5 minutes. The beads were collected on a magnet for 5 minutes, and the liquid was removed. The above DNA spike HPLM sample (1 ml) was added to the beads, the tube was placed on a vortex adapter, and stirred at medium speed for 10 minutes. The beads were collected on a magnet for 5 minutes, and the supernatant was removed and retained. 100 μL of 10 mM sodium acetate pH 4.5 was added to the beads, and the beads were washed by vortexing, and then the beads were collected on a magnet. The liquid was removed, and the beads were eluted in 20 μL of 10 mM Tris-HCl pH 8.5 / 10 mM NaCl for 10 minutes. The eluate was transferred to a new tube. The beads were resuspended in 100 μL of 10 mM TE preservative buffer. DNA in the sample was quantified using qPCR.

[0083] Binding and elution method 3: RPMI medium Samples were prepared at Roswell Park Memorial Institute (RPMI) 1640 by adding the above cfDNA standards at biologically relevant concentrations (approximately 5 ng / ml and 1 × 10¹⁰ copies / ml for each fragment). For each sample to be tested, 790 μL of RPMI 1640 and 10 μL of DNA standard were mixed. Then, 100 microliters (100 μL) of 1% Triton® X-100 was added and the tube was vortexed at high speed for 5 seconds. 100 microliters (100 μL) of 1 M sodium acetate pH 4.5 was added and the tube was vortexed at high speed for 5 seconds. The samples were kept on ice. K22 beads (10 mg / ml) were equilibrated with 10 mM sodium acetate pH 4.5 (1:100 v / v beads:sample ratio). 10 mM sodium acetate pH 4.5 (100 μL) was pipetteed into a 1.5 ml microcentrifuge tube. The bead stock was resuspended by vortexing each sample five times for 10 seconds. 10 microliters (10 μL) of the bead stock was pipetteed into 10 mM sodium acetate pH 4.5, the tube was vortexed at high speed for 10 seconds, and then allowed to stand at room temperature for 5 minutes. The beads were collected on a magnet for 5 minutes, and the liquid was removed. The above DNA spike RPMI1640 sample (1 ml) was added to the beads, the tube was placed on a vortex adapter, and stirred at medium speed for 10 minutes. The beads were collected on a magnet for 5 minutes, and the supernatant was removed and retained. 100 μL of 10 mM sodium acetate pH 4.5 was added to the beads, and the beads were washed by vortexing, and then the beads were collected on a magnet. The liquid was removed, and the beads were eluted in 20 μL of 10 mM Tris-HCl pH 8.5 / 10 mM NaCl for 10 minutes. The eluate was transferred to a new tube. The beads were resuspended in 100 μL of 10 mM TE buffer. DNA in the sample was quantified using qPCR.

[0084] qPCR method The above synthetic cfDNA standard was placed in a low-binding 1.5 mL microcentrifuge tube (Eppendorf) and 10 ml of molecular biology-grade water. 7The samples were diluted to molecules / μL. These dilutions were combined in equal proportions to prepare 1:10 serial dilutions of the multiplexed samples, facilitating the creation of standard curves for qPCR. Each qPCR reaction product contained a total of 25 μL and included 1X Brilliant III Ultra-Fast qPCR Master Mix (Agilent #600880, Santa Clara, CA, USA), 0.5 μM of each primer, and 0.2 μM of each probe (75 bp CFstd probe, 167 bp CFstd probe, 334 bp CFstd probe, 501 bp CFstd probe). qPCR was performed using an Agilent AriaMx instrument in skirted PCR plates sealed with optically clear strip caps (Agilent #401490 and #401425, Agilent Technologies, Santa Clara, CA) with the following parameters: 40 cycles of 10 minutes at 95°C, 15 seconds at 95°C, and 1 minute at 57°C.

[0085] Zeta potential measurement Zeta potential measurements were performed using a Malvern Zetasizer Nano ZSP (Malvern Panalytical, Malvern, United Kingdom). A 10 mg / mL solution of 2.5 μL of functionalized beads was added to 5 mL of acetic acid buffer (pH=4.5, 10 mM) or Tris buffer (pH=8.5, 10 mM) and loaded into a disposable foldable capillary cell (part number DTS1070). The cell was equilibrated at room temperature for 60 seconds and then analyzed using the Smoluchowski approximation with the Zetasizer instrument software. Three measurements were performed and averaged, with each measurement having a minimum of 10 and a maximum of 100 runs.

[0086] DNA fragment analysis test method DNA solutions were characterized using an automated pulsed-field capillary electrophoresis system, the Femto Pulse System from Agilent Technologies, Inc. (Santa Clara, CA). The system and kit were used as directed. Electrophoretic maps were integrated at the indicated DNA fragment sizes, which are representative of the DNA mixtures used in the examples. Relative peak areas between fragment sizes are reported as %.

[0087] Lambda DNA SYBR qPCR Test Method qPCR standards were prepared by creating a 10-fold dilution of 500 μg / mL lambda DNA (New England Biolabs, #N3011) in molecular-grade water (Invitrogen, #10977015). A 1:100 dilution (5 ng / μL) containing 1.91 × 10⁸ lambda DNA copies was used as a high standard concentration. A total of seven standard dilutions were made, including an untemplated control (NTC), up to 5 femtograms / microliter (fg / μL) (191 copies in 5 μL). All samples from DNA capture treatment (bead suspension and eluate) were diluted 10-fold and 100-fold with molecular-grade water. The qPCR reaction product was prepared using SYBR® Green PCR Master Mix (Thermo Fisher, #4364344) containing a final concentration of 0.625 micromolar (μM) F primer, a 0.625 μM R primer, and 5 μL of sample template or DNA standard. The PCR cycle was as follows: 40 cycles of 10 minutes at 95°C, followed by 15 seconds at 95°C and 1 minute at 60°C. Primers were used to target HindIII 23kb fragments of HindIII-digested lambda DNA (Lambda 1F: CGG CGT CAA AAA GAA CTT CC, Lambda 1 R: CAG TCA ACC ACC AGG GAA TAA) and HindIII 4kb fragments (Lambda 4F: TGG CAT TCT GGA GGG AAA TAC, Lambda 4 R: CAG TCA ACC ACC AGG GAA TAA).

[0088] Preparation example Preparation Example 1 (PE1): MyOne silane beads modified with acrylooxypropyltrimethoxysilane The magnetic beads were completely resuspended by vortexing a stock bottle of Dynabeads MyOne Silane (40 mg / mL) suspension for 1 minute. 0.5 mL of the MyOne Silane suspension was transferred to a 2 mL centrifuge tube and pelletized by magnetism. The aqueous phase was replaced with 2 mL of condensing solvent. A condensing solvent composition for a 95:5 MeOH:H2O (w / w) sample was prepared by adding 95.08 g of MeOH and 5.186 g of DI H2O. The pH was adjusted to 4.5 by adding 17 drops of glacial acetic acid from a glass Pasteur pipette. The beads were completely resuspended by vortexing to obtain a 10 mg / mL suspension. The beads were again pelletized by magnetism, and the solvent was replaced with 2 mL of condensing solvent. 60 μL of 3-acrylooxypropyltrimethoxysilane was added, and the solution was stirred with a tube rotator for 25.5 hours.

[0089] Preparation Example 2 (PE2): Acetyl Passivated SpeedBeads The procedure was adapted from the passivation strategy detailed in U.S. Patent Application Publication No. 20190071662(A1). 2 mL of S-bead solution (100 mg beads) was added to a 50 mL Falcon tube and diluted with 8 mL of water. The beads were isolated magnetically, and the supernatant was discarded. The isolated beads were washed with 2 × 10 mL of DMF, and the supernatant was discarded after each wash. The beads were resuspended in 7.8 mL of DMF, and 2 mL of AAH and 200 μL of DIPEA were added. The tube was shaken at 250 rpm for 30 minutes. The beads were isolated for 10 minutes, and the brown supernatant was discarded. The beads were then washed with 3 × 10 mL of DMF and finally resuspended in 2 mL of DMF for storage.

[0090] Examples Example 1 (EX1): Kryptofix 22 Functionalized Dynabeads MyOne Carboxylic Acid 1.5 mL of Dynabeads MyOne Carboxylic Acid magnetic bead suspension (lot 01158205, 9 μmol) was added to a 2 mL tube (USA Scientific, Ocala, FL, polypropylene), and the bottle was rotated for 30 minutes to resuspend the beads. The beads were isolated by magnetism, and the supernatant was replaced with 1.5 mL of MES buffer (25 mM, pH=6). The mixture was vortexed, equilibrated for 10 minutes, and isolated by magnetism for 2 minutes. The supernatant was again replaced with 1.5 mL of MES buffer (25 mM, pH=6), the sample was vortexed, equilibrated for another 10 minutes, and isolated by magnetism for 2 minutes. 0.0630 g (240 μmol) of K22 and 2 mL of MES buffer (25 mM, pH=6) were added to a 15 mL centrifuge tube and stirred to dissolve. To the isolated magnetic beads, 150 μL (18 μmol) of K22 solution was added, and the suspension was stirred with a rotisserie bar at room temperature for 30 minutes. 344 μL (18 μmol) of freshly prepared EDC solution (10 mg / mL of 25 mM MES buffer) and 6.5 μL of 25 mM MES buffer were added to the sample, and the mixture was stirred with a rotisserie bar at room temperature for 2 hours. During the reaction, the mixture was vortexed every 30 minutes. The functionalized beads were isolated magnetically and washed three times with 800 μL of 50 mM pH 7.5 Tris solution and once with 800 μL of PBS pH 7.4 (resuspended and isolated magnetically). The functionalized beads were finally resuspended in 1500 μL of PBS pH 7.4 buffer and stored in a refrigerator (4°C to 6°C).

[0091] Example 2 (EX2): Homopiperazine-functionalized Dynabeads MyOne Carboxylic Acid Instead of Kryptofix 22, homopiperazine was used and the solution was prepared according to the synthesis method described (Error! Reference source not found). The homopiperazine reagent solution was prepared at 120 μM.

[0092] Example 3 (EX3): 5% Morpholine, K22-functionalized Dyna Beads, MyOne Carboxylic Acid The beads were prepared according to the synthesis method described in (Error! Reference source not found), except that instead of 150 μL of 120 μM K22 in MES, the beads were functionalized with 7.5 μL of 120 μM morpholine in MES and 142.5 μL of K22 in MES.

[0093] Example 4 (EX4): 10% morpholine, K22-functionalized Dynabeads MyOne Carboxylic Acid The beads were prepared according to the synthesis method described in (Error! Reference source not found), except that the beads were functionalized with 15 μL of 120 μM morpholine in MES and 135 μL of K22 in MES instead of 150 μL of 120 μM K22 in MES.

[0094] Example 5 (EX5): 20% morpholine, K22-functionalized Dynabeads MyOne Carboxylic Acid The beads were prepared according to the synthesis method described in (Error! Reference source not found), except that the beads were functionalized with 30 μL of 120 μM morpholine in MES and 120 μL of K22 in MES instead of 150 μL of 120 μM K22 in MES.

[0095] Example 6 (EX6): 40% Morpholine, K22 Functionalized Dynabeads MyOne Carboxylic Acid The beads were prepared according to the synthesis method described in (Error! Reference source not found), except that the beads were functionalized with 60 μL of 120 μM morpholine in MES and 90 μL of K22 in MES instead of 150 μL of 120 μM K22 in MES.

[0096] Example 7 (EX7): 80% morpholine, K22-functionalized Dynabeads MyOne Carboxylic Acid The beads were prepared according to the synthesis method described in (Error! Reference source not found), except that instead of 150 μL of 120 μM K22 in MES, the beads were functionalized with 120 μL of 120 μM morpholine in MES and 30 μL of K22 in MES.

[0097] Example 8 (EX8): Preparation of K22-functionalized Dynabeads MyOne Tosylactivated 150 μL of Dynabeads Tosylactivated suspension was added to a 4 mL glass vial after resuspending by rotating the reagent bottle. A K22 solution, prepared by dissolving 0.0392 g of K22 in 0.5 mL of anhydrous DMF solvent, was added to this vial. The mixture was sonicated at room temperature for 2 minutes to resuspend the beads. The sample was heated to 100°C in an oil bath, and removed every 30–45 minutes for gentle shaking to resuspend the beads. The reaction mixture was heated for a total of 6 hours. After cooling to room temperature, the beads were isolated magnetically, and the reaction solution was replaced with 2 mL of 0.1 M HCl(aq). The beads were resuspended, isolated magnetically, and the solution was again replaced with 1.5 mL of 0.1 M HCl(aq). The solution was stored for approximately 18 hours. The beads were resuspended and transferred to a 2 mL centrifuge tube. The beads were isolated by magnetic means, and the solution was replaced with 1 mL of 50 mM Tris 7.5 pH buffer. The sample was vortexed and resuspended, and the Tris buffer was replaced two more times. The beads were isolated by magnetic means, the Tris solution was replaced with 1 mL of PBS buffer, and the beads were vortexed and resuspended. The beads were isolated by magnetic means, the PBS buffer was replaced with 1.5 mL of PBS buffer, and a 10 mg / mL suspension was obtained after vortexing. The sample was immediately refrigerated for later use.

[0098] Example 9 (EX9): Preparation of K22-functionalized Dynabeads MyOne Silane A suspension of silane-functionalized magnetic beads in 1 mL of methanol was added to a 2 mL centrifuge tube and pelletized by magnetism. To this pellet, 0.5 mL of methanol and 0.050 g of K22 were added to the centrifuge tube, and approximately 0.2 M K22 was added to a 0.5 mL methanol solution prepared by dissolving K22 in 1 mL of methanol. The pellet was resuspended by vortexing and placed in a 50°C oven for 26 hours. The sample was vortexed, followed by magnetic precipitation of the beads, and the reaction mixture was replaced with 1 mL of methanol. The sample was vortexed and resuspended, followed by magnetic precipitation, and the liquid phase was replaced with deionized water. The same procedure was repeated once more with deionized water, and the liquid phase was replaced twice with PBS to obtain a suspension with a concentration of 10 mg / mL. The sample was refrigerated for later use.

[0099] Example 10 (EX10): Preparation of K22-functionalized Dynabeads Epoxy M-270 15.4 mg of Dynabeads M270 Epoxy lyophilized beads were added to a 4 mL glass vial and suspended in 1 mL of anhydrous DMF. In a separate vial, 0.0402 g of K22 was gently dissolved in 0.5 mL of DMF while heating, and cooled to room temperature. The entire K22 solution was added to the bead suspension and sonicated for 1 minute. The capped vial was heated in an oil bath at 100 °C for 4 hours, stirring approximately every 30 minutes. After removal from the oil bath, the bead suspension was precipitated by magnetism, and the solvent was replaced with 1.5 mL of 0.1 M HCl(aq). The beads were then resuspended, precipitated by magnetism, and the solution was again replaced with 1.5 mL of 0.1 M HCl(aq), and stored under these conditions for approximately 18 hours. The beads were resuspended, transferred to a 2 mL centrifuge tube, precipitated by magnetism, and the solution was replaced with 1 mL of 50 mM Tris 7.5 pH buffer. The sample was vortexed to resuspend the beads, and the Tris buffer was replaced two more times. The beads were precipitated by magnetism, the solution was replaced with 1 mL of PBS buffer, and the beads were vortexed to resuspend them. The precipitation method was repeated once more, the solution was replaced with 1.54 mL of PBS buffer, and a 10 mg / mL suspension was obtained after vortexing. The sample was refrigerated immediately.

[0100] Example 11 (EX11) - Preparation of Cyclene-Functionalized S Beads 0.2 mL of passivated S bead stock solution (10 mg, 5.8 μm active functional group) from PE2 was added to a 1.5 mL Eppendorf tube, and the beads were isolated. 300 μL of TEA solution (5.5 mg / mL, 16.2 μmol in DMF) and 229 μL of EDC solution (10 mg / mL, 12 μmol in DMF) were added to the beads and mixed for 5 minutes. 100 μL of cyclenetetrahydrochloride stock solution, prepared at 120 μmol / mL in DMF, was added, and the solution was mixed overnight at room temperature. The beads were isolated and washed with 2 × 500 μL of Tris buffer and 2 × 500 μL of PBS. The beads were left to stand in 1000 μL of PBS (10 mg / mL) and stored at 4°C until further use.

[0101] Example 12 (EX12) - Preparation of triazacyclononane-functionalized S beads 0.2 mL of passivated S bead stock solution (10 mg, 5.8 μm active functional group) from PE2 was added to a 1.5 mL Eppendorf tube, and the beads were isolated. 300 μL of TEA solution (5.5 mg / mL, 16.2 μmol in DMF) and 229 μL of EDC solution (10 mg / mL, 12 μmol in DMF) were added to the beads and mixed for 5 minutes. 100 μL of 1,4,7-triazacyclononane stock solution, prepared at 120 μmol / mL in DMF, was added, and the solution was mixed overnight at room temperature. The beads were isolated and washed with 2 × 500 μL of Tris buffer and 2 × 500 μL of PBS. The beads were left to stand in 1000 μL of PBS (10 mg / mL) and stored at 4°C until further use.

[0102] Comparative Example Comparative Example 1 (CE1) - Dynabeads MyOne Carboxylic acid It was used without any further modifications.

[0103] Comparative Example 2 (CE2)-Dynabeads MyOne Silane It was used without any further modifications.

[0104] Comparative Example 3 (CE3) - Morpholine Functionalized Dynabeads MyOne Carboxylic Acid Instead of Kryptofix 22, morpholine was used, and the solution was prepared according to the synthesis method described (Error! Reference source not found). A morpholine reagent solution was prepared at 120 μM.

[0105] Comparative Example 5 (CE4) - Preparation of hydrolyzed Dynabeads Epoxy M-270 14.9 mg of Dynabeads M27 Epoxy lyophilized beads and 1 mL of 0.1 M HCl (aq) were added to a 4 mL glass vial. The sample was capped and sonicated for 1 minute, after which another 1 mL of 0.1 M HCl (aqueous solution) was added. The sample was stirred by inversion in a secondary container on a jar roller at room temperature for 20.5 hours. The beads were then precipitated by magnetic means, and the aqueous solution was replaced with 1 mL of 50 mM Tris pH 7 buffer. The beads were resuspended by vortexing, precipitated by magnetic means, and the solution was replaced with 1 mL of Tris. The sample was resuspended, transferred to a 2 mL centrifuge tube, and another 1 mL of Tris buffer was added. The sample was vortexed, precipitated by magnetic means, and the solution was replaced with 1 mL of PBS buffer. The sample was vortexed to resuspend the beads, precipitated by magnetic means, and the solution was replaced with 1.490 mL of PBS to obtain a 10 mg / mL suspension after vortexing. The sample was refrigerated immediately afterward.

[0106] Comparative Example 5 (CE5) - Qiagen beads from the Qiagen QIAamp kit Qiagen beads were used without any further modifications.

[0107] Comparative Example 6: (CE6)-Propyltrimethylammonium chloride on Dynabeads MyOne Silane It was prepared using the same method as PE1, but with propyltrimethylammonium chloride trimethoxysilane in methanol instead of 3-acrylooxypropyltrimethoxysilane.

[0108] Comparative Example 7 (CE7) - S Beads The S beads were used without any further modifications.

[0109] [Table 2]

[0110] [Table 3]

[0111] [Table 4]

[0112] [Table 5] * ND = Not detected

[0113] [Table 6] * ND = Not detected

[0114] Tables 5A and 5B report the amount of unbound DNA (supernatant), the amount of DNA that remained bound after elution (beads), and the amount of eluted bound DNA.

[0115] [Table 7]

[0116] [Table 8]

[0117] [Table 9] Higher values ​​than the initial values ​​indicate concentration.

[0118] [Table 10]

[0119] [Table 11]

Claims

1. A method for processing polynucleotides, a) To provide a solid support containing a ligand, wherein at least a portion of the ligand contains a cyclic amine group having more than six ring members, b) Exposing the solid support to polynucleic acid molecules in a buffer having a pH of less than 5.5, thereby binding at least a portion of the polynucleic acid molecules to the ligand, c) Exposing the solid support having the bound polynucleic acid molecules to a pH greater than 6 to release a portion of the bound polynucleic acid molecules from the ligands of the solid support, and optionally retaining a portion of the bound polynucleic acid molecules on the solid support. d) Optionally, wash the solid support containing the bound and / or retained bound polynucleic acid molecules, e) Optionally, prepare a suspension from the solid support containing the retained bound polynucleic acid molecules, f) The released portion of the polynucleic acid molecule bound to c), and / or A method comprising using a retained portion or suspension thereof of a polynucleic acid molecule bound to the solid support.

2. The method according to claim 1, wherein the cyclic amine group comprises at least 7, 8, 10, 12, 14, 16, or 18 members.

3. The method according to claim 1 or 2, wherein the cyclic amine group comprises at least two nitrogen atoms.

4. The method according to any one of claims 1 to 3, wherein the cyclic amine group comprises at least two heteroatoms selected from nitrogen and oxygen.

5. The method according to claim 4, wherein the cyclic amine group comprises at least two, three, or four oxygen atoms.

6. The method according to any one of claims 1 to 5, wherein the ligand is a reaction product of a cyclic amine compound and a functional group on the surface of the solid support, and the functional group is selected from an acidic group or a salt thereof, an epoxy group, a (meth)acrylic group, and p-toluenesulfonyl.

7. The method according to any one of claims 1 to 6, wherein the cyclic amine group comprises at least one amide group.

8. The method according to any one of claims 1 to 7, wherein the cyclic amine group further comprises at least one ether moiety.

9. The method according to claim 8, wherein the amine compound is a homopiperazine or an azacrown compound containing an azacrown ether.

10. The method according to any one of claims 1 to 9, wherein the solid support further comprises a second different ligand comprising an amine group, an ether group, or a combination thereof.

11. The method according to claim 10, wherein the second different ligand is annular.

12. The method according to claim 10 or 11, wherein the second different ligand is morpholine.

13. The method according to any one of claims 10 to 12, wherein the second different ligand is present in an amount of less than 50% by weight based on the total amount of ligands.

14. The method according to any one of claims 1 to 13, wherein the solid support is particles, magnetic particles, or non-magnetic particles.

15. The method according to any one of claims 1 to 14, further comprising washing the solid support with a buffer having a first pH after step b).

16. The method according to any one of claims 1 to 15, wherein f) utilizes the released portion of the bound polynucleic acid molecule.

17. The method according to any one of claims 1 to 16, wherein the use includes size separation, purification, quantification, and amplification.

18. The method according to any one of claims 1 to 17, wherein the polynucleic acid molecule is cell-free DNA.

19. The method according to claim 18, wherein at least 70, 80, or 90% of the 167 bp or 334 bp fragments are released during step c).

20. Magnetic particles comprising ligands bonded to magnetic particles, wherein at least a portion of the ligands comprises a cyclic amine group having more than six ring members.

21. The magnetic particle according to claim 20, wherein the cyclic amine group is an ionizable group.

22. The magnetic particle according to claim 20 or 21, wherein the solid support containing the ligand has a negative or positive zeta potential at pH 4.

5.

23. The magnetic particle according to any one of claims 20 to 22, wherein the solid support containing the ligand has a zeta potential lower at pH 8.5 than at pH 4.

5.

24. Magnetic particles according to any one of claims 20 to 23, wherein the difference between the zeta potential at pH 4.5 and the zeta potential at pH 8.5 is at least 10, 20, 30, 40, or 50 mV.

25. A solid support comprising a ligand bonded to the solid support, wherein at least a portion of the ligand comprises a cyclic amine group having more than six ring members.

26. A solid support or magnetic particle according to claims 20 to 25, further characterized by claims 2 to 14.

27. A kit comprising a solid support or magnetic beads according to claims 20 to 26, and a buffer having a pH of less than 5.

5.

28. The kit according to claim 27, further comprising a buffer having a pH greater than 6.

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