EP4695390A1 - Solid support comprising cyclic amine ligands suitable for polynucleic acid processing, articles and methods - Google Patents

Abstract

A method of processing polynucleic acids is described comprising providing a solid support comprising ligands, wherein at least a portion of the ligands comprise a cyclic amine group comprising greater than 6 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 with 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 polynucleic acid molecules bound to the solid support; and utilizing the released portion of bound polynucleic acid molecules and/or the retained portion of the polynucleic acid molecules bound to the solid support or the suspension thereof. Also described are solid supports (e.g. magnetic beads) comprising ligands bound to the solid support; wherein at least a portion of the ligands comprise a cyclic amine group comprising greater than 6 members; and kits comprising such solid supports.

Description

SOLID SUPPORT COMPRISING CYCLIC AMINE LIGANDS SUITABLE FOR POLYNUCLEIC ACID PROCESSING, ARTICLES AND METHODS

Summary

In one embodiment, a method of processing polynucleic acids is described comprising: a) providing a solid support comprising ligands, wherein at least a portion of the ligands comprise a cyclic amine group comprising greater than 6 members; b) 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; c) exposing the solid support with 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 polynucleic acid molecules bound to the solid support; d) optionally washing the solid support comprising the bound and/or retained bound polynucleic acid molecules; e) optionally preparing a suspension from the solid support comprising the retained bound polynucleic acid molecules; f) utilizing the released portion of bound polynucleic acid molecules of c), and/or the retained portion of the polynucleic acid molecules bound to the solid support or the suspension thereof.

In another embodiment, a solid support (e.g. magnetic beads) are described comprising ligands bound to the solid support; wherein at least a portion of the ligands comprise a cyclic amine group comprising greater than 6 members.

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

Detailed Description

The methods and articles described herein comprise a solid support. The surface of the solid support comprises ligands. The ligands can reversibly bond or otherwise interact with the polynucleic acids, including for example ionic/electrostatic interactions, hydrogen-bonding interactions, hydrophobic interactions, and combinations thereof.

Various solid supports have been described in the literature. The solid support is typically comprised of organic polymers (e.g. plastics) or inorganic materials or combinations of both. Examples of suitable solid support materials include metal oxides such as A1₂O₃, TiO₂, ZrO₂, Ta₂O₃; as well as silica materials such as SiO₂ and polysilicic acid. The solid supports can be magnetic materials such as iron, cobalt or nickel and oxides, alloys, ceramics or amalgams thereof. Suitable organic polymers include polystyrene, poly(meth)acryl polymers including poly(meth)acrylates and poly(meth)acrylamides, polyurethanes, polyamides such as nylon; polyolefins, such as polyethylene, polypropylene, polybutadiene, and copolymers thereof. Other solid support materials include polysaccharides, and in particular hydrogels such as agarose, cellulose, dextran, SEPHADEX®, SEPHACRYL®, and chitosan. Inorganic supports include, for example, glass or metal surfaces such as gold. In some embodiments, the ligands described herein (e.g. covalently) bond to the solid support material. In some embodiments, the solid support is not gold nanoparticles.

Various (e.g. magnetic and non-magnetic) particles can be utilized as a solid support. In some embodiments, the particles have a mean particle size of at least 0.5 or 1 micron. In some embodiments, the particles typically have a mean particle size no greater than 500, 250, 100, 75, 50, 25, 15, 10, or 5 microns. Although the particles are typically spherical, other shaped particles can also be utilized.

In some embodiments, the solid support is a plurality of particles including magnetic beads and in particular paramagnetic beads. In typical embodiments, the (e.g. ionizable) ligand is formed by (e.g. covalently) bonding cyclic amine compounds, as described herein, to (para)magnetic particles (also referred to as beads) comprising functional groups on the surface of the particles.

A representative reaction scheme is as follows: wherein X is a functional group on the surface of the support that reacts with the cyclic amine compound and Y is the reaction product of X and the cyclic amine compound.

It is appreciated that an organic linking group (e.g. alkylene) may be present between the solid support (e.g. magnetic particles) and the functional group X. In some embodiments, the cyclic amine ligand is bonded to the solid support (e.g. magnetic particles) with an organic linking group that lacks a sulfur moiety.

In some embodiments, solid supports comprising carboxylic acid groups or salts thereof (e.g. carboxylate groups) on the surface are commercially available. Some commercially available beads are described in the forthcoming examples. Gold nanoparticles (10 nm carboxylic acid functionalized polyethylene glycol 3000 g/mole) are commercially available from MilliporeSigma (Product No. 765457). Other commercially available products are carboxyl multiwell plates (Corning® PureCoat™ Carboxyl plate), carboxyl modified polystyrene (Polybead® Carboxylate Microspheres), carboxyterminated biosensor surface (Octet® Amine Reactive 2nd-Generation Biosensors), carboxylic acid silica gels (SiliaBond Carboxylic Acid (WCX), product number R70030B), and carboxylate polystyrene monodisperse microspheres, commercially available from Polysciences as Polybead® Carboxylate Sampler Kit. As described for other amine compounds in US2019/0071662, the carboxylic acid groups or salts thereof on the surface of the solid support (e.g. particles) covalently bond with an amine group of the cyclic amine compound forming an amide linking group. An illustrative reaction scheme is as follows:

An amide group can also be the reaction product of an amine and an ester. In yet other embodiments, a ligand with an amide group may be the reaction product of an amine and an acid halide.

Although the above solid support particle is depicted as having a single ligand, it is appreciated that the solid supports (e.g. each particle) comprises a plurality of ligands. For example, the solid support particles utilized in the examples are surmised to have approximately 0.6 mmol of carboxylic acid or carboxylate groups per gram of particles. Thus, if all such carboxylic acid or carboxylate groups were reacted with an amine compound as described herein, the number of ligands would be about equal to the number of carboxylic acid or carboxylate groups. When the particle is 1 um in diameter on average, the particles may contain up to approximately 1.8 xlOe-4 picomoles of ligand/pm². When the particle comprises a second ligand or unreacted carboxylic acid/carboxylate groups, the particles may contain lower amounts of ligand per surface area of the particle.

Cyclic amine compound can also react with other functional groups on the surface of the support (e.g. magnetic beads).

In some embodiments, the functional groups on the surface of the support are p-toluene-sulfonate (i.e. Tosylate) groups. Magnetic beads with surface p-toluene-sulfonate (i.e. Tosylate) groups are available from Thermo Fisher Scientific Inc obtained under the trade name “DYNABEADS MYONE TOSYLACTIVATED”, product number 65502.

In some embodiments, the functional groups on the surface of the support are epoxy groups. Magnetic beads with surface epoxy groups are available from Thermo Fisher Scientific Inc obtained under the trade name “DYNABEADS M-270 EPOXY”, product number 14301. The amine group can react, ring-opening an epoxy group, thereby covalently bonding the cyclic amine group to the support (e.g. magnetic beads).

In other embodiments, the functional groups on the surface of the support are (meth)acryloxy groups. The amine group can react with a (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) comprising silanol groups are reacted via condensation with a (meth)acryloxy silane compound (such as acryloxypropyltrimethoxysilane) forming a (meth)acryloxy terminal group. The linking group (L) between the support (e.g. magnetic beads) and (meth)acryloxy terminal group comprises the condensation reaction product of the silanol with the alkoxy silane. Magnetic beads with surface silanol groups are available from Thermo Fisher Scientific Inc obtained under the trade name “DYNABEADS MYONE SILANE”, product number 37002D.

Other supports comprising functional groups that can react with a cyclic amine compound are commercially available or described in the literature.

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

The functional groups on the surface of the solid support (e.g. particles) are reacted with a cyclic amine compound comprising greater than 6 members. The resulting ligand is a cyclic amine group comprising greater than 6 members. In some embodiments, the cyclic group of the ligand bonded to the support may comprise one less amine group than the cyclic amine compound. The cyclic amine compound and cyclic amine group comprises at least 7, 8, 9, 10, 12, 14, 15, 16, or 18 members. The total number of members of the heterocycle is typically no greater than 24. In some embodiments, the cyclic amine compound comprises at least two nitrogen atoms. In some embodiments, the cyclic amine comprises at least 2, 3, or 4 amine groups. The total number of amine groups is typically no greater than 4, 5, 6, 7 or 8. At least one of the amine groups is reacted with a functional group of the solid support. In some embodiments, the cyclic amine compound/group comprises at least 2 heteroatoms selected from nitrogen and oxygen. In some embodiments, the cyclic amine compound/group comprises at least 2, 3, or 4 oxygen atoms (e.g. ether groups). The total number of oxygen atoms (e.g. ether groups) is typically no greater than 4. Some representative cyclic amine compounds are depicted as follows.

Various other cyclic amine compounds having greater than 6 members have been described in the literature. See for example W092/05804; incorporated herein by reference.

The cyclic amine compounds and cyclic amine group of the ligand may optionally further comprise substituents provided the presence thereof does not substantially detract from the binding and/or release of the polynucleic acid molecules at different pHs.

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

The cyclic amine compounds and cyclic amine group of the ligand typically does not comprise immobilized metal ions, such as in the case of metal chelates.

In some embodiments, the cyclic amine group lacks carboxylic acid or carboxylate functional groups.

In typical embodiments, a single cyclic amine group having greater than 6 members is bonded to the solid support (e.g. magnetic particles). Thus, the cyclic amine group having greater than 6 members is not a polymerized unit of a polymer such as a polyamide, poly(organo)phosphazene, or poly aery lie acid (e.g. superabsorbent) polymer.

In some embodiments, the surface of the solid support (e.g. particles) is subject to passivation prior to reaction with the cyclic amine compounds. Passivation involves reacting surface functionality present on the solid support capable of positive ionization in aqueous buffer with a chemistry that prevents such ionization. In some embodiments, the solid supports are passivated with acetic anhydride.

Without intending to be bound by theory, it is surmised that the amine, oxygen, and/or amide linking group may be involved in binding nucleic acids. With reference to Table 2 of the forthcoming examples, when cyclic amine compounds having greater than 6 members was reacted with the functional group (e.g. carboxylic acid groups or salts) thereof on the surface of a solid support (e.g. particles), the resulting solid support (e.g. particles) had a greater zeta potential at a pH of 4.5 than at a pH of 8.5. This change in zeta potential is indicative of a change in the electrical potential near the electrical double layer. The electrical double layer includes any unreacted negative carboxylic acid/carboxylate surface charge of the particle and (e.g. positive) counterions in the solution that associate with the surface of the particle. Such change in electrical potential likely contributes to the ability of the ligand to bind and release DNA which can be beneficial for subsequent processing including size selection, amplification or modification of the DNA.

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

In other embodiments, the ligand further comprises an ionizable amine group. For example, when a cyclic amine compound comprising at least two amine groups is reacted with the carboxylic acid groups or salts thereof on the surface of the solid support (e.g. particles), one of the amine groups forms an amide linkage and the other amine group of the ligand can reversibly ionize. The above cyclic amine compounds and ligands formed from such compounds each comprise a secondary amine or a tertiary amine (when substituents are present). It has been described in the literature that amine groups function as ionizable groups.

In some embodiments, the solid support (e.g. particles) comprising functional groups is reacted with a single cyclic amine compound comprising greater than 6 members, as described above, forming a ligand. In other embodiments, the solid support (e.g. particles) is reacted with at least two different amine compounds, forming a first and second ligand.

In some embodiments, the first and second ligands are both cyclic amine compounds having more than 6 members as described herein.

In other embodiments, the second ligand is a cyclic amine compound with 6 members or less than 6 members in the heterocycle. In some embodiments, the cyclic amine compound further comprise an ether group, such as in the case of morpholine. With reference to Table 9, supports that further comprised morpholine as a second ligand had higher eluate concentrations of smaller cell free DNA with sizes ranging from about 75 to 500 base pairs as compared to the first ligand alone. In some embodiments, the eluate 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.

In other embodiments, the second different ligand is piperazine or a derivative thereof, such as described in PCT/IB2022/060641 and PCT/IB2022/060640; incorporated herein by reference. Representative compounds include for example N-methyl piperazine, N-phenyl piperazine, N-(2- hydroxyethyl)piperazine, N-(4-methoxyphenyl)piperazine, N-(4-trifluoromethylphenyl)piperazine, and 1- (4-bromophenyl)piperzine. In yet other embodiments, the second ligand is a fluorinated amine (e.g. monoamine or diamine. Various other ligands for processing polynucleic acids have been described in the art.

In some embodiments, the second amine compound may not function to bind polynucleic acids, but functions to simply lower the concentration of the first ligand.

The molar ratio of the first amine compound to the second amine compound typically ranges from 1 : 10 to 10:1. Likewise, the molar ratio of first ligand to second ligand ranges from 1 : 10 to 10: 1. In some embodiments, the molar ratio of the first amine compound or first ligand to second amine compound or ligand is at least 2:10, 3:10, 4:10, 5:10, 6:10, 7:10, 8:10, 9:10. In some embodiments, the molar ratio of the first amine compound or first ligand to second amine compound or ligand is no greater than 9:10, 8:10, 7:10, 6:10, 5:10, 4:10, 3:10, 2:10.

The solid support (e.g. particles) comprising the described ligands and kits can be utilized in a variety of polynucleic acid processing techniques including for example size separation, purification, quantification, amplification, tagmentation, digestion and preparation of libraries (e.g. for nucleic acid sequencing).

The method of processing polynucleic acid typically comprises a) providing a solid support comprising ligands, wherein at least a portion of the ligands comprise a cyclic amine group comprising greater than 6 members, as described herein. The method further comprises b) exposing the solid support to polynucleic acid molecules (e.g. in a buffer) at a first pH to bind at least a portion of the polynucleic acid molecules to the ligands. The method typically further comprises c) exposing the solid support with 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 comprises retaining a portion of the polynucleic acid molecules bound to the solid support. In some embodiments, the method comprises washing the solid support comprising the bound and/or retained bound polynucleic acid molecules. In some embodiments, the method comprises preparing a suspension from the solid support comprising the retained bound polynucleic acid molecules. The method comprises utilizing the released portion of bound polynucleic acid molecules (e.g. the eluate) and/or the retained portion of the polynucleic acid molecules bound to the solid support or the suspension thereof.

With reference to 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 no greater than 5.5, 5, 4.5, 4, or 3.5. 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).

With reference to step c), releasing of polynucleic acids 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 no greater than 11, 10.5, 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, or 6. In some embodiments, the solid support is exposed to polynucleic acid molecules in a buffer having a high pH for 10 minutes at ambient temperature (e.g. 25°C).

Various low and high pH biological buffers for use in the method and kit are known.

Some suitable buffers include for example citrate buffer (sodium citrate and citric acid monohydrate), acetate buffer, and TE buffer as further described in the examples; phosphate-buff ered saline (PBS); N-2-acetamido-2-aminoethanesulfonic acid (ACES); N-2-acetamido-2-iminodiacetic acid (ADA); amino methyl propanediol (AMP); 3-l,l-dimethyl-2-hydroxyethylamino-2-hydroxy propanesulfonic acid (AMPSO); N,N-bis2-hydroxyethyl-2-aminoethanesulfonic acid (BES); N,N-bis-2- hydroxyethylglycine (BICINE); bis-2-hydroxyethyliminotrishydroxymethylmethane (Bis-Tris); 1,3- bistrishydroxymethylmethylaminopropane (BIS-TRIS Propane); 4-cyclohexylamino-l -butane sulfonic acid (CABS); 3 -cyclohexylamino- 1 -propane sulfonic acid (CAPS); 3-cyclohexylamino-2-hydroxy-l- propane sulfonic 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-N-N-bis-2-ethanesulfonic acid (PIPES); piperazine-N-N-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); N-trishydroxymethyl- methyl-2-aminoethanesulfonic acid (TES); N-trishydroxymethylmethylglycine (TRICINE); trishydroxymethylaminomethane (TRIS); histidine and polyhistidine; imidazole and derivatives thereof; triethanolamine dimers, oligomers and polymers; and di/tri/oligo amino acids, for example Gly-Gly; and Ser-Ser, Gly-Gly-Gly, and Ser-Gly.

In some embodiments, the low pH buffer is citrate buffer (sodium citrate and citric acid monohydrate) or acetate buffer. In some embodiments, the high pH buffer is PBS or TRIS.

The method may optionally comprise one or more washing steps. In one embodiment, the method comprises washing the solid support comprising the bound polynucleic acid molecules after step b). This washing step typically utilizes a low pH buffer. In some embodiments, this wash step utilizes the same buffer as step b). In another embodiment, the method comprises washing the solid support comprising the retained polynucleic acid molecules after step c). This washing step typically utilizes a high pH. Illustrative suspension buffers, also described as storage buffers, include PBS and TE buffer. In some embodiments, the suspension buffer has a pH of about 8. Neutral pH buffers, or water, can also be used.

The buffers generally have an ion 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. A common suitable salt for use in (e.g. (poly)nucleic acid capture) buffers is sodium chloride. In one embodiment, the buffer has a pH greater than 6 and a salt concentration of less than IM, 500 mM, 250 mM, 100 mM, 50 mM, 25 mM or 10 mM.

In some embodiments, at least some of the method steps may be performed with a kit. The kit typically comprises the solid support comprising ligands and a low pH buffer (e.g. having a pH of less than 5.5), as previously described. In some embodiments, the kit further comprises a high pH buffer, as previously described suitable for releasing a portion of polynucleic acid molecules. In some embodiments, the kits further comprise a wash and/or suspension buffer, as previously described.

A variety of polynucleic acids can be processed using the solid supports comprising ligands, methods, and kits described herein. In some embodiments, the polynucleic acids comprise at least 100, 200, 300, 400 or 500 base pairs. In some embodiments, the polynucleic acids comprise 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 acids comprise 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 acids comprise 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 greater. In some embodiments, the polynucleic acid comprises a distribution of sizes having a minimum and maximum defined by an interval of the number of base pairs just described.

In some embodiments, the following performance criteria described herein were obtained with respect to a DNA standard or a mixture of standards, as described in greater detail in the forthcoming examples. Representative commercially available DNA standards include for example I DNA ( i.e. duplex DNA isolated from bacteriophage lambda that is 48,502 base pairs in length), I DNA-Hindlll digest (i.e. DNA isolated from bacteriophage lambda digested with the restriction endonuclease Hindlll to produce 8 DNA fragments of sizes ranging from 125 bp to 23,130 bp), cell free DNA (“cfDNA’) (double-stranded DNA fragments 75 bp, 167 bp, 334 bp, and 501 bp).

Unless stated otherwise, the processing of a DNA standard was conducted at a weight ratio of polynucleic acids (e.g. DNA):solid support (e.g. particles) of 1:250 w/w. Other ratios are also suitable. For example, the weight ratio of polynucleic acids (e.g. DNA):solid support (e.g. particles) may range from 1:10 to 1:2500. In some embodiments, the weight ratio of polynucleic acids (e.g. DNA):solid support (e.g. particles) is at least 1:25, 1:50, 1:100, 1:150, or 1:200. In some embodiments, the weight ratio of polynucleic acids (e.g. DNA):solid support (e.g. particles) is no greater than 1:2500; 1:2000; 1:1500, 1:1000, or 1:500. In some embodiments, the size and distribution of the polynucleic acids is known, such as in the case of the standards. In other embodiments, the size and distribution of the polynucleic acids can be determined with methods known in the art, such as pulsed-field gel electrophoresis or a Qubit fluorometer.

Unless specified otherwise, the following terms are defined as follows:

“Supernatant” refers to the solution left behind after polynucleic acid molecules (e.g. DNA) are bound to the solid support (e.g. particles). Thus, characterization of supernatant pertains to the polynucleic acid molecules (e.g. DNA) that don’t bind; Percent bound polynucleic acid molecules (e.g. DNA) can be calculated according to the formula 100 x (Initial DNA ng - Supernatant DNA ng)/Initial DNA ng.

“Eluate” refers to the solution of polynucleic acids molecules (e.g. DNA) initially bound to the beads, but released when exposed to a higher pH buffer for 10 minutes at room temperature;

“Solid support (e.g. particle) suspension” refers to providing the solid support (e.g. particles) in an aqueous liquid after separation of eluate containing the released polynucleic acid molecules (e.g. DNA). The aqueous liquid may be characterized as a carrier liquid that conveys the solid support (e.g. particles) with the bound polynucleic acids to subsequent processing and analysis steps. Some of the polynucleic acids 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 acids bond 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 polynucelic acids released into the aqueous liquid of the suspension may be less than 10, 5, or 1 wt.% as compared to the total amount of polynucelic acids of the suspension (i.e. the sum of polynucleic acids bound to the solid support and released into the aqueous liquid of the suspension).

In some embodiments, the solid support (e.g. particles), methods, and kits comprising the described ligands can be utilized to process (e.g. bind) polynucleic acids (e.g. DNA) ranging in size from 100 to 50,000 base pairs, such as I DNA and I DNA-Hindlll Digest mix in an 80:20 v:v ratio or cell free DNA, as described above.

In some embodiments, the DNA of the supernatant and eluate were quantified. The bound DNA was calculated as described above.

With reference to Tables 3 and 4, in some embodiments, the unbound polynucleic acid molecules of the supernatant have a mass of polynucleic acid molecules less than 80, 70, 60, 50, 40, 30, or 20% of the total initial polynucleic acid molecules. In some embodiments, the retained bound polynucleic acid molecules have a mass of polynucleic acid molecules greater than 50, 60, 70, 80, 90% of the total initial polynucleic acid molecules. In some embodiments, the mass of released polynucleic acid molecules is greater than 10, 20, 30, 40, 50, 60, 70, or 80% of the total initial polynucleic acid molecules. In some embodiments, the solid support comprises a greater amount of retained bound polynucleic acid molecules than released polynucleic acid molecules. In contrast, when the solid support (e.g. particles) comprised carboxylic acid/carboxylate (i.e. CE1) in the absence of the described ligands, the amount of bound polynucleic acid was 4% and no polynucleic acids were released. Further, when the solid support (e.g. particles) comprised morpholine ligands (i.e. CE3), the amount of bound polynucleic acid was 27% and 12% polynucleic acids were released.

Tables 11 and 12 demonstrate that high amount of polynucleic acids can be bound and released for cyclic amines lacking one or more ether moieties.

In other embodiments, after steps a-c), the method further comprises quantifying the size and distribution of the polynucleic acids (e.g. DNA) fragment sizes with methods known in the art, such as pulsed-field gel electrophoresis. With reference to Table 8, the initial DNA (e.g. of the standard) contains a specific concentration of certain DNA fragments ranging in size from 125 base pairs to 48,505 base pairs. Notably, a greater amount of DNA of a particular size fragment in the eluate as compared to the initial DNA, is indicative of enrichment for that DNA fragment size. Notably, the amount of DNA in the eluate for the fragment sizes of 125, 564, 2027, 2322, 4361, 6557, and 9416 or in other words fragments in a size range from 125 to about 10,000 were enriched.

These results indicate that the steps of binding and releasing polynucleic acid molecules is amenable for size selection or size separation of polynucleic acids, especially higher molecular weight fragments. The ability to perform size selection by eluting a biased distribution of molecular weight fragments while still being able to utilize both populations (bound and eluted nucleic acid) was unexpected and beneficial because all of the nucleic molecules, whether they were eluted or not, are now accessible for utilization. This enables size selection with direct utilization of both populations without buffer exchange or desalting. Both populations can be utilized in the same way or taken on to different process steps. This also maximizes the usable and accessible nucleic acids after size selection because both populations can be utilized.

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

With reference to Table 10, high amounts of 167 bp and 334 bp fragments can be bound and released. After elution, the amount remaining on the beads was 10% or less.

In some embodiments, the solid support (e.g. particles), methods, and kits comprising the described ligands can be utilized for amplifying polynucleic acids. Numerous techniques are available for amplifying nucleic acids. These techniques include polymerase chain reaction (PCR), ligase chain reaction (LCR), self-sustained sequence replication (3SR), nucleic-acid-sequence-based amplification (NASBA), strand displacement amplification (SDA), transcription-mediated isothermal CR cycling probe technology, cascade rolling circle amplification (CRCA), nicking endonuclease amplification reaction (NEAR), transcription mediated amplification (TMA), loop- mediated isothermal amplification (LAMP), helicase-dependent amplification (HD A), CRISPR-Cas- based amplification, and in vitro transcription (IVT).

PCR (Polymerase Chain Reaction) is a method for amplifying a target DNA sequence using a heat-stable 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. Because the newly synthesized DNA strands can subsequently serve as additional templates for the same primer sequences, successive rounds of primer annealing, strand elongation, and dissociation produce rapid and highly specific amplification of the desired sequence.

Rolling Circle Amplification (RCA) is an amplification process driven by a DNA polymerase which can replicate with either linear or geometric kinetics under isothermal (single temperature) conditions. In the presence of two suitably designed primers, a geometric amplification occurs via DNA strand displacement and hyperbranching to generate 10¹² or more copies of DNA template in 1 hour.

With reference to Tables 6 and 7, lower Ct values demonstrate greater (e.g. PCR) amplification. Notably, a difference of 6 in a Ct value is generally equivalent to a 100-fold difference in the amount of the target polynucleic acid. Thus, even small reductions in Ct values are of significance. In some embodiments, the reduction in Ct values is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 as compared to buffer lacking the functionalized support (e.g. beads) with cyclic amine ligands. Reduction in Ct values are evident in the eluate as well as the bead suspension at dilutions of 1:10 and 1:100. Thus, the solid support (e.g. particles), methods, and kits comprising the described ligands are advantageous for amplifying higher molecular weight fragments, such as the 23 kb Hindlll digest fragment of X DNA and for amplifying lower molecular weight fragments, such as the 4 kb -Hindlll digest fragment of X DNA.

The solid support (e.g. particles) comprising the described ligands, methods, and kits can be utilized for library preparation for nucleic acid sequencing. A sequencing library is a collection of DNA fragments that have been modified with nucleic acid adaptors into a format that is compatible with the sequencing technology and instrument under use. Each sequencing instrument has its own library preparation workflows to add the necessary barcodes and adaptors and modify the fragments to enable sequencing. Libraries can be made for high-throughput sequencing instruments, which rely on methods such as sequencing by synthesis, sequencing by ligation, sequencing by binding, pyrosequencing or impedance-based sequencing, or real-time long-read instruments which rely on methods such as sequencing by synthesis in zero-mode waveguide or nanopore sequencing. In some embodiments, this sequencing includes tagmentation and enzymatic reactions to fragment the DNA. In some embodiments, the enzyme may be bonded to a bead such as exemplified by bead-linked transposomes of the “Illumina DNA Prep” reference guide.

Utilizing the released and/or retained and/or suspended polynucleic acid molecules may comprise processing or analyzing the polynucleic acids including for example size separation, purification, quantification, detection or modification (e.g. via exposure to at least one enzyme) such as amplification, transcription, tagmentation, digestion, ligation, or preparation of libraries (e.g. for nucleic acid sequencing).

In view of the favorable test results obtained by testing of DNA standard, it is surmised that the solid support (e.g. particles) comprising the described ligands, methods, and kits can be utilized 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 or otherwise wholly or partially disrupted. In some embodiments, the solid support (e.g. particles) comprising the described ligands, methods, and kits can be utilized for testing for target DNA, such as from microbes, or samples of food.

Various polynucleic acid molecules can be processed including bacteria and those obtained from microbes, fungi, plants, or animals. A biological sample comprising polynucleic acids can be a naturally- occurring sample or deliberately designed or synthesized sample or library. In one embodiment, the sample contains a population of cells or cell fragments, including without limitation cell membrane components, exosomes, and sub-cellular components. The cells may be a homogenous population of cells, such as isolated cells of a particular type, or a mixture of different cell types, such as from a biological fluid or tissue of a human or mammalian or other species subject. The biological sample can be simple, for example containing isolated DNA or deriving from a homogeneous cell culture or tissue source, or can be complex, such as deriving from tumor, blood, or whole organ samples. The biological sample can be from any suitable source, such as a healthy tissue or cell source, a diseased tissue or cell source, a cell culture or line, cell extracts or lysates, a biopsy, and the like.

Other biological samples comprising 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 fluids, mucosal secretions (e.g., buccal, vaginal, or rectal). Still other samples include a blood-derived or biopsy-derived biological sample of tissue or a cell lysate (i.e., a mixture derived from tissue and/or cells). Other suitable tissues include hair, fingernails, and the like. Additional samples include libraries of antibodies, antibody fragments and antibody mimetics like affibodies. Other samples can be synthesized or engineered collections of chemical molecules, proteins, antibodies or any other of the polyanions described herein.

EXAMPLES

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

Synthetic cDNA standards ( cfDNA )

A synthetic cfDNA standard comprised of short double-stranded DNA fragments (75 bp, 167 bp, 334 bp, and 501 bp) and qPCR primers/probe sets were utilized for the simultaneous detection and quantitation of each individual fragment in low copy number. Each fragment was derived from a different random DNA sequence of appropriate length, shared identical primer binding sights to facilitate amplification of ~75 bp and contained unique fragment-specific probe sequences. Each probe had a different fluorophore that facilitated the quantitation of these standards in a multiplexed reaction, either as the only DNA input or as a spike-in for complex samples such as human-like plasma medium or bovine plasma. These standards were used in experiments to determine the efficiency of recovery short DNA fragments with functional beads of interest.

Test Methods

BINDING OF DNA ONTO BEADS Functionalized magnetic beads (25 pL) were added to the bottom of a non-binding 96-well plate (Greiner Bio-One, Frickenhausen, Germany). The binding buffer (75 pL), 10 mM citrate buffer at pH 4, was added to the well and mixed thoroughly. The plate was placed on a plate magnet (Invitrogen, Waltham, MA) for 2 min. The liquid was removed, then DNA (specific DNA indicated in each example) and 10 mM citrate pH 4 buffer was added to the well. The beads were resuspended by aspirating with a pipette and then left to sit at room temperature (RT) for 10 min. After placing on a plate magnet, the residual liquid was collected as the supernatant (also abbeviated “super” in tables below). Wash buffer (citrate buffer pH 4 at 10 mM (100 pL)) was added to the wells and the beads were resuspended in the liquid by aspirating with a pipette. After placing the plate on the magnet, the residual liquid was removed. The elution buffer (100 pL), 10 mM Tris/10 mM NaCl pH 8.5, was added and the beads were resuspended by pipette aspiration. After letting sit at RT for 10 min, beads were separated from the liquid using the plate magnet and the liquid was collected as eluate. Separated beads were resuspended in 100 pL storage solution (TE buffer) as bead suspension. If needed, samples were stored at -20 °C until further use.

DNA QUANTIFICATION METHOD (QUBIT)

Qubit assay kits (Qubit dsDNA HS kit, Invitrogen) were used as instructed. All assay components were equilibrated to room temperature. A stock solution of “working solution” was prepared by mixing 20 pL of “reagent” with 3980 pL of “buffer” and vortexed on low speed to ensure complete mixing. For each sample, 10 pL was mixed with 190 pL of working solution in Qubit tubes and vortexed. Standards were prepared by mixing 10 pL of the 2 standards with 190 pL of the working solution in a Qubit tube, then vortexed. The tubes were then read individually in the Qubit fluorometer, which reports the concentration of analyte in the sample. Based on this concentration and final volume of the sample, the amount of DNA bound and recovered was calculated and reported in this report as %. When the DNA concentration was too low for detection in the Qubit fluorometer, the DNA % is reported as 0.

BIND AND ELUTE METHOD 1: Hindlll Digest and 20% Lambda “high MW” DNA:

Materials Used:

DNA mixture: 80% Hindlll Digest + 20% Lambda "high MW" DNA at 100 ng/pl

Buffer 1 (rinse, bind, wash): lOmM Citrate pH 4.0

Buffer 2 (elution): lOrnM Tris-HCl/10 mM NaCl pH 8.5

Procedure:

To a 1.5 mL tube pre-rinsed with 10 mM citrate buffer pH 4.0 (Buffer 1), was transferred 12.5 pL bead suspension immediately after obtaining a uniform suspension by vortexing and pipetting. 87.5 pL of Buffer 1 was added and mixed by pipetting to homogenize the mixture. The beads were magnetically precipitated over 2 minutes and the liquid was removed and replaced with 95 pL Buffer 1 and 5 pL 80% Hindlll Digest + 20% Lambda “high MW” DNA at 100 ng/pL. The sample was mixed well by pipette and allowed to sit for 10 minutes. The beads were magnetically precipitated for 2 minutes and the liquid was collected as supernatant. The beads were washed with 100 pL Buffer 1 by mixing with pipette followed by atwo-minute magnetic precipitation. The wash liquid was collected and replaced with 100 pL Buffer 2 and mixed with the pipette and let sit for 10 minutes. The beads were magnetically precipitated for 2 minutes and the liquid was collected as the eluate. The beads were suspended for storage by adding 100 pL TE buffer and mixed with the pipette to suspend. Control samples were prepared by adding 5 uL of DNA directly to 10 mM citrate buffer pH 4 and directly to 10 mM Tris-HCl/NaCl pH 8.5. DNA recovered was quantified using Qubit method.

BIND AND ELUTE METHOD 2: CONTRIVED HUMAN PLASMA-LIKE MEDIA (HPLM)

Samples were prepared in Human plasma-like medium by adding the cfDNA standards described above at biologically relevant concentrations (~5 ng/ml and 1 x 1010 copies/ml for each fragment). The bind and elute testing were performed as follows. For each sample to be tested, 790 pL of HPLM and 10 pL of DNA standard were mixed. One hundred microliters (100 pL) of 1% Triton X-100 was then added and tubes were vortexed at high speed for 5 seconds. One hundred microliters (100 pL) of IM sodium acetate pH 4.5 was added and tubes were vortexed at high speed for 5 seconds. Samples were kept on ice. K22 beads (10 mg/ml) were equilibrated with 10 mM sodium acetate pH 4.5 (1 : 100 v/v bead: sample ratio). 10 mM sodium acetate pH 4.5 (100 pL) was pipetted into a 1.5 ml microcentrifuge tube. Bead stock was resuspended by vortexing 5 times for 10 seconds each. Ten microliters (10 pL) of bead stock was pipetted into the 10 mM sodium acetate pH 4.5 and tubes were vortexed for 10 seconds on high speed then let sit are room temperature for 5 min. Beads were collected on a magnet for 5 min and liquid removed. The DNA spiked HPLM samples described above (1 ml) were added to the beads and tubes were placed on a vortex adapter and agitated at medium speed for 10 min. Beads were collected with a magnet for 5 min and supernatant was removed and retained. Beads were washed by addinglOO pL of 10 mM sodium acetate pH 4.5 to the beads and vortexing then beads were collected with a magnet. Liquid was removed and beads were eluted with 20 pL of 10 mM Tris-HCl pH 8.5/10 mM NaCl for 10 min. Eluate was transferred to a fresh tube. Beads were resuspended in 100 pL of 10 mM TE storage buffer. qPCR was used to quantify DNA in samples.

BIND AND ELUTE METHOD 3: RPMI MEDIA

Samples were prepared in Roswell Park Memorial Institute (RPMI) 1640 by adding the cfDNA standards described above at biologically relevant concentrations (~5 ng/ml and 1 x 1010 copies/ml for each fragment). For each sample to be tested, 790 pL of RPMI 1640 and 10 pL of DNA standard were mixed. One hundred microliters (100 pL) of 1% Triton X-100 was then added and tubes were vortexed at high speed for 5 seconds. One hundred microliters (100 pL) of IM sodium acetate pH 4.5 was added and tubes were vortexed at high speed for 5 seconds. Samples were kept on ice. K22 beads (10 mg/ml) were equilibrated with 10 mM sodium acetate pH 4.5 (1:100 v/v bead: sample ratio). 10 mM sodium acetate pH 4.5 (100 j L) was pipeted into a 1.5 ml microcentrifuge tube. Bead stock was resuspended by vortexing 5 times for 10 seconds each. Ten microliters (10 pL) of bead stock was pipeted into the 10 mM sodium acetate pH 4.5 and tubes were vortexed for 10 seconds on high speed then let sit are room temperature for 5 min. Beads were collected on a magnet for 5 min and liquid removed. The DNA spiked RPMI 1640 samples described above (1 ml) were added to the beads and tubes were placed on a vortex adapter and agitated at medium speed for 10 min. Beads were collected with a magnet for 5 min and supernatant was removed and retained. Beads were washed by addinglOO pL of 10 mM sodium acetate pH 4.5 to the beads and vortexing then beads were collected with a magnet. Liquid was removed and beads were eluted with 20 pL of 10 mM Tris-HCl pH 8.5/10 mM NaCl for 10 min. Eluate was transferred to a fresh tube. Beads were resuspended in 100 pL of 10 mM TE storage buffer. qPCR was used to quantify DNA in samples. qPCR METHOD

Synthetic cfDNA standards described above were diluted in low-bind 1.5 mL microcentrifuge tubes (Eppendorf) to 10⁷molecules/pL in molecular biology-grade water; these dilutions were combined in equal proportions, and 1:10 serial dilutions of the multiplexed sample were created to facilitate generation of a standard curve for qPCR. Each qPCR reaction contained 25 pL total and contained IX Brilliant III Ultra-Fast qPCR MasterMix (Agilent #600880, Santa Clara, CA, USA), 0.5 pM of each primer 0.2 pM of each probe (75bp_CFstd_probe, 167bp_CFstd_probe, 334bp_CFstd_probe, 501bp_CFstd_probe). The qPCR was run in a skirted PCR plate sealed with optically-clear strip caps (Agilent #401490 and #401425 respectively, Agilent Technologies, Santa Clara, CA) using an Agilent AriaMx instrument with the following parameters: 10 min at 95 °C, 40 cycles of: 15 seconds at 95 °C and 1 min at 57 °C.

ZETA POTENTIAL MEASUREMENTS

Zeta potential measurements were taken on a Malvern Zetasizer Nano ZSP (Malvern Panalytical, Malvern, United Kingdom). 2.5 pL of a 10 mg/mL solution of functionalized beads was added to 5 mL acetate buffer (pH = 4.5, 10 mM) or Tris buffer (pH = 8.5, 10 mM) and loaded onto a disposable folded capillary cell (part #DTS1070). The cell was equilibrated at room temperature for 60 s and then analyzed with the Smoluchowski approximation in the Zetasizer instrument software. Three measurements were taken and averaged, with each measurement having a minimum of 10 and maximum of 100 runs.

DNA FRAGMENT ANALYSIS TEST METHOD

Solutions of DNA 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 instructed. The electropherogram was integrated at the indicated DNA fragment size representative of the DNA mixture used in the Examples. The relative peak areas between the fragment sizes are reported as %.

LAMBDA DNA SYBR qPCR TEST METHOD The qPCR standard was prepared by making 10-fold dilutions of Lambda DNA 500 pg/mL (New England Biolabs, #N3011) in molecular grade water (Invitrogen, #10977015). The 1:100 dilution (5 ng/pL) was used as the high standard concentration which contained 1.91x108 Lambda DNA copies in 5 pL. A total of 7 standard dilutions were run, down to 5 femtograms/microliter (fg/pL) (191 copies in 5 pL), included a no-template control (NTC). Samples from DNA capture processing (bead suspensions and eluates) were all diluted 10-fold and 100-fold in molecular grade water. The qPCR reactions were prepared with SYBR™ Green PCR Master Mix (Thermo Fisher, #4364344), with a final concentration of 0.625 micromolar (pM) F and 0.625 pM R primers and 5 pL of sample template or DNA standard. The PCR cycles were as follows: 10 min at 95 °C, followed by 40 cycles of 15 seconds at 95 °C and 1 min at 60 °C. Primers were used that targeted the Hindlll 23 kb fragment (LambdalF: CGG CGT CAA AAA GAA CTT CC, Lambda 1R: CAG TCA ACC ACC AGG GAA TAA) and the Hindlll 4 kb fragment (Lambda4F: TGG CAT TCT GGA GGG AAA TAC, Lambda4R: CAG TCA ACC ACC AGG GAA TAA) of Hindlll digested Lambda DNA.

Preparatory Examples

PREPARATORY EXAMPLE 1 (PEI): MyOne Silane beads modified with acryloxypropyltrimethoxysilane

A stock bottle of Dynabeads MyOne Silane (40 mg/mL) suspension was vortexed for one minute to completely resuspend the magnetic beads. To a 2 mL centrifuge tube, 0.5 mL of the MyOne Silane suspension was transferred and magnetically pelleted. Aqueous phase was replaced with 2 mL condensation solvent. The condensation solvent composition of 95:5 Me0H:H20 (w/w) sample was prepared by adding 95.08 g MeOH and 5.186 g DI H2O. pH was adjusted to 4.5 by addition of 17 drops glacial acetic acid from a glass Pasteur pipette. The beads were fully resuspended by vortex to obtain a suspension at 10 mg/mL. The beads were again magnetically pelleted and solvent was exchanged with 2 mL condensation solvent. 60 pL 3 -acryloxypropyltrimethoxysilane was added and solution was agitated by tube rotator for 25.5 h.

PREPARATORY EXAMPLE 2 (PE2): Acetyl Passivated SpeedBeads

Procedure adapted from passivation strategy detailed in U.S. Patent Publication 20190071662A1. 2 mL of SBead solution (100 mg beads) was added to a 50 mL Falcon tube and diluted with 8 mL water. The beads were magnetically isolated, and the supernatant was discarded. The isolated beads were washed with 2 x 10 mL DMF and the supernatant was discarded after each wash. The beads were resuspended in 7.8 mL DMF, and 2 mL AAH and 200 pL DIPEA were added. The tube was shaken at 250 revolutions per minute (rpm) for 30 minutes (min). The beads were isolated over 10 min and the brown supernatant was discarded. Then the beads were washed with 3 x 10 mL DMF and finally resuspended in 2 mL DMF for storage.

Examples EXAMPLE 1 (EXI): Kryptofix 22- functionalized Dynabeads MyOne Carboxylic Acid

To a 2 mL tube (USA Scientific, Ocala, FL, polypropylene) was added 1.5 mL Dynabeads MyOne Carboxylic Acid magnetic bead suspension (Lot 01158205, 9 pmol) wherein the bottle was rolled for 30 minutes to resuspend. The beads were magnetically isolated, and the supernatant was replaced with 1.5 mL MES buffer (25 mM, pH = 6). The mixture was vortexed, allowed to equilibrate for 10 minutes, and magnetically isolated for two minutes. The supernatant was replaced again with 1.5 mL MES buffer (25 mM, pH = 6) and the sample was vortexed, allowed to equilibrate another 10 minutes, and magnetically isolated for two minutes. To a 15 mL centrifuge tube was added 0.0630 g (240 pmol) K22 and 2 mL MES buffer (25 mM, pH = 6) and agitated to dissolve. To the isolated magnetic beads was added 150 pL (18 pmol) K22 solution and the suspension was agitated in a rotator for 30 minutes at room temperature. A 344 pL (18 pmol) freshly prepared EDC solution (10 mg/mL 25 mM MES buffer) and 6.5 pL 25 mM MES buffer was added to the sample, and the mixture was allowed to agitate by rotator for 2 hours at room temperature. The mixture was vortexed every 30 minutes throughout the reaction. The functionalized beads were magnetically isolated and washed (resuspended and magnetically isolated) three times with 800 pL 50 mM pH 7.5 Tris solution and once with 800 pL PBS pH 7.4. Functionalized beads were finally resuspended in 1500 pL PBS pH 7.4 buffer and stored in a refrigerator (4-6 °C).

EXAMPLE 2 (EX2): Homopiperazine-functionalized Dynabeads MyOne Carboxylic Acid

Prepared by synthetic method described in Error! Reference source not found, using homopiperazine instead of Kryptofix 22. Homopiperazine reagent solution was prepared at 120 pM.

EXAMPLE 3 (EX3): 5% Morpholine, K22 functionalized Dynabeads MyOne Carboxylic Acid

Prepared by synthetic method described in Error! Reference source not found, except beads were functionalized with 7.5 pL 120 pM morpholine in MES and 142.5 pL K22 in MES instead of 150 pL 120 pM K22 in MES.

EXAMPLE 4 (EX4): 10% Morpholine, K22 functionalized Dynabeads MyOne Carboxylic Acid

Prepared by synthetic method described in Error! Reference source not found, except beads were functionalized with 15 pL 120 pM morpholine in MES and 135 pL K22 in MES instead of 150 pL 120 pM K22 in MES.

EXAMPLE 5 (EX5): 20% Morpholine, K22 functionalized Dynabeads MyOne Carboxylic Acid

Prepared by synthetic method described in Error! Reference source not found, except beads were functionalized with 30 pL 120 pM morpholine in MES and 120 pL K22 in MES instead of 150 pL 120 pM K22 in MES.

EXAMPLE 6 (EX6): 40% Morpholine, K22 functionalized Dynabeads MyOne Carboxylic Acid Prepared by synthetic method described in Error! Reference source not found, except beads were functionalized with 60 pL 120 pM morpholine in MES and 90 pL K22 in MES instead of 150 pL 120 pM K22 in MES.

EXAMPLE 7 (EX7): 80% Morpholine, K22 functionalized Dynabeads MyOne Carboxylic Acid

Prepared by synthetic method described in Error! Reference source not found, except beads were functionalized with 120 pL 120 pM morpholine in MES and 30 pL K22 in MES instead of 150 pL 120 pM K22 in MES.

EXAMPLE 8 (EX8): Preparation of K22 functionalized Dynabeads MyOne Tosylactivated

To a 4 mL glass vial was added 150 pL Dynabeads Tosylactivated suspension after resuspension by rolling the reagent bottle. To the vial was added K22 solution that was prepared by dissolving 0.0392 g K22 in 0.5 mL anhydrous DMF solvent. The mixture was sonicated for two minutes at room temperature to resuspend beads. The sample was heated to 100 °C in an oil bath and remove every 30-45 minutes to gently shake to resuspend beads. The reaction mixture was heated for a total of 6 hours. After cooling to room temperature, the beads were magnetically isolated, and the reaction solution was replaced with 2 mL 0.1M HCl(aq). The beads were resuspended, magnetically isolated, and the solution was replaced again with 1.5 mL 0.1M HCl(aq). The solution was stored for approximately 18 hours. The beads were resuspended, transferred to a 2 mL centrifuge tube, and the beads were magnetically isolated, and the solution was replaced with 1 mL 50 mM Tris 7.5 pH buffer. The sample was vortexed to resuspend, and the Tris buffer was replaced two more times. The beads were magnetically isolated, and the Tris solution was replaced with 1 mL PBS buffer and vortexed to resuspend beads. The beads were magnetically isolated, and the PBS buffer was replaced with 1.5 mL PBS buffer to obtain a 10 mg/mL suspension after vortexing. The sample was refrigerated immediately for later use.

EXAMPLE 9 (EX9): Preparation of K22 functionalized Dynabeads MyOne Silane

To a 2 mL centrifuge tube, 1 mL Error! Reference source not found, silane-functionalized magnetic bead suspension in methanol was added and magnetically pelleted. To the pellet was added 0.5 mL methanol and 0.5 mL of an approximately 0.2M K22 in methanol solution prepared by adding 0.050 g K22 to a centrifuge tube and dissolving in 1 mL methanol. The pellet was resuspended by vortex and placed in a 50 °C oven for 26 hours. Sample was vortexed, followed by magnetically precipitating the beads and replacing the reaction liquid with 1 mL methanol. Sample was vortexed to resuspend followed by magnetic precipitation and liquid phase was replaced with deionized water. Liquid replacement followed the same procedure once more with deionized water and twice with PBS to obtain a suspension at 10 mg/mL concentration. Sample was refrigerated for later use.

EXAMPLE 10 (EX10): Preparation of K22 functionalized Dynabeads Epoxy M-270 To a 4 mL glass vial, added 15.4 mg Dynabeads M270 Epoxy freeze-dried beads and suspended in 1 mL anhydrous DMF. In a separate vial, 0.0402 g K22 was dissolved in 0.5 mL DMF with gentle heat and allowed to cool to room temperature. The entire K22 solution was added to the bead suspension and sonicated for 1 minute. The capped vial was heated in a 100 °C oil bath for 4 hours, agitating approximately every 30 minutes. Upon removal from the oil bath, the bead suspension was magnetically precipitated, and solvent replaced with 1.5 mL 0.1M HC1 (aq). The beads were then resuspended, magnetically precipitated, and replaced solution again with 1.5 mL 0.1M HCl(aq), storing for approximately 18 h at this condition. The beads were resuspended, transferred to 2 mL centrifuge tube, magnetically precipitated, and the solution was replaced with 1 mL 50 mM Tris 7.5 pH buffer. The samples were vortexed to resuspend the beads and the Tris buffer replacement was repeated twice more. The beads were magnetically precipitated, and the solution was replaced with 1 mL PBS buffer and vortexed to resuspend beads. The precipitation method was repeated once more, and the solution was replaced with 1.54 mL PBS buffer to obtain a 10 mg/mL suspension after vortexing. Sample was refrigerated immediately after.

EXAMPLE 11 (EX11) - Preparation of cyclen functionalized SBeads

0.2 mL of the passivated SBead stock solution from PE2 (10 mg, 5.8 pm active functionality) was added to a 1.5 mL Eppendorf tube and the beads were isolated. 300 pL TEA solution (5.5 mg/mL in DMF, 16.2 pmol) and 229 pL EDC solution (10 mg/mL in DMF, 12 pmol) were added to the beads and mixed for 5 min. 100 pL of cyclen tetrahydrochloride stock solution, prepared at 120 pmol/mL in DMF, was added and the solution was mixed at room temperature overnight. The beads were isolated and washed with 2 x 500 pL Tris buffer and 2 x 500 pL PBS. The beads were left in 1000 pL PBS (10 mg/mL) and stored at 4 °C until further use.

EXAMPLE 12 (EX12) - Preparation of triazacyclononane functionalized SBeads

0.2 mL of the passivated SBead stock solution from PE2 (10 mg, 5.8 pm active functionality) was added to a 1.5 mL Eppendorf tube and the beads were isolated. 300 pL TEA solution (5.5 mg/mL in DMF, 16.2 pmol) and 229 pL EDC solution (10 mg/mL in DMF, 12 pmol) were added to the beads and mixed for 5 min. 100 pL of 1,4,7-triazacyclononane stock solution, prepared at 120 pmol/mL in DMF, was added and the solution was mixed at room temperature overnight. The beads were isolated and washed with 2 x 500 pL Tris buffer and 2 x 500 pL PBS. The beads were left in 1000 pL PBS (10 mg/mL) and stored at 4 °C until further use.

Comparative Examples

COMPARATIVE EXAMPLE 1 (CE1) - Dynabeads MyOne Carboxylic acid

Used without further modification.

COMPARATIVE EXAMPLE 2 (CE2) - Dynabeads MyOne Silane Used without further modification

COMPARATIVE EXAMPLE 3 (CE3) - Morpholine-functionalized Dynabeads MyOne Carboxylic Acid

Prepared by synthetic method described in Error! Reference source not found, using morpholine instead of Kryptofix 22. Morpholine reagent solution was prepared at 120 pM.

COMPARATIVE EXAMPLE 5 (CE4) - Preparation of Hydrolyzed Dynabeads Epoxy M-270

To a 4 mL glass vial was added 14.9 mg Dynabeads M27 Epoxy freeze-dried beads and 1 mL 0.1M HC1 (aq). The sample was capped and sonicated for 1 minute before adding another 1 mL 0.1M HC1 (aq). The sample was agitated by inversion in a secondary container on ajar roller at room temperature for 20.5 hours. The beads were then magnetically precipitated and the aqueous solution was replaced with 1 mL 50 mM Tris pH 7 buffer. The beads were the resuspended by vortex, magnetically precipitated, and solution exchanged with 1 mL Tris. The sample was resuspended, transferred to a 2 mL centrifuge tube, and an additional 1 mL Tris buffer was added. The sample was vortexed, magnetically precipitated, and the solution was replaced with 1 mL PBS buffer. The sample was vortexed to resuspend beads, magnetically precipitated, and solution replaced with 1.490 mL PBS to obtain a 10 mg/mL suspension after vortexing. Sample was refrigerated immediately after.

COMPARATIVE EXAMPLE 5 (CE5) - Qiagen Beads from Qiagen QIAamp Kit

Used without further modifications to Qiagen Beads.

COMPARATIVE EXAMPLE 6 (CE6) - Propyltrimethylammonium chloride on Dynabeads MyOne Silane

Prepared by the same method as PEI using propyltrimethylammonium chloride trimethoxysilane in methanol instead of 3-acryloxypropyltrimethoxysilane.

COMPARATIVE EXAMPLE 7 (CE7) - SBeads

Used without further modification to SBeads.

Table 2. Magnetic beads generated and corresponding Zeta Potentials in various buffers

Table 3. Bind and elute results of cfDNA standard sample using BIND AND ELUTE METHOD 1 Table 4. Qubit fluorescence measurements of supernatant to determine bound DNA % and of eluate for % recovered after elution using BIND AND ELUTE METHOD 1

Table 1A. DNA fragment analysis of DNA (in eluate, super, and beads) using BIND AND ELUTE

METHOD 3

*ND = Not Detected

Table 2B. DNA fragment analysis of DNA - Avg % Recovery (in eluate, super, and beads) using BIND

AND ELUTE METHOD 3

*ND = Not Detected Tables 5A and 5B report the amount of DNA that did not bind (Super), the amount of DNA that stayed bound after elution (Beads), and the amount of bound DNA that was eluted. Table 3. Lambda DNA SYBR qPCR targeting the 4 kb Hindlll digest fragment of full-length Lambda

DNA of eluate and bead suspension diluted 1:10 and 1:100. (Low molecular weight)

Table 4. Lambda DNA SYBR qPCR targeting the 23 kb Hindlll digest fragment of full-length Lambda DNA of eluate and bead suspension diluted 1:10 and 1:100. (High molecular weight)

Claims

Table 5. DNA fragment analysis of DNA (in supernatant and eluate) of Homopiperazine using BIND AND ELUTE METHOD 1 Values higher than initial demonstrate enrichment. Table 9. Bind and elute with cfDNA in BIND AND ELUTE METHOD 2 (Triton X- 100) Table 10 - Bind and elute in Qiagen QIAamp protocol using cfDNA in bovine plasma What is claimed is:

1. A method of processing polynucleic acids comprising: a) providing a solid support comprising ligands, wherein at least a portion of the ligands comprise a cyclic amine group comprising greater than 6 members; b) 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; c) exposing the solid support with 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 polynucleic acid molecules bound to the solid support; d) optionally washing the solid support comprising the bound and/or retained bound polynucleic acid molecules; e) optionally preparing a suspension from the solid support comprising the retained bound polynucleic acid molecules; f) utilizing the released portion of bound polynucleic acid molecules of c), and/or the retained portion of the polynucleic acid molecules bound to the solid support or the suspension thereof.

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

3. The method of claims 1-2 wherein the cyclic amine group comprises at least two nitrogen atoms.

4. The method of claims 1-3 wherein the cyclic amine group comprises at least 2 heteroatoms selected from nitrogen and oxygen.

5. The method of claim 4 wherein the cyclic amine group comprises at least 2, 3, or 4 oxygen atoms.

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

7. The method of claims 1-6 wherein the cyclic amine group comprises at least one amide group.

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

9. The method of claim 8 wherein the amine compound is homopiperazine or an azacrown compound including azacrown ethers.

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

11. The method of claim 10 wherein the second different ligand is cyclic.

12. The method of claims 10-11 wherein the second different ligand is morpholine.

13. The method of claims 10-12 wherein the second different ligand is present in an amount less than 50 wt.% based on the total amount of ligands.

14. The method of claims 1-13 wherein the solid support is particles, magnetic particles, or nonmagnetic particles.

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

16. The method of claims 1-15 wherein f) comprises utilizing the released portion of bound polynucleic acid molecules.

17. The method of claims 1-16 wherein utilizing comprises size separation, purification, quantification, and amplification.

18. The method of claims 1-17 wherein the polynucleic acid molecules is cell free DNA.

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

20. Magnetic particles comprising ligands bound to the magnetic particles; wherein at least a portion of the ligands comprise a cyclic amine group comprising greater than 6 members.

21. The magnetic particles of claim 20 wherein the cyclic amine group is an ionizable group.

22. The magnetic particles of claims 20-21 wherein the solid support comprising ligands has a negative or positive zeta potential at a pH of 4.5.

23. The magnetic particles of claims 20-22 wherein the solid support comprising ligands has a lower zeta potential at a pH of 8.5 than at a pH of 4.5.

24. The magnetic particles of claims 20-23 wherein the difference between a zeta potential at a pH of 4.5 and a zeta potential at a pH of 8.5 is at least 10, 20, 30, 40 or 50 mV.

25. A solid support comprising ligands bound to the solid support; wherein at least a portion of the ligands comprise a cyclic amine group comprising greater than 6 members.

26. The solid support or magnetic particles of claims 20-25 wherein the ligand or solid support is further characterized by claims 2-14.

27. A kit comprising the solid support or magnetic beads of claims 20-26 and a buffer having a pH of less than 5.5.

28. The kit of claim 27 further comprising a buffer having a pH greater than 6.