Method for separating nucleic acids using zigzag chromatography

Abstract

Disclosed herein are methods for separating nucleic acids using zigzag chromatography. The methods provided enable robust separation of nucleic acid species, including DNA, RNA, and mixtures thereof. In addition, the methods are suitable for separating topologically distinct analog nucleic acid species. Separation of nucleic acid species using the disclosed chromatographic methods can be accomplished in 10 minutes (e.g., between 1 and 4 minutes), while maintaining high levels of efficiency and peak resolution.

Description

[0001] Cross-references to related applications This application claims priority and benefit to U.S. Provisional Application No. 63 / 559,084, filed February 28, 2024, entitled "Methods for the Separation of Nucleic Acids with Slalom Chromatography". This application also claims priority and benefit to U.S. Provisional Application No. 63 / 701,042, filed September 30, 2024, entitled "Methods for the Separation of Nucleic Acids with Slalom Chromatography". The contents of both U.S. Provisional Applications Nos. 63 / 559,084 and 63 / 701,042 are incorporated herein by reference in their entirety. Technical Field

[0002] This disclosure relates in general to methods for separating nucleic acids using liquid chromatography, particularly zigzag chromatography. Background Technology

[0003] Nucleic acids form the basis of molecular cloning technology, are crucial in the manufacture of biotherapeutic agents such as antibodies, and are at the forefront of novel therapeutic approaches such as cell / gene therapy and RNA-based vaccines. Therefore, the ability to manipulate, isolate, and characterize nucleic acids is fundamental to academia, the biotechnology, and the pharmaceutical industry. Existing nucleic acid isolation and characterization methods are slow (typically requiring 30 minutes or more to several days) and lack the high resolution achieved when isolating other macromolecules such as proteins. Gel electrophoresis, as the primary method for separating nucleic acids, requires sample manipulation for visualization (e.g., adding dyes such as ethidium bromide), optimization of gel concentration based on nucleic acid size, and hinders downstream processing of target nucleic acid species (e.g., extraction from the gel itself, which significantly reduces yield and introduces contaminants). Therefore, there is a need in the art for methods that can rapidly isolate nucleic acids with limited sample manipulation and high resolution. Summary of the Invention

[0004] The ability to robustly and efficiently characterize nucleic acids is crucial for the development of therapeutic agents across multiple therapeutic modalities, including but not limited to antibodies, cell / gene therapies, and mRNA vaccines. This invention provides a method for column-based nucleic acid isolation within 10 minutes or less (e.g., 10 minutes, 9 minutes, 8 minutes, 6 minutes, 5 minutes, 3 minutes, 2 minutes, etc.).

[0005] Therefore, in one aspect, this document discloses a method for separating DNA and RNA, the method comprising: a) loading a sample containing multiple single-stranded RNA (ssRNA) molecules and multiple double-stranded DNA (dsDNA) molecules onto a chromatographic column, wherein the column comprises multiple porous or non-porous particles; b) eluting the sample from the column, wherein elution is performed between 0.6 min and 10 min; and c) detecting multiple ssRNA molecules and / or multiple dsDNA molecules in the eluent, wherein the multiple ssRNA molecules form at least one independent peak, and wherein the multiple dsDNA molecules form at least one independent peak. In some embodiments, elution is performed between 0.6 min and 6 min. In some embodiments, elution is performed between 1 min and 3 min. In some embodiments, elution produces a band retention factor of less than 0.35. In some embodiments, the sample further comprises multiple single-stranded DNA and / or multiple double-stranded RNA.

[0006] In one aspect, this document discloses a method for separating double-stranded DNA (dsDNA), the method comprising: a) loading a sample containing multiple dsDNA molecules onto a chromatographic column, wherein the column comprises multiple porous or non-porous particles; b) eluting the sample from the column, wherein the elution produces a band retention factor (k1) of less than 0.35; and c) detecting the multiple dsDNA molecules in the eluent. In some embodiments, the sample further comprises multiple single-stranded RNA (ssRNA) molecules, multiple double-stranded RNA (dsRNA) molecules, and / or multiple single-stranded DNA (ssDNA) molecules. In some embodiments, the dsDNA is linear, circular, or supercoiled. In some embodiments, the ssRNA is linear or circular. In some embodiments, step c) further includes detecting the multiple ssRNA molecules, multiple dsRNA molecules, and / or multiple ssDNA molecules in the eluent. In some implementations, elution with k1 less than 0.35 (e.g., 0.33 or less) can be controlled, customized, or adjusted to a desired k1 value by selecting the ionic strength conditions for eluting the sample from the column. During elution from the column, the ionic strength conditions can be varied in several ways, such as increasing or decreasing the salt concentration of the mobile phase. Alternatively or additionally, the surface of the stationary phase or the material within the column flow path can be modified to affect the net charge (e.g., increase the net negative charge on the surface).

[0007] In one aspect, this article discloses a method for separating DNA, the method comprising: a) loading a sample containing a plurality of dsDNA molecules and ssDNA molecules onto a chromatographic column, wherein the column comprises a plurality of porous or non-porous particles; b) eluting the sample from the column, wherein the elution produces a band retention factor (k1) of less than 0.35; and c) detecting the plurality of dsDNA molecules and ssDNA molecules in the eluent.

[0008] In one aspect, this article discloses a method for isolating RNA, comprising: a) loading a sample containing multiple ssRNA molecules and multiple dsRNA molecules onto a chromatographic column, wherein the column contains multiple porous or non-porous particles; b) eluting the sample from the column, wherein the elution produces a band retention factor (k1) of less than 0.35; and c) detecting multiple ssRNA molecules and / or multiple dsRNA molecules in the eluent.

[0009] In one aspect, this article discloses a method for separating DNA and RNA, the method comprising: a) loading a sample containing multiple ssRNA molecules and multiple dsDNA molecules onto a chromatographic column, wherein the column contains multiple porous or non-porous particles; b) eluting the sample from the column, wherein the elution produces a band retention factor (k1) of less than 0.35; and c) detecting multiple ssRNA molecules and / or multiple dsDNA molecules in the eluent.

[0010] In some embodiments of the above method, elution is performed between 0.6 minutes and 10 minutes. In some embodiments, elution is performed between 0.6 minutes and 6 minutes. In some embodiments, elution is performed between 1 minute and 3 minutes. In some embodiments, step b) is performed with an efficiency (N) >40,000. In some embodiments, the eluent is collected.

[0011] In some implementations, dsDNA, ssDNA, ssRNA and / or dsRNA contain about 1,000 base pairs to about 100,000 base pairs.

[0012] In some embodiments, the diameter of the plurality of particles is between about 1 µm and about 10 µm. In some embodiments, the particles are porous. In some embodiments, the plurality of particles have an average pore size between about 100 Å and 900 Å. In some embodiments, the plurality of particles have an average pore size of about 125 Å. In some embodiments, the plurality of particles have an average pore size of about 250 Å. In some embodiments, the plurality of particles have a specific pore volume between 0.5 cc / g and 1.3 cc / g.

[0013] In some embodiments, at least a portion of the inner surface of the column body is coated with an alkylsilane material. In some embodiments, the alkylsilane material is a hydrophilic nonionic layer of polyethylene glycol silane. In some embodiments, the alkylsilane material is a vapor-phase deposition product of precursor bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane.

[0014] In some embodiments, the chromatographic column is connected to a high-performance liquid chromatography (HPLC) system or an ultra-high-performance liquid chromatography (UHPLC) system. In some embodiments, step c) is performed using a UV detector or an adjustable UV (TUV) detector. In some embodiments, step c) is performed using a multi-angle light scattering (MALS) detector.

[0015] In some embodiments, step b) is performed at a flow rate greater than 0.5 mL / min, and wherein the dsDNA, ssDNA, ssRNA, and / or dsRNA comprises about 2,000 to about 25,000 base pairs. In some embodiments, the plurality of porous or non-porous particles have a diameter between 1.5 µm and 1.9 µm.

[0016] In some embodiments, step b) is performed at a flow rate greater than 0.75 mL / min, and wherein the dsDNA, ssDNA, ssRNA, and / or dsRNA comprises about 5,000 to about 50,000 base pairs. In some embodiments, the plurality of porous or non-porous particles have a diameter between 2.8 µm and 3.2 µm.

[0017] In some embodiments, step b) is performed at a flow rate greater than 1.0 mL / min, and wherein the dsDNA, ssDNA, ssRNA, and / or dsRNA comprises about 25,000 to about 50,000 base pairs. In some embodiments, the plurality of porous or non-porous particles have a diameter between 4.8 µm and 5.2 µm.

[0018] In some embodiments, step b) is performed at a flow rate greater than 2.5 mL / min, and wherein the dsDNA, ssDNA, ssRNA, and / or dsRNA comprises about 25,000 to about 100,000 base pairs. In some embodiments, the plurality of porous or non-porous particles have a diameter between 9.8 µm and 10.2 µm. Attached Figure Description

[0019] The present technology will be more fully understood through the following detailed description in conjunction with the accompanying drawings.

[0020] Figure 1 This is a diagram illustrating a method for isolating nucleic acid molecules according to some implementation schemes of this technology.

[0021] Figure 2 Chromatograms of double-stranded DNA species isolated according to some embodiments of this technology are provided.

[0022] Figure 3Chromatograms of circular and linear DNA species isolated according to some embodiments of this technology are provided.

[0023] Figure 4 Chromatograms of single-stranded and double-stranded RNA isolated according to some embodiments of this technology are provided.

[0024] Figure 5 Chromatograms of double-stranded DNA separated using mobile phases of different ionic strengths are provided.

[0025] Figure 6 Chromatograms of double-stranded RNA separated at low and high shear flow rates are provided.

[0026] Figure 7A and Figure 7B Chromatograms of double-stranded DNA samples separated at various flow rates are provided. Figure 7A Chromatograms of supercoiled dsDNA isolated from linear dsDNA are provided. Figure 7B Chromatograms of circular dsDNA isolated from linear dsDNA are provided.

[0027] Figure 8A and Figure 8B The correlation between nucleic acid length and k coefficient of dsDNA isolated using methods according to some embodiments of this technology is shown.

[0028] Figure 9 Chromatograms of a mixture of DNA and RNA separated according to the present technical embodiment are provided.

[0029] Figure 10 Chromatograms of DNA digestion products isolated according to some embodiments of this technology are provided.

[0030] Figure 11 Chromatograms of small interfering RNA (siRNA) calibrators isolated and detected using multi-angle light scattering (MALS) according to some embodiments of this technology are provided.

[0031] Figures 12A to 12F Chromatograms of DNA digestion products isolated according to some embodiments of this technology and detected using multi-angle light scattering (MALS) are provided.

[0032] Figure 12A A chromatogram before UV broadening is provided.

[0033] Figure 12B A chromatogram after UV broadening is provided.

[0034] Figure 12C Zimm plot of the first of two peaks detected by MALS is provided.

[0035] Figure 12D Zimm plot of the second of the two peaks detected by MALS is provided.

[0036] Figure 12E A random coil plot of the first of two peaks detected by MALS is provided.

[0037] Figure 12F A random coil plot of the second of two peaks detected by MALS is provided.

[0038] Figure 13 Chromatograms of DNA digestion products in the range of 0.1 µg to 5 µg at concentrations isolated and detected using MALS according to some embodiments of this technology are provided.

[0039] Figure 14 Representative chromatograms of DNA digestion products separated using the method according to this technique and detected using multi-angle light scattering (MALS) and dynamic light scattering (DLS) detectors are provided.

[0040] Figure 15 Representative chromatograms are provided for the separation of DNA from additional mixture components using particles with a pore size of 125 Å (top) or 250 Å (bottom).

[0041] Figure 16 Chromatograms showing the theoretical separation of dsRNA and ssRNA present in the mixture relative to standard nucleic acid gradient standards are provided.

[0042] Figure 17 An exemplary graph depicting the separation degree of nucleic acid species according to an embodiment of the present technology is provided.

[0043] Figure 18A , Figure 18B , Figure 18C and Figure 18D The method provides a depiction using 3.0µm nonporous silica particles at a flow rate of 0.05mL / min. Figure 18A ), 0.1 mL / min Figure 18B ), 0.2 mL / min Figure 18C ) or 0.3 mL / min ( Figure 18D Example data on the separation of nucleic acid types under ().

[0044] Figures 19A to 19D Exemplary data are provided to depict the nucleic acid species separation using 2.6µm nonporous silica particles.

[0045] Figure 19A Use λ-DNA-HindIII digestion products without additives in the mobile phase.

[0046] Figure 19B The λ-DNA-HindIII digestion product was prepared using 0.5% by volume 2-[2-(diethylamino)ethoxy]ethanol as a mobile phase additive.

[0047] Figure 19C Use an additive-free λ-DNA single-enzyme digestion mixture in the mobile phase.

[0048] Figure 19D The λ-DNA single-enzyme digestion mixture was prepared using 0.5% by volume 2-[2-(diethylamino)ethoxy]ethanol as a mobile phase additive.

[0049] Figure 20A , Figure 20B and Figure 20C A description is provided of using non-porous silica particles in an acidic environment (pH = 6); Figure 20A ), neutral (pH = 7; Figure 20B ) and alkaline (pH = 8; Figure 20C Example data on the separation efficiency of nucleic acid species at different pH levels.

[0050] Figures 21A to 21C An exemplary graph is provided depicting the resolution of nucleic acid species separation using nonporous polymer particles at different ionic intensities.

[0051] Figure 21A The chromatogram is shown in 1X PBS.

[0052] Figure 21B The chromatogram is shown in 2x PBS.

[0053] Figure 21C The chromatogram is shown in 4x PBS.

[0054] Figure 22 An exemplary graph depicting the resolution of nucleic acid species separation using surface-porous 3.0µm hybrid solid core particles is provided. Figure 22 ).

[0055] Figure 23 An exemplary graph is provided depicting the resolution of nucleic acid species separation using 2.7µm solid-core silica particles.

[0056] Figure 24A and Figure 24B An exemplary graph depicting the resolution of nucleic acid species separation using 1.6 µm solid silica core particles bonded with a T3-bonded phase is provided. Figures 24A to 24B ). Figure 24AThe results are shown using 1,000 base pairs of DNA gradient standards. Figure 24B The results of digestion with λ-DNA BstEII are shown.

[0057] Figure 25A and Figure 25B An exemplary graph is provided depicting the resolution of nucleic acid species separated using 1.6µm solid-core silica particles (90Å pore size). Figure 25A The results are shown using 1,000 base pairs of DNA gradient standards. Figure 25B The results of digestion with λ-DNA BstEII are shown.

[0058] Figure 26A and Figure 26B An exemplary graph is provided depicting the resolution of nucleic acid species separation using 1.6µm solid silica particles (pore size 120Å). Figure 26A The results are shown using 1,000 base pairs of DNA gradient standards. Figure 26B The results of digestion with λ-DNA BstEII are shown.

[0059] Figure 27 An exemplary graph is provided depicting the resolution of nucleic acid species separation using the surface of diol-functionalized particles at different flow rates.

[0060] Figure 28A An exemplary graph depicting the resolution of nucleic acid species separated using a sulfate-functionalized surface is provided. The oligonucleotides separated by the gel electrophoresis experiment in the illustration match the corresponding peaks.

[0061] Figure 28B , Figure 28C and Figure 28D Showing Figure 28A The dependence of chromatographic peaks on flow rate and ionic strength observed in the study.

[0062] Figure 29A , Figure 29B , Figure 29C and Figure 29D An exemplary graph depicting the separation degree of nucleic acid species using amide-functionalized surfaces is provided.

[0063] Figure 29E The oligonucleotides isolated by gel electrophoresis are shown.

[0064] Figure 30A and Figure 30B An exemplary graph depicting the separation degree of nucleic acid species using hydrophobic surface modification is provided. Figure 30A The chromatograms obtained using unfunctionalized surfaces are shown. Figure 30BThe chromatograms obtained using C1 XT functionalized surfaces are shown. Detailed Implementation

[0065] This document discloses a method for separating nucleic acids under high flow rate conditions (i.e., zigzag chromatography). To facilitate understanding of this technique, certain terms are first defined. It should be noted that whenever a value or range of a parameter is described, the values ​​and ranges within that range are also intended to be part of this disclosure. Unless otherwise defined, the word "about" means ±5%. It should also be noted that, as used herein and in the claims, the singular forms "an," "a," and "the" include plural references unless the context clearly specifies otherwise. Additional terms are defined throughout the specification.

[0066] definition As used herein, the term "zigzag chromatography" refers to a chromatographic method that separates molecules by size based on hydrodynamic phenomena under high flow conditions. The principle of zigzag chromatography is further described in Hirabayashi and Kasai, J. Chromatogr. A (1996) 722(1-2):135-42 and Hirabayashi et al., Biochemistry (1990) 29(41):9515-21.

[0067] The high flow rates used in zigzag chromatography (SC) provide sufficiently high shear rates, resulting in sustained elongation of molecules (e.g., nucleic acids), which is important for separating molecules by size. This elongation can be assessed using the Weissenberg number. The Weissenberg number is a dimensionless measure that is a function of relaxation time and shear rate. in The relaxation time (in seconds) is expressed by the following formula: Where h is the dynamic velocity (Pa*s); L C It is the outline length (m); It is the duration (m); k B It is the Boltzmann constant (1.38 × 10⁻⁶). -23 J.K. -1 T is temperature (Kelvin); and Shear rate (seconds) -1 ), is expressed by the following formula: in It is the velocity difference between two points in a direction perpendicular to the flow direction, and the distance between these two points is... ; It is the average linear gap velocity along the packed bed; and d is the average flow diameter.

[0068] Relaxation time Part of the relaxation time depends on the intrinsic properties of the target nucleic acid. In some cases, the relaxation time of a molecule under specific conditions is known in the art. For example, the L... C It equals the number of base pairs * 3.4 Å; the dynamic velocity of linear dsDNA is 1.2 cP; and the persistence length of linear dsDNA is 450 Å. Alternatively, the relaxation time of a molecule under specific conditions can be determined empirically using known methods readily understood by those skilled in the art. (See, for example, Bouchiat et al., Biophysical Journal (1999) 76:409-413).

[0069] As used herein, the term "nucleic acid" or "nucleic acid molecule" refers to a polymeric molecule containing two or more nucleotides. Nucleic acids can contain deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or combinations thereof. DNA contains nucleotides such as cytidine, guanosine, adenosine, and thymidine. RNA contains nucleotides such as cytidine, guanosine, adenosine, and uridine. In some embodiments, nucleic acid molecules can contain nucleic acid analogs (i.e., non-naturally occurring nucleic acids or analogs thereof). Examples of nucleic acid analogs include peptide nucleic acids, locked nucleic acids, diol nucleic acids, threon nucleic acids, and hexitol nucleic acids. Nucleic acid analogs are further reviewed in Wang et al., Molecules (2023) 28(20):7043. Nucleic acid molecules can also be modified with a range of chemical modifications at the nucleobase, sugar, or phosphodiester backbone, which are further reviewed in Epple et al., Emerg. Top. Life. Sci. (2021) 5(5):691-697.

[0070] The length of nucleic acid molecules ranges from approximately 5,000 base pairs to approximately 100,000 base pairs. Nucleic acids can be single-stranded (e.g., single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA)). In some embodiments, ssRNA is mRNA. Nucleic acids can be double-stranded (e.g., double-stranded DNA (dsDNA) or double-stranded RNA (dsRNA)). Double-stranded nucleic acids consist of complementary sequences (e.g., base-pairing sequences), as is known in the art and will be readily understood by those skilled in the art.

[0071] Nucleic acids can exist in different topological structures, including linear and circular conformations. In some embodiments, circular nucleic acids can be supercoiled, where the circular nucleic acid molecule undergoes additional torsional strain.

[0072] As used herein, the term "porous" refers to a material having a pore volume greater than 0.1 cc / g. Preferably, the porous polymer has a pore volume greater than 0.1 cc / g (e.g., 0.5 cc / g). As used herein, the term "non-porous" refers to a material having a pore volume less than 0.1 cc / g. Preferably, the non-porous polymer has a pore volume less than 0.10 cc / g (e.g., 0.05 cc / g), and in some embodiments, a pore volume less than 0.02 cc / g is preferred. Pore volume is determined using methods known in the art based on multi-point nitrogen adsorption experiments (Micromeritics ASAP 2400; Micromeritics Instruments Inc., Norcross, GA). In some embodiments, the pore volume is between 0.5 cc / g and 1.3 cc / g.

[0073] As used herein, the term "surface porous particle" refers to a material having a solid or non-porous core and an outer layer surrounding the core, the outer layer being porous or having a higher degree of porosity than a non-porous core. In some embodiments, the porous or surface porous particles described herein comprise a pore volume of about 20 Å to about 900 Å (e.g., about 45 Å).

[0074] As used herein, the term "band retention factor," also known as k1, refers to the ratio of the volume of analyte (e.g., nucleic acid molecules) retained on the column to the volume of voids retained. k1 can be defined using the following formula: in This refers to the retention of molecules (e.g., nucleic acids) without stretching (e.g., at low flow rates); in This refers to the characteristic compressive stress of molecules (e.g., nucleic acids); and in This refers to the maximum stretching of a molecule (e.g., nucleic acid).

[0075] And the shear stress pressure variable is t= h / d p The above-mentioned measures can be determined empirically and can be variables such as flow rate, viscosity (e.g., viscosity of the mobile phase or temperature), and particle size.

[0076] As used in this article, the term "efficiency" or "N" in chromatographic analysis refers to a measured value of peak dispersion. N can be defined using the following formula: Where L is the column length; Where d p It is the particle size; and Where k is the zone retention factor.

[0077] As used herein, the term "independent peak" refers to a peak in a chromatogram (e.g., a detected analyte) that achieves baseline separation.

[0078] As used herein, the term "selectivity factor" or "α" refers to the ability of a chromatographic method or system to distinguish analytes within a sample, typically expressed as the ratio of two peaks. α can be defined using the following formula: Where t0 refers to the gap elution time; Where t R1 and t R2 These refer to the elution times of the first and second peaks, respectively. In some implementations, the selectivity factor is a comparison between two nucleic acid species.

[0079] Band retention factor (where t) R As defined above, t i (retention time between particles) as The function is plotted to determine the value of k1 empirically.

[0080] The method described above allows for the separation of nucleic acids with high resolution by manipulating particle size, column length, and column pressure. For example, but not limited to, a particle size of 1.7 µm, a column pressure of 10,000 psi, and a column length of 15 cm can achieve maximum resolution by separating DNA between 4 kb and 6 kb within 1.1 minutes (see example). Figure 17 By adapting the variables according to the teachings described herein and as shown in the examples (see, for example, Example 4), maximum separation of DNA with different nucleotide lengths can be achieved.

[0081] Methods and conditions for isolating nucleic acids In one aspect, this paper discloses a method for separating nucleic acid species by size using zigzag chromatography. Typically, nucleic acids are separated based on electrophoretic mobility using gel electrophoresis or similar methods. These methods can be time-consuming (ranging from hours to days) and lack sufficient sensitivity to detect nucleic acid heterogeneity. They also hinder downstream sample processing because they require extraction of nucleic acids from the gel itself (significantly reducing yield and increasing contamination). This technique utilizes zigzag chromatography, which allows for rapid separation of nucleic acid species by size. Because these methods utilize high-performance liquid chromatography (HPLC) or ultra-high-performance liquid chromatography (UHPLC) systems, they provide separation and characterization of complex mixtures of nucleic acid species. The method disclosed in this invention enables highly efficient separation of nucleic acids by size within 10 minutes.

[0082] Figure 1 An embodiment of this disclosure is illustrated. Sample 100, which may contain double-stranded DNA (dsDNA; 101), single-stranded DNA (ssDNA; 102), single-stranded RNA (ssRNA; 103), double-stranded RNA (dsRNA; 104), or any combination thereof, is loaded (110) onto column (120). Column 120 comprises a plurality of porous or non-porous particles. The sample is flowed through the column using a mobile phase and eluted (130). In a preferred embodiment, the time between loading (110) and elution (130) is between 1 minute and 10 minutes. The sample is detected using a detector (140), such as a UV detector, and a resulting chromatogram (150) is generated. Peaks in the chromatogram, such as peaks 151 and 152, correspond to different sizes of nucleic acid species.

[0083] The method disclosed herein utilizes conditions leading to high shear rates, causing nucleic acid molecules in the column to be stretched / extended under a shear flow. The stretching or extension of nucleic acid molecules can be determined by the Weissenberg number, which compares the elastic and viscous forces applied to the molecules. With a Weissenberg number greater than 1, nucleic acid molecules are stretched under a shear flow and will be separated by size based on the zigzag chromatography principle. With a Weissenberg number less than 1, nucleic acid molecules are not stretched under a shear flow and will not be separated by size based on the zigzag chromatography principle.

[0084] Therefore, in one aspect, the method disclosed herein utilizes a condition that causes the nucleic acid molecule to exhibit a Weissenberg number (Wi) greater than 1. In some embodiments, Wi is between 1 and 100, 5 and 100, 10 and 100, 15 and 100, 20 and 100, 25 and 100, 30 and 100, 35 and 100, 40 and 100, 45 and 100, 55 and 100, 60 and 100, 65 and 100, 70 and 100, 75 and 100, 80 and 100, 85 and 100, 90 and 100, or 95 and 100.

[0085] The shear rate, and consequently the Weissenberg number (Wi), can be affected by several parameters, including flow rate, particle size, mobile phase viscosity, and temperature. Therefore, these variables can be adjusted to achieve a sufficient Wi number for separating the desired nucleic acid molecules.

[0086] In some embodiments, the flow rate is between 0.1 mL / min and 15 mL / min. In some embodiments, the flow rate is 0.1 mL / min to 0.5 mL / min, 0.5 mL / min to 1.0 mL / min, 1.0 mL / min to 1.5 mL / min, 1.5 mL / min to 2.0 mL / min, 2.0 mL / min to 2.5 mL / min, 2.5 mL / min to 3.0 mL / min, 3.0 mL / min to 3.5 mL / min, 3.5 mL / min to 4.0 mL / min, 4.0 mL / min to 4.5 mL / min, 4.5 mL / min to 5.0 mL / min, 5.0 mL / min to 5.5 mL / min, 5.5 mL / min to 6.0 mL / min, 6.0 mL / min to 6.5 mL / min, 6.5 mL / min to 7.0 mL / min, 7.0 mL / min. The flow rates are: L / min to 7.5 mL / min, 7.5 mL / min to 8.0 mL / min, 8.0 mL / min to 8.5 mL / min, 8.5 mL / min to 9.0 mL / min, 9.0 mL / min to 9.5 mL / min, 9.5 mL / min to 10 mL / min, 10 mL / min to 10.5 mL / min, 10.5 mL / min to 11 mL / min, 11.5 mL / min to 12 mL / min, 12 mL / min to 12.5 mL / min, 12.5 mL / min to 13 mL / min, 13 mL / min to 13.5 mL / min, 13.5 mL / min to 14 mL / min, 14 mL / min to 14.5 mL / min, or 14.5 mL / min to 15 mL / min. Generally speaking, increasing the flow rate increases the shear rate.

[0087] In some embodiments, the plurality of particles have a diameter between about 1 µm and about 10.5 µm. In some embodiments, the plurality of particles have a diameter between about 1 µm and 2 µm, 2 µm and 3 µm, 3 µm and 4 µm, 4 µm and 5 µm, 5 µm and 6 µm, 6 µm and 7 µm, 7 µm and 8 µm, 8 µm and 9 µm, 9 µm and 10 µm, or 10 µm and 10.5 µm. In some embodiments, the plurality of particles have a diameter between about 1.5 µm and 1.9 µm. In some embodiments, the plurality of particles have a diameter between about 2.8 µm and 3.2 µm. In some embodiments, the plurality of particles have a diameter between about 4.8 µm and 5.2 µm. In some embodiments, the plurality of particles have a diameter between about 9.8 µm and 10.2 µm. In some embodiments, the plurality of particles have a diameter of about 1.7 µm. In some embodiments, the plurality of particles have a diameter of about 3.0 µm. In some embodiments, multiple particles have a diameter of approximately 5.0 µm. In other embodiments, multiple particles have a diameter of approximately 10 µm. Generally, larger particle sizes are used to separate larger nucleic acid molecules, and smaller particle sizes are used to separate smaller nucleic acid molecules. Example 4 and Tables 2 to 5 illustrate the effect of particle size and flow rate on the ability to separate nucleic acid molecules of different sizes.

[0088] In some implementations, the particles have an average pore size between 100 Å and 900 Å. It is not desirable to be bound by any particular theory, as increasing the pore size alters the retention of larger molecules relative to smaller nucleic acid species. Example 9 and Figure 15 The effect of pore size on the retention of nucleic acid species as well as additional larger components such as proteins and buffer components is demonstrated.

[0089] In some embodiments, the viscosity of the mobile phase is adjusted. Methods for changing the viscosity of the mobile phase are known in the art and include, for example, the addition of sugar (e.g., sucrose). Generally, increased viscosity increases the shear rate. A variety of mobile phase buffers are suitable for use with the methods disclosed in this invention, as will be understood by those skilled in the art. In some embodiments, the buffer is a phosphate-buffered saline solution. In some embodiments, the phosphate-buffered saline solution is at a concentration of 1x, 2x, 0.1x, 0.01x, or any value between 0.01x and 2x.

[0090] In some embodiments, the temperature at which the separation takes place is varied. In some embodiments, the temperature is between 25°C and 50°C. In some embodiments, the temperature is between 25°C and 30°C, 30°C and 35°C, 35°C and 40°C, 40°C and 45°C and 50°C. Generally speaking, increasing the temperature decreases the shear rate.

[0091] Separation DNA , RNA or their mixtures The methods disclosed herein can be used to isolate nucleic acids, including DNA, RNA, or mixtures thereof. Nucleic acids can be ssDNA, dsDNA, ssRNA, or dsRNA, and can exist in any known topological structure, including linear, circular, or supercoiled. The length of nucleic acids can range from 1,000 to 100,000 base pairs. In some implementation schemes, the number of nucleic acids ranges from 1000 to 2000, 2000 to 3000, 3000 to 4000, 4000 to 5000, 5000 to 7500, 7500 to 10,000, 10,000 to 12,500, 12,500 to 15,000, 15,000 to 17,500, 17,500 to 20,000, 20,000 to 25,000, 25,000 to 30,000, 30,000 to 35,000, and 35,000. Between 40,000, 40,000 to 45,000, 45,000 to 50,000, 50,000 to 55,000, 55,000 to 60,000, 60,000 to 65,000, 65,000 to 70,000, 70,000 to 75,000, 75,000 to 80,000, 80,000 to 85,000, 85,000 to 90,000, 90,000 to 95,000, or 95,000 to 100,000.

[0092] Therefore, in one aspect, this document discloses a method for separating samples containing DNA and RNA. The method includes loading a sample containing DNA and RNA onto a chromatographic column comprising a plurality of porous or non-porous particles, eluting the DNA and RNA from the column, and detecting the DNA and RNA in the eluent. In some embodiments, the DNA is dsDNA and / or ssDNA. In some embodiments, the RNA is dsRNA or ssRNA. In some embodiments, the DNA and RNA are eluted from the column in a time interval of about 1 minute to 10 minutes, more preferably 1 minute to 3 minutes. In some embodiments, the DNA and RNA are eluted with a band retention factor of less than 0.3. In some embodiments, elution is performed with an efficiency greater than 40,000. Example 4 describes a method for separating samples containing DNA and RNA according to some embodiments of the present technology.

[0093] On the other hand, this document discloses a method for separating samples containing DNA. The method includes loading a DNA-containing sample onto a chromatographic column comprising multiple porous or non-porous particles, eluting the DNA from the column, and detecting the DNA in the eluent. In some embodiments, the DNA is dsDNA and / or ssDNA. In some embodiments, the DNA is eluted from the column in a time interval of about 1 minute to 10 minutes, more preferably 1 minute to 3 minutes. In some embodiments, the DNA is eluted with a band retention factor of less than 0.3. In some embodiments, elution is performed with an efficiency greater than 40,000. Examples 1 to 2 and... Figures 2 to 3 A method for separating DNA-containing samples according to some embodiments of the present technology is described.

[0094] On the other hand, this document discloses a method for separating samples containing RNA. The method includes loading an RNA-containing sample onto a chromatographic column comprising multiple porous or non-porous particles, eluting the RNA from the column, and detecting the RNA in the eluent. In some embodiments, the RNA is dsRNA and / or ssRNA. In some embodiments, DNA is eluted from the column in a time interval of about 1 to 10 minutes, more preferably 1 to 3 minutes. In some embodiments, the DNA is eluted with a band retention factor of less than 0.3. In some embodiments, elution is performed with an efficiency greater than 40,000. Example 3 and... Figure 4 A method for isolating RNA-containing samples according to some embodiments of this technology is described. Example 6 and Figure 6 A method for isolating linear dsRNA from other dsRNA impurity conformational isoforms present in a sample is described. Example 7 and... Figures 7A to 7B A method for isolating supercoiled DNA or circular DNA from linear DNA impurities is described. Example 8 and... Figures 10 to 11 , Figures 12A to 12D and Figures 13 to 14 A method for isolating DNA fragments and detecting said fragments using multi-angle light scattering (MALS) and / or dynamic light scattering (DLS) is described.

[0095] For example, but not limited to, the methods described herein can be used to isolate double-stranded RNA (dsRNA) from single-stranded RNA (ssRNA) present in a mixture. Figure 16 As shown, samples containing 4.2kb dsRNA and 4.2kb ssRNA will be isolated using the method described herein.

[0096] In some embodiments of the above methods, the eluent is collected for downstream processing. Examples of downstream processing include, but are not limited to, sequencing or cloning nucleic acid molecules using methods known in the art.

[0097] Chromatography system The methods disclosed herein can be used with any high-performance liquid chromatography (HPLC) or ultra-high-performance liquid chromatography (UHPLC) system. The system comprises a column containing a plurality of particles. These particles can be porous or non-porous. In some embodiments, the porous or non-porous particles are inorganic silica particles, organic particles, or inorganic / organic hybrid particles. Examples of suitable particles include, but are not limited to, ethylene-bridged hybrid particles (BEH; comprising tetraethoxysilane (TEOS) and bis(triethoxysilyl)ethane, as described in U.S. Patent Nos. 6,686,035 and 7,250,214, which are incorporated herein by reference). Additional particle compositions include, for example, hybrid inorganic / organic particles, such as those described in U.S. Patent Nos. 11,291,974 and 9,145,481. Additional particles may include polystyrene, divinylbenzene, polyacrylamide, and polymethacrylates with different degrees of crosslinking.

[0098] As described above, the plurality of particles can have a diameter between about 1 µm and about 10.5 µm. In some embodiments, the plurality of particles have a diameter between about 1 µm and 2 µm, 2 µm and 3 µm, 3 µm and 4 µm, 4 µm and 5 µm, 5 µm and 6 µm, 6 µm and 7 µm, 7 µm and 8 µm, 8 µm and 9 µm, 9 µm and 10 µm, or 10 µm and 10.5 µm. In some embodiments, the plurality of particles have a diameter between about 1.5 µm and 1.9 µm. In some embodiments, the plurality of particles have a diameter between about 2.8 µm and 3.2 µm. In some embodiments, the plurality of particles have a diameter between about 4.8 µm and 5.2 µm. In some embodiments, the plurality of particles have a diameter between about 9.8 µm and 10.2 µm. In some embodiments, the plurality of particles have a diameter of about 1.7 µm. In some embodiments, the plurality of particles have a diameter of about 3.0 µm. In some embodiments, the multiple particles have a diameter of about 5.0 µm. In some embodiments, the multiple particles have a diameter of about 10 µm.

[0099] In some implementations, the particles contain negatively charged surfaces, which facilitates electrostatic repulsion between negatively charged nucleic acid molecules and the particle surface. Without being bound by any particular theory, the distance between the nucleic acid molecules and the particle surface narrows the peak width and increases the separation rate at a constant resolution. This electrostatic repulsion can be further tuned by changing the ionic strength of the mobile phase, for example, by increasing or decreasing the salt content of the mobile phase. Example 5 and... Figure 5 This demonstrates the effect of ionic strength on elution time and peak resolution (i.e., the change in the k1 value of the peak) when isolating nucleic acids (such as linear double-stranded DNA). Examples of negatively charged surfaces include silanol groups, and those containing negatively charged side groups (e.g., COO2). - or SO3 - ) Silane is used for surface modification, grafting negatively charged groups onto polymer particles, or chemically modifying polymer particles to produce negatively charged groups on the surface, as will be understood by those skilled in the art.

[0100] In some embodiments, the column uses non-porous particles. Other embodiments use porous particles. In these embodiments, the analyte size is relevant to the consideration of k1. For example, in embodiments where the analyte size (e.g., length, width, volume, etc.) is larger than the size of a single pore within the particle, the k1 value can be calculated and evaluated as described above. However, if the analyte size is smaller than the pore size (e.g., DNA size is smaller than the pore size (e.g., 3000 Å)), a correction is required to account for the possibility of the analyte penetrating into the particle volume. In cases involving particle pore sizes larger than the analyte, a mixed retention mechanism will exist, altering the zonal retention factor value calculated by the "universal retention factor formula," which is defined herein and throughout: In the above formula, k 1,SEC For example, the reservation in which the SEC principle applies can be defined by the following formula: Where R g It is the radius of gyration of DNA as defined below. D meso It refers to the size of the mesopores on the particles, and Where ε e It is the porosity between the particles of the column, ε p It is the internal porosity of the particles.

[0101] In the above formula, k 1,HDC For example, the reservation in which the HDC principle takes effect can be defined by the following formula: Where D pore It refers to the average distance between particles, where D rep Defined by the following formula: And R g The radius of gyration is defined by the following formula: and <L ext > is the average length of DNA spread under shear flow conditions.

[0102] In the above formula, k 1,SC For example, the reservation where the SC principle takes effect can be defined by the following formula: Where α is an empirical proportionality constant independent of the DNA outline length (α = 0.1 mm). -1 ), It is the characteristic shear stress of the DNA strand (for dsDNA, (0.25 Pa), with the remaining variables defined above. In the context of the general retention factor formula, k... 1,SC Define variables <L ext >

[0103] Without being bound by any particular theory, it should be understood that various chromatographic principles can affect the retention of analytes (e.g., DNA or RNA molecules) based on analyte size and relaxation time (ms). For example, at increased flow rates (i.e., high shear rates), analytes with longer relaxation times will have increased retention on the column. In this context, k1 can be defined using the general retention factor formula described above.

[0104] The column material can be stainless steel, polyetheretherketone (PEEK) lined steel, titanium, or a stainless steel alloy. The column inner diameter can range from about 2.1 mm to about 7.8 mm. The column length can range from about 10 mm to about 300 mm. Exemplary column dimensions include, but are not limited to, 2.1 mm × 20 mm, 2.1 mm × 50 mm, 2.1 mm × 100 mm, 2.1 mm × 150 mm, 4.6 mm × 50 mm, 4.6 mm × 100 mm, 4.6 mm × 150 mm, and 4.6 mm × 300 mm.

[0105] The choice of column and particles, particularly regarding column length and particle size, depends in part on the size of the nucleic acid molecules to be separated and detected, as will be readily understood by one of ordinary skill in the art.

[0106] According to embodiments of this disclosure, the column is connected to the detector in a fluid series configuration. In one aspect, the detector is an ultraviolet (UV) or tunable ultraviolet (TUV) detector. In some embodiments, the detector is a multi-angle light scattering (MALS) detector. In some embodiments, the UV or TUV detector measures between 210 nm and 300 nm. In a preferred embodiment, the UV or TUV detector measures between 230 nm and 260 nm, or more preferably between 230 nm and 260 nm. These wavelengths are known in the art for detecting nucleic acid molecules, including DNA and RNA. Additional detectors, such as fluorescence spectroscopy or mass spectrometry detectors, may be used in conjunction with the methods disclosed in this invention. Detectors may be used individually or in series and may be further tuned to detect molecules of interest. For example, but not limited to, a fluorescence detector may be used if the sample contains fluorescent molecules of interest.

[0107] In some implementations, the inner surface of the column is treated to reduce nonspecific binding and improve the overall efficiency of the chromatographic system. Specifically, alkylsilane coatings or other high-performance surfaces are provided to limit or reduce nonspecific binding of the sample to the wall or inner surface of the column body. It is not desirable to be bound by theory; it is believed that alkylsilane coatings covering metal surfaces prevent or minimize contact between fluid flowing through the column body and the inner surface of the column. Typically, an alkylsilane coating is applied to a metal surface that defines the so-called column wetting path. The metal wetting path includes all metal-formed surfaces exposed to fluids during column operation. The metal wetting path includes not only the column body wall but also metal frits disposed within the column.

[0108] Generally, alkylsilane coatings are applied using vapor deposition. A precursor is loaded into a reactor containing the part to be coated. The vaporized precursor reacts on the surface of the part to be coated to form a first layer of deposited material. A step function can be used to apply vapor deposition to apply multiple layers of deposited material to the surface to increase the coating thickness and / or to apply layers of different materials (e.g., alternating between a first and a second material) to form a coating.

[0109] In one embodiment, the alkylsilane coating comprises a hydrophilic nonionic layer of polyethylene glycol silane. In another embodiment, the alkylsilane coating is formed from one or more precursor materials selected from bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane. In some embodiments, the high-performance surface is C2-PEG. The C2-PEG coating can be prepared as described in U.S. Patent Publication No. 2022 / 0118443. For example, organosilane precursors, such as bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane, can be first vapor-deposited onto a metal surface, including the inner surface of a column or a metal sieve plate of a column. After the organosilane precursor vapor-deposition, the coated metal component can be coated with a toluene solution of polyethylene glycol (PEG) (e.g., 2-[methoxy(polyoxyethylene ether)]). 6-9 Treatment with [propyl]tris(dimethylaminosilane)). The reagent solution can be reacted with the metal part for about 3 days, washed with toluene, then washed with isopropanol, and then vacuum dried at 70°C to obtain a C2-PEG coating. Other embodiments of alkylsilyl coatings suitable for use with this technology are described in U.S. Patent Publication No. 2019 / 0086371 and U.S. Patent Application Publication No. 2022 / 0118443 (which are incorporated herein by reference). Example

[0110] Example 1: Isolation of linear double-stranded DNA Linear double-stranded DNA was isolated using the methods disclosed herein. Lambda DNA ((λ-DNA) double-stranded DNA (dsDNA) samples (48,502 base pairs / nucleotides in length) were used (purchased from Thermo Scientific). ™ The sample was digested using restriction enzymes to produce six linear dsDNA fragments with lengths of approximately 2,027, 2,322, 4,361, 6,557, 9,416, and 23,130 base pairs, respectively. The sample was loaded onto a 4.6 mm × 15 cm column containing porous 1.7 µm BEH particles with a pore size of 45 Å. The column hardware was coated with a C2-PEG high-performance surface. The sample was flowed through the column using 100 mM phosphate-buffered saline (pH 8) at a flow rate of 1 mL / min. Figure 2 As shown, this method achieved robust separation of the expected dsDNA fragments and baseline separation peaks (with fragments 2,027 and 2,322 co-eluted) within 2.25 minutes. Peaks labeled as DNA-free are attributed to small molecules present in the sample entering the mesopores of the 1.7 µm BEH45 particles. The k1 values ​​for the 2,027 and 2,322 bp peaks were -0.16; for the 4,361 bp peak, 0.01; for the 6,557 bp peak, 0.24; for the 9,416 bp peak, 0.31; and for the 23,130 bp peak, 0.8. Increasing the flow rate or particle size can provide lower k1 values ​​for the 23,130 bp fragment.

[0111] Example 2: Isolation of Circular and Linear Double-Stranded DNA The ability to isolate DNA molecules with different topologies using the methods disclosed herein was established. 5,386 samples of nicked circular double-stranded DNA fX174 RF II (5,386 base pairs / nucleotides in length) (purchased from NEB) were used. Approximately 90% of the samples contained nicked circular DNA, with the remainder being supercoiled or linear.

[0112] The sample was loaded onto a 4.6 mm × 15 cm column containing porous 1.7 µm BEH particles with a pore size of 45 Å. The column hardware was coated with a C2-PEG high-performance surface. The sample was flowed through the column using 100 mM phosphate-buffered saline (pH 8) at a flow rate of 1 mL / min. Figure 3 As shown, this method achieves robust selectivity between circular and linear forms of DNA samples within 1.2 minutes. In contrast, separation of the same sample using size exclusion chromatography results in poor selectivity between these two forms, with elution times as long as 12 to 15 minutes (data not shown). Therefore, the method disclosed herein can separate samples of similar lengths with different topologies. For this separation, the k1 value for linear DNA was 0.15. The unlabeled third peak did not contain DNA.

[0113] Example 3: Isolation of single-stranded and double-stranded RNA The ability to separate single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA) using the methods disclosed herein was established. KH20 RNA samples with a length of approximately 2,400 base pairs / nucleotide (purchased from New England Biolabs) were used.

[0114] The sample was loaded onto a 4.6 mm × 150 mm column containing porous 1.7 µm BEH particles with a pore size of 45 Å. The column hardware was coated with a C2-PEG high-performance surface. The sample was flowed through the column using 100 mM phosphate-buffered saline (pH 8) at a flow rate of 1 mL / min. Figure 4 As shown, this method can distinguish between ssRNA and dsRNA forms in an RNA sample within 1 minute (the dashed box indicates a magnified area of ​​the chromatogram). Therefore, the method disclosed in this invention provides a rapid detection of ssRNA purity in a sample. For this separation, the k1 value of the 2.4 kb dsRNA sample was -0.24.

[0115] Example 4: Separation of DNA / RNA by flow rate and particle size The relationship between particle size, flow rate, and nucleic acid size was determined. Single-stranded RNA (ssRNA), single-stranded DNA (ssDNA), double-stranded RNA (dsRNA), and double-stranded DNA (dsDNA) of different lengths were tested, and the selectivity factor (α) for separating double-stranded molecules from single-stranded molecules was determined. ds / ss ) or a selectivity factor (α) for separating double-stranded DNA from double-stranded RNA. ds2 / ds1 Experiments were repeated at a series of flow rates using particles with sizes of 1.7 µm, 3.0 µm, 5.0 µm, and 10 µm on a 4.6 mm inner diameter column. The particles were BEH porous particles (average pore size approximately 125 Å). The column hardware was coated with a C2-PEG high-performance surface.

[0116] The relaxation time (τ) of the tested DNA / RNA molecules R ) and radius of gyration (R) G The summary is shown in Table 1.

[0117] Table 1: Summary of parameters of the tested nucleic acids The ability to separate nucleic acid molecules was first tested using 1.7µm particles at flow rates of 0.1 mL / min, 0.5 mL / min, and 1.0 mL / min. In relevant cases, the principle of hydrodynamic chromatography (HDC) was also used. The selectivity factor was calculated when the shear rate approached zero. The selectivity factor was determined as described above. As shown in Table 2, generally speaking, at a particle size of 1.7 µm and a flow rate >0.5 mL / min, good separation was provided by the selectivity factor for nucleic acid molecules between 5,000 bp / nt and 25,000 bp / nt, including both double-stranded and single-stranded molecules.

[0118] Table 2: Specificity factor using 1.7µm particles The ability to separate nucleic acid molecules was then tested using 3.0 µm particles at flow rates of 0.25 mL / min, 0.75 mL / min, or 1.5 mL / min. Selectivity factors were also calculated using hydrodynamic chromatography (HDC; a method different from zigzag chromatography due to its low shear rate) where appropriate. The selectivity factors were determined as described above. As shown in Table 3, generally, a particle size of 3 µm and a flow rate >0.25 mL / min provided good separation of nucleic acid molecules between 5,000 bp / nt and 50,000 bp / nt, including both double-stranded and single-stranded types, as measured by the selectivity factor. A flow rate of 0.25 mL / min was able to separate nucleic acid types up to 100 kbp / knt in length.

[0119] Table 3: Specificity factors using 3.0µm particles The ability to separate nucleic acid molecules was then tested using 5.0 µm particles at flow rates of 1.0 mL / min, 2.5 mL / min, or 5.0 mL / min. Selectivity factors were also calculated using hydrodynamic chromatography (HDC; a method different from zigzag chromatography due to its low shear rate) where appropriate. The selectivity factors were determined as described above. As shown in Table 4, generally, a particle size of 5 µm and a flow rate >1.0 mL / min provided good separation of nucleic acid molecules between 5,000 bp / nt and 50,000 bp / nt, including both double-stranded and single-stranded types, as measured by the selectivity factor. A flow rate of 1.0 mL / min was able to separate nucleic acid types up to 100 kbp / knt in length.

[0120] Table 4: Specificity Factors Using 5.0µm Particles Finally, the ability to separate nucleic acid molecules was tested using 10 µm particles at flow rates of 2.5 mL / min, 7.5 mL / min, or 15 mL / min. In relevant cases, the selectivity factor was also calculated using hydrodynamic chromatography (HDC; a method different from zigzag chromatography due to its low shear rate). The selectivity factor was determined as described above. As shown in Table 5, generally, at a particle size of 10 µm and a flow rate >2.5 mL / min, good separation was provided for nucleic acid molecules between 25,000 bp / nt and 100,000 bp / nt, including both double-stranded and single-stranded molecules, as measured by the selectivity factor.

[0121] Table 5: Specificity Factors Using 10µm Particles In summary, the methods disclosed in this invention are capable of separating nucleic acid molecules and mixtures thereof spanning a range of nucleotide lengths (from 1,000 to 100,000 base pairs / nucleotide). Generally, larger particle sizes can separate larger nucleic acid molecules, and vice versa (e.g., smaller particle sizes can separate smaller nucleic acid molecules). These methods further demonstrate the ability to separate single-stranded and double-stranded molecules.

[0122] The above method also allows for the measurement of dsDNA length using known standards, such as... Figure 8A (For 1.7µm particles) and Figure 8B As depicted (for 2.5µm particles). Figures 8A to 8B The correlation between dsDNA length, measured in base pairs, and the k coefficient is shown.

[0123] The above method also allows for the separation of DNA and RNA using 1.7 µm diethylene-bridged hybrid particles (BEH) with a diol-bonded surface and an average pore size of 130 Å, such as... Figure 9 As shown.

[0124] Example 5: Effect of ionic strength on the separation of double-stranded DNA The effect of mobile phase ionic strength on the separation of double-stranded DNA was evaluated. Double-stranded DNA (dsDNA) plasmid digestion products were loaded onto a 4.6 mm × 300 mm column containing 2.6 µm non-porous silica particles, and eluted using mobile phases of 1X PBS (high ionic strength), 0.1X PBS, 0.01X PBS, or 0.001X PBS (low ionic strength). The column hardware was coated with a C2-PEG high-performance surface.

[0125] like Figure 5 As shown, reducing the ionic strength of the mobile phase shortens retention time (e.g., faster peak elution) while improving peak separation and the k1 value of the peaks. At 1X PBS, higher molecular weight peaks eluted after 10 minutes, with k1 factors higher than 0.35. In this embodiment, using 2.6µm non-porous silica particles, reducing the ionic strength of the mobile phase from 1X PBS to 0.1X PBS or more preferably 0.01X PBS resulted in improved overall separation of the dsDNA sample. The results shown for 0.1X PBS and 0.01X PBS indicate elution peaks with k1 factors equal to or lower than 0.35. Reducing the ionic strength to 0.001X PBS resulted in DNA melting due to sample instability in the mobile phase.

[0126] The ionic strength of the mobile phase can be further adjusted to improve peak shape and change retention time, taking into account the net negative charge of the sample being tested and the particles used in the column. For example, the adjustment or modification of the particle surface can be controlled to generate a net negative charge on the particle surface, thereby affecting the ionic strength conditions for elution in the column.

[0127] Example 6: Isolation of double-stranded RNA (dsRNA) species Double-stranded RNA (dsRNA) samples were loaded onto a 4.6 mm × 300 mm column containing diethylene-bridged hybrid (BEH) diol particles and eluted using either a low-shear flow rate (0.025 mL / min) or a high-shear flow rate (1.2 mL / min). The column hardware was coated with a C2-PEG high-performance surface. Figure 6 As shown, increasing the flow rate from 0.025 mL / min to 1.2 mL / min altered the retention of dsRNA species, thereby allowing the separation of impurity conformational isoforms and target linear dsRNA in less than 1 minute.

[0128] Example 7: Isolation of Circular / Supercoiled and Linear DNA Species Samples containing supercoiled DNA (ΦX 174 RF I DNA) or circular DNA (ΦX 174 RF II DNA) were loaded onto a 4.6 mm × 150 mm column containing 1.7 µm ethylene-bridged hybrid (BEH) particles with an average pore size of 45 Å. The column hardware was coated with a C2-PEG high-performance surface. Samples were flowed through the column using 100 mM phosphate buffer (pH 8) at flow rates of 0.2 mL / min, 0.3 mL / min, 0.5 mL / min, or 1 mL / min. Figures 7A to 7B As shown, increasing the flow rate allows for the extraction of supercoiled DNA samples ( Figure 7A ) and circular DNA samples ( Figure 7B Linear dsDNA impurities were isolated from the sample.

[0129] Example 8: Detection of isolated nucleic acids using multi-angle light scattering (MALS) Samples containing λ DNA-HindIII digestion products (purchased from Promega Corporation) were loaded onto a 4.6 mm × 300 mm column containing 2.5 µm diethylene-bridged hybrid (BEH) particles with an average pore size of 125 Å. The column hardware was coated with a C2-PEG high-performance surface. Samples were loaded at concentrations of 5 µg or 0.5 µg and flowed through the column with 1X phosphate-buffered saline (PBS) at a flow rate of 0.2 mL / min. The eluent was detected using a multi-angle light scattering (MALS) detector. Figure 10 As shown, DNA digestion products can be separated and detected using MALS, especially at higher concentrations, where 0.5 µg represents the detection limit for light scattering.

[0130] As a calibrator, samples containing small interfering RNA (siRNA) were loaded onto a 4.6 mm × 300 mm column containing 2.5 µm diethylene-bridged hybrid (BEH) particles with an average pore size of 125 Å. The column hardware was coated with a C2-PEG high-performance surface. Samples were loaded at a concentration of 1 µg and 1X PBS was flowed through the column at a flow rate of 0.2 mL / min. The eluent was detected using a MALS detector. Figure 11 Chromatograms of samples detected using MALS are provided. The traces labeled LS and UV represent light scattering and UV measurements, respectively. Black squares represent molar mass (MM) measurements. The expected monomer mass of siRNA is 13.2 kDa, and the observed fitted monomer mass is approximately 12.4 kDa. Therefore, siRNA is determined to be a suitable calibrator for MALS detection in combination with the provided method.

[0131] siRNA was used as a calibrator to model DNA-HindIII digestion products to determine their molar mass. Figures 12A to 12B Provided before UV signal broadening ( Figure 12A ) and after expansion ( Figure 12B Chromatogram of DNA-HindIII digestion products. Small peaks (corresponding to lower molecular weight DNA fragments) conform to the Zimm pattern, such as... Figure 12C (Peak X) and Figure 12D As shown in (peak Y), and in the form of random coils, as... Figure 12E (Peak X) and Figure 12F As shown in (peak Y). Figure 12C and Figure 12D Provided the corresponding Figures 12A to 12B Zim plot of peaks X and Y marked in the middle. Figure 12E and Figure 12F Provided the corresponding Figures 12A to 12B The random coil plot of peaks X and Y marked in the figure. Methods for modeling MALS-derived data and determining relevant parameters for said models are known in the art and are described, for example, in U.S. Patent No. 6,651,009, which is incorporated herein by reference. Figure 13 The light scattering and molar mass measurements of samples detected at 0.1 µg, 0.5 µg, 2 µg, and 5 µg are shown.

[0132] In addition to MALS detection, dynamic light scattering (DLS) can also be used to detect samples to determine the hydration radius (R). H Samples containing λ DNA digestion products were loaded onto a 4.6 mm × 300 mm column containing 2.5 µm diethylene-bridged hybrid (BEH) particles with an average pore size of 125 Å. Samples were flowed through the column at a flow rate of 0.2 mL / min and detected using MALS and DLS detectors. Figure 14 Representative chromatograms of the detected nucleic acid types are provided, including UV and light scattering traces, molar mass measurements (points), and calculated hydration radius values ​​(bar chart in the upper right corner).

[0133] Example 9: Isolation of nucleic acids from macromolecules This technique can be used to separate nucleic acids (such as dsDNA, ssDNA, dsRNA, or ssRNA) from additional macromolecules (including biomolecules (proteins, such as enzymes)) and buffer components. A sample containing dsDNA gradient standards with DNA fragments of 2kb, 4.4kb, 6.6kb, 9.4kb, and 23.1kb, RNase, pyrophosphatase, NTP, buffer, and T7 RNA polymerase is separated on a column containing 2.5µm diethylene-bridged hybrid (BEH) particles (pore size 125Å or 250Å). Figure 15 As shown in the figure above, the 125 Å pore size results in the co-elution of DNA with additional components present in the sample. In contrast, as... Figure 15 As shown in the figure below, when using 250 Å pore size particles, DNA fragments in the range of 2 kb to 9.4 kb are eluted from the column before additional components present in the sample. Therefore, 250 Å pore size particles can be used in conjunction with the provided method to isolate nucleic acids, such as DNA or RNA, from additional components present in a mixture.

[0134] Example 10: Zigzag Chromatography Using Non-porous Particles Zigzag chromatography analysis was performed using columns containing nonporous silica-based particles of different sizes at different flow rates. Figures 18A to 18D Chromatographic experiments were performed on a sample containing λ-DNA-HindIII digestion products. The column used was a 4.6 mm × 300 mm column packed with 3 µm non-porous silica particles. The mobile phase was water, and various flow rates were employed. At a low flow rate (0.05 mL / min), the experiment took approximately 52 minutes in total. Many peaks were observed, but several overlapped and were not independent. Increasing the flow rate to 0.1 mL / min... Figure 18B ), 0.2 mL / min Figure 18C ) or 0.3 mL / min ( Figure 18D Better separation was observed between the main peaks in the spectrum. Furthermore, the time required to completely elute all components of the sample was significantly reduced.

[0135] The experiment was then repeated using 2.6µm non-porous silica particles. Figures 19A to 19D Each experiment was performed at pH 7.4 using 1M phosphate-buffered saline as the mobile phase. In both experiments, the samples were λ-DNA-HindIII digestion products (…). Figure 19A and Figure 19B In the other two experiments, the samples were mixtures of λ-DNA single enzyme digestions (…). Figure 19C and Figure 19D In addition, two experiments ( Figure 19B and Figure 19D The mobile phase additive included 0.5 vol% 2-[2-(diethylamino)ethoxy]ethanol. Good separation was observed in each experiment.

[0136] Example 11: The effect of pH in zigzag chromatography using non-porous particles The performance of a 4.6 × 150 mm column containing 2.6 µm nonporous silica particles was then examined at different pH values. Figures 20A to 20C In each experiment, the sample was the λ-DNA-HindIII digestion product, and the mobile phase was water containing 0.5 v / v tetramethylammonium chloride and 0.5 v / v tris(hydroxymethyl)aminomethane at a flow rate of 0.1 mL / min. The experiment was conducted at an acidic pH (…). Figure 20A At neutral pH (), only two peaks were observed, partly due to aggregate formation, with the aggregates eluting as a single peak at approximately 11.65 min. Figure 20B At alkaline pH (), aggregates are better separated as multiple overlapping peaks. Figure 20C At this pH level, the entire chromatogram is better separated, and multiple independent peaks can be distinguished. This experiment shows that zigzag chromatography of oligonucleotides is particularly effective at alkaline pH.

[0137] Example 12: Effect of ionic strength on the separation of non-porous particles The effect of solution ionic strength on separation using non-porous particles was then examined. Samples of λ-DNA single-enzyme digestion mixtures were studied using a 2.1 × 300 mm column containing 2.2 µm BEH particles at a flow rate of 0.05 mL / min, pH 7.4, and three different concentrations of buffer solution. Figure 21A The chromatogram under 1X PBS is shown. Figure 21B The chromatogram under 2X PBS is shown. Figure 21C Chromatograms under 4X PBS are shown. Good separation was observed in each experiment.

[0138] Example 13: Zigzag Chromatography Using Porous Surface Particles The study investigated the use of zigzag chromatography of oligonucleotides with various porous surface particles. Figures 22 to 2 6). Particle types include 3.0µm hybrid (inorganic-organic) particles with a solid core (such as particles sold by Waters Corporation under the CORTECS trademark). Figure 22 ); 2.7µm solid silica particles (e.g., CORTECS silica particles) Figure 23 ), 1.6µm solid-core silica particles with T3 bonded phase (e.g., Cortecs-T3 from Waters Corporation) Figures 24A to 24B ), 1.6µm solid-core silica particles with an average pore size of 90Å (e.g., CORTECS90Å) Figures 25A to 25B ), and 1.6µm solid-core silica particles with an average pore size of 120Å (e.g., CORTECS120Å) ( Figures 26A to 26B The sample included a mixture of λ-DNA single enzyme digestions (…). Figure 22 ), λ-DNA BstEII digestion product ( Figure 23 , Figure 24B , Figure 25B , Figure 26B ) or 1 kbps DNA gradient standards ( Figure 24A , Figure 24B , Figure 25B Experiments using a 1.6 µm silica core with a T3 bonded phase (e.g., Cortecs-T3) also included 20% acetonitrile in the mobile phase. Good separation was observed in each experiment.

[0139] Example 14: Zigzag Chromatography Using Diol-Functioned Particles The study investigated oligonucleotide zigzag chromatography using particles with diol-functionalized surfaces. Figure 27 The separation of λ-DNA HindIII digestion products using a 4.6 × 300 mm column containing non-porous 3.5 µm divinylbenzene (DVB) organic polymer particles surface-modified with ethylene glycol is shown. The mobile phase was phosphate at pH 8. Good separation was observed at various flow rates. Different oligonucleotides were better separated at different flow rates.

[0140] Example 15: Using sulfate esters base Zigzag Chromatography of Functionalized Particles A zigzag chromatography method for oligonucleotides using particles with sulfate-functionalized surfaces was investigated. Figures 28A to 28C The particles described in Example 14 are shown in further functionalization to include SO3. − Separation efficiency after functionalization. Particles were packed into a 4.6 × 300 mm column with 100 mM phosphate (pH 8) as the mobile phase. A flow rate of 0.8 mL / min was used in the initial study. Compared to unfunctionalized particles, sulfate-functionalized particles showed significantly narrowed peaks (…). Figure 28A This experiment demonstrates that particles functionalized with negatively charged surface groups exhibit particular effectiveness in DNA separation. Figures 28B to 28D This indicates that these SO3 − The functionalized particles exhibited a similar dependence on ionic strength and flow rate as observed in similar experiments.

[0141] Example 16: Zigzag Chromatography Using Amide-Functionalized Particles The study investigated oligonucleotide zigzag chromatography using particles containing amide-functionalized surfaces. Figures 29A to 29D The separation efficiency of a 2.1 × 150 mm column containing 1.7 µm BEH130 particles functionalized to include an amide surface is shown. These particles were investigated at various flow rates (0.375 mL / min). Figure 29A ), 0.20 mL / min Figure 29B ), 0.10 mL / min Figure 29C ) and 0.05 mL / min ( Figure 29D )). Figure 29E The gel electrophoretic separation of the samples is then shown, indicating the types of oligonucleotides that could be separated in the experiments. Good separation was observed in each experiment. These experiments demonstrate that the reversed-phase liquid chromatography (RPLC) method is compatible with the zigzag chromatography method disclosed herein.

[0142] Example 17: Zigzag Chromatography Using Particles with Hydrophobic Surfaces Zigzag chromatography of oligonucleotides using particles with surfaces functionalized with hydrophobic groups was investigated. 1.7 µm BEH particles with an average pore size of 45 Å and functionalized with trimethylchlorosilane surface groups were packed into a 4.6 × 300 mm column. Separation was performed using a 70%:30% (v / v) water / acetonitrile solution of 100 mM phosphate (pH 8) as the mobile phase at a flow rate of 0.9 mL / min. Figure 30A The separation results were compared with those of the unfunctionalized surface ( ). Figure 30B The comparison was performed at a flow rate of 1.0 mL / min. Due to the difference in flow pressure resistance, a flow rate of 0.9 mL / min (as used in the functionalized surface experiment) and a flow rate of 1.0 mL / min (as used in the unfunctionalized surface experiment) produced similar pressure drops along the column length in both experiments.

[0143] Unlike the hydrophilic surfaces used in Examples 14 to 16, the hydrophobic C1 surfaces exhibit broader peaks and lower separation compared to the unfunctionalized surfaces.

Claims

1. A method for separating DNA and RNA, the method comprising: a) A sample containing multiple single-stranded RNA (ssRNA) molecules and multiple double-stranded DNA (dsDNA) molecules is loaded onto a chromatographic column, wherein the column contains multiple particles; b) Elute the sample from the column, wherein the elution is performed between 0.6 minutes and 10 minutes; and c) Detect the plurality of ssRNA molecules and / or the plurality of dsDNA molecules in the eluent, wherein the plurality of ssRNA molecules form at least one independent peak, and wherein the plurality of dsDNA molecules form at least one independent peak.

2. The method of claim 1, wherein the elution is performed between 1 minute and 3 minutes.

3. The method according to claim 1 or 2, wherein the elution produces a band retention factor of less than 0.

35.

4. A method for isolating double-stranded DNA (dsDNA), the method comprising: a) Loading a sample containing multiple dsDNA molecules onto a chromatographic column, wherein the column contains multiple particles; b) Elute the sample from the column, wherein the elution produces a zonal retention factor (k1) less than 0.35; and c) Detect the plurality of dsDNA molecules in the elution buffer.

5. The method according to claim 4, wherein the sample further comprises a plurality of single-stranded RNA (ssRNA) molecules, a plurality of double-stranded RNA (dsRNA) molecules and / or a plurality of single-stranded DNA (ssDNA) molecules.

6. The method according to claim 4 or 5, wherein the dsDNA is linear, circular, circular, or supercoiled, and / or wherein the ssRNA is linear, circular, or circular.

7. The method of claim 5, wherein step c) further comprises detecting the plurality of ssRNA molecules, the plurality of dsRNA molecules and / or the plurality of ssDNA molecules in the elution buffer.

8. The method according to any one of claims 4 to 7, wherein the ionic strength conditions for eluting the sample from the column are tailored such that the band retention factor is less than 0.

33.

9. A method for isolating DNA, the method comprising: a) Loading a sample containing multiple double-stranded DNA (dsDNA) molecules and single-stranded DNA (ssDNA) molecules onto a chromatographic column, wherein the column contains multiple particles; b) Elute the sample from the column, wherein the elution produces a zonal retention factor (k1) less than 0.35; and c) Detect the plurality of dsDNA molecules and ssDNA molecules in the elution buffer.

10. A method for isolating RNA, the method comprising: a) Loading a sample containing multiple single-stranded RNA (ssRNA) molecules and multiple double-stranded RNA (dsRNA) molecules onto a chromatographic column, wherein the column contains multiple particles; b) Elute the sample from the column, wherein the elution produces a zonal retention factor (k1) less than 0.35; and c) Detect the plurality of ssRNA molecules and / or the plurality of dsRNA molecules in the eluent.

11. A method for separating DNA and RNA, the method comprising: a) A sample containing multiple single-stranded RNA (ssRNA) molecules and multiple double-stranded DNA (dsDNA) molecules is loaded onto a chromatographic column, wherein the column contains multiple particles; b) Elute the sample from the column, wherein the elution produces a zonal retention factor (k1) less than 0.35; and c) Detect the plurality of ssRNA molecules and / or the plurality of dsDNA molecules in the eluent.

12. The method according to any one of claims 8 to 10, wherein the elution is performed within 10 minutes; preferably between 1 minute and 3 minutes.

13. The method according to any one of claims 1 to 12, wherein step b) is performed with an efficiency (N) of >40,000.

14. The method according to any one of claims 1 to 13, wherein the eluent is collected.

15. The method according to any one of claims 1 to 14, wherein the dsDNA, the ssDNA, the ssRNA and / or the dsRNA comprises about 1,000 base pairs to about 100,000 base pairs.

16. The method according to any one of claims 1 to 15, wherein the diameter of said plurality of particles is between about 1 µm and about 10 µm; preferably between 1.5 µm and 5 µm.

17. The method according to any one of claims 1 to 16, wherein at least a portion of the inner surface of the column body is coated with an alkylsilyl material.

18. The method of claim 17, wherein the alkylsilane material is a hydrophilic nonionic layer of polyethylene glycol silane.

19. The method of claim 17, wherein the alkylsilyl material is a vapor-deposited product of bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane.

20. The method according to any one of claims 1 to 19, wherein the chromatographic column is connected to a high-performance liquid chromatography (HPLC) system or an ultra-high-performance liquid chromatography (UHPLC) system.

21. The method according to any one of claims 1 to 20, wherein step c) is performed using an ultraviolet (UV) detector, an adjustable ultraviolet (TUV) detector, a multi-angle light scattering (MALS) detector, or a dynamic light scattering (DLS) detector.

22. The method according to any one of claims 1 to 21, wherein step b) is performed at a flow rate greater than 0.5 mL / min, and wherein the dsDNA, the ssDNA, the ssRNA and / or the dsRNA comprises about 2,000 base pairs to about 25,000 base pairs.

23. The method of claim 22, wherein the plurality of particles have a diameter between 1.5 µm and 1.9 µm.

24. The method according to any one of claims 1 to 21, wherein step b) is performed at a flow rate greater than 0.75 mL / min, and wherein the dsDNA, the ssDNA, the ssRNA and / or the dsRNA comprises about 5,000 base pairs to about 50,000 base pairs.

25. The method of claim 24, wherein the plurality of particles have a diameter between 2.8 µm and 3.2 µm.

26. The method according to any one of claims 1 to 21, wherein step b) is performed at a flow rate greater than 1.0 mL / min, and wherein the dsDNA, the ssDNA, the ssRNA and / or the dsRNA comprises about 25,000 base pairs to about 50,000 base pairs.

27. The method of claim 26, wherein the plurality of particles have a diameter between 4.8 µm and 5.2 µm.

28. The method according to any one of claims 1 to 21, wherein step b) is performed at a flow rate greater than 2.5 mL / min, and wherein the dsDNA, the ssDNA, the ssRNA and / or the dsRNA comprises about 25,000 base pairs to about 100,000 base pairs.

29. The method of claim 28, wherein the plurality of particles have a diameter between 9.8 µm and 10.2 µm.

30. The method according to any one of claims 3 to 29, wherein k1 is calculated using a general retention factor formula.

31. The method according to any one of claims 9 to 11, wherein the ionic strength conditions for eluting the sample from the column are tailored such that the band retention factor is less than 0.

33.

32. The method according to any one of claims 1 to 31, wherein the plurality of particles comprises a plurality of non-porous particles.

33. The method according to any one of claims 1 to 31, wherein the plurality of particles comprises a plurality of porous particles.

34. The method according to any one of claims 1 to 31, wherein the plurality of particles comprises a plurality of surface-porous particles.

35. The method of claim 33 or 34, wherein the plurality of porous particles or the plurality of surface porous particles have an average pore size between 20 Å and 900 Å.

36. The method of claim 35, wherein the average pore size is about 45 Å.

37. The method of claim 35, wherein the average pore size is about 125 Å.

38. The method of claim 35, wherein the average pore size is about 250 Å.

39. The method according to any one of claims 33 to 38, wherein the plurality of particles have a size between 0.5 cm 3 / g to 1.3cm 3 Specific pore volume between / g.

40. The method according to any one of claims 1 to 39, wherein the plurality of particles comprise inorganic materials.

41. The method of claim 40, wherein the inorganic material comprises silicon dioxide.

42. The method according to any one of claims 1 to 39, wherein the plurality of particles comprise an organic polymer.

43. The method of claim 42, wherein the organic polymer comprises divinylbenzene.

44. The method according to any one of claims 1 to 39, wherein the plurality of particles comprise a hybrid organic-inorganic material.

45. The method of claim 44, wherein the hybrid organic-inorganic material comprises a bridged ethylene hybrid (BEH) material.

46. ​​The method according to any one of claims 1 to 45, wherein the plurality of particles comprises functional groups bound to the surface of the particles, wherein the functional groups are hydrophilic functional groups.

47. The method of claim 46, wherein the hydrophilic functional group is a negatively charged functional group.

48. The method according to claim 46 or 47, wherein the hydrophilic functional group is a sulfate ester group.

49. The method of claim 46, wherein the hydrophilic functional group comprises a diol.

50. The method of claim 46, wherein the hydrophilic functional group comprises an amide.

51. The method according to any one of claims 1 to 45, wherein the plurality of particles comprise functional groups bound to the surface of the particles, wherein the functional groups are hydrophobic functional groups.

52. A method for isolating RNA, the method comprising: a) Loading a sample containing multiple single-stranded RNA (ssRNA) molecules and multiple double-stranded RNA (dsRNA) molecules onto a chromatographic column, wherein the column contains multiple particles; b) Elute the sample from the column, wherein the elution is performed over a period of no more than 3 minutes; and c) Detect the plurality of ssRNA molecules and / or the plurality of dsRNA molecules in the eluent.

53. The method of claim 52, wherein the elution is performed within a maximum of 1 minute.

54. The method of claim 52, wherein the plurality of particles comprise bridged ethylene hybrid (BEH) particles with a pore size of about 45 Å.

55. The method according to claim 52, wherein the elution in step (b) has a flow rate of about 1 mL / min.

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