Separation of long oligonucleotides

Abstract

A method for separating and analyzing oligonucleotides or nucleic acid biopolymers includes the following steps: loading a sample containing multiple oligonucleotide species onto a column; injecting a mobile phase consisting of a combination of a first solution and a second solution into the column to elute the multiple oligonucleotide species; and analyzing at least one of the first oligonucleotide species and the second oligonucleotide species using mass spectrometry. The multiple oligonucleotide species include a first oligonucleotide species and a second oligonucleotide species. The first solution contains an ion-pairing reagent. The ion-pairing reagent contains a primary amine or a secondary amine. The ratio of the first solution to the second solution in the mobile phase is changed over time to separate the first oligonucleotide species from the second oligonucleotide species.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Non-provisional Application No. 63 / 482,151, filed January 30, 2023, the entire contents of which are incorporated herein by reference.

[0002] Technical Field FIELD OF THE DISCLOSURE The present disclosure relates generally to the technical fields of chromatography and mass spectrometry, including methods for separating long oligonucleotides. [Background technology]

[0003] Introduction Recent events, particularly the COVID-19 pandemic, have led to the widespread adoption of mRNA as a useful therapeutic approach, including various mRNA-based COVID vaccines. The production of artificial transcripts by in vitro transcription requires analytical testing to determine the outcome and yield of the reaction. Ion-pair reversed-phase chromatography is widely used for the characterization of proteins and nucleic acids, including pharmaceutical analysis. Ion-pair reversed-phase chromatography, coupled with UV detection and mass spectrometry, can be used to characterize primary drug products and assess impurities in pharmaceuticals. However, nucleic acids are composed of many structurally and chemically related variants. These variants are often not fully characterized. The above highlights the need for improved separation methods for long oligonucleotides in the future. Summary of the Invention [Means for solving the problem]

[0004] Summary of the Invention In a first aspect, a method for separation of analyzing oligonucleotides or nucleic acid biopolymers can include loading a sample containing multiple oligonucleotide species onto a column. The multiple oligonucleotide species can include a first oligonucleotide species and a second oligonucleotide species. The method can further include injecting a mobile phase comprising a combination of a first solution and a second solution onto the column to elute the multiple oligonucleotide species. The first solution can include an ion pairing reagent. The ion pairing reagent can include a primary amine or a secondary amine. The ratio of the first solution to the second solution in the mobile phase can be varied over time to separate the first oligonucleotide species from the second oligonucleotide species. The method can further include analyzing at least one of the first oligonucleotide species and the second oligonucleotide species using a mass spectrometer.

[0005] In various embodiments of the first aspect, the sample can include a solution of multiple oligonucleotide species in a first solution, a second solution, or a combination thereof.

[0006] In various embodiments of the first aspect, the second solution can include an ion-pairing reagent.

[0007] In various embodiments of the first aspect, the ion-pairing reagent can include at least four carbons. In certain embodiments, the primary amine of the ion-pairing reagent can include an alkyl chain of at least four carbons, such as amylamine or hexylamine. In certain embodiments, the secondary amine of the ion-pairing reagent can include two alkyl chains, at least one of which can include at least three carbons (e.g., N-ethylpropylamine, dipropylamine (DPA), N-ethylbutylamine, N-propylbutylamine, dibutylamine (DBA), dipentalamine, or dihexylamine).

[0008] In various embodiments of the first aspect, the first solution can include a fluoroalcohol, such as hexafluoroisopropanol (HFIP).

[0009] In various embodiments of the first aspect, the first solution can include formic acid or formate, acetic acid or acetate, or any combination thereof.

[0010] In various embodiments of the first aspect, the first solution can include a first organic solvent such as methanol, ethanol, acetone, acetonitrile, tetrahydrofuran, isopropanol, or any combination thereof.

[0011] In various embodiments of the first aspect, the second solution can include a second organic solvent, such as methanol, ethanol, acetone, acetonitrile, tetrahydrofuran, isopropanol, or a combination thereof.

[0012] In various embodiments of the first aspect, the column can include a stationary phase such as divinylbenzene or a derivative thereof. In certain embodiments, the stationary phase can be porous with an average pore size between about 50 angstroms and about 200 angstroms, and the first and second oligonucleotide species can have a size of less than 10 kDa. In certain embodiments, the stationary phase can be porous with an average pore size between about 200 angstroms and about 500 angstroms, and the first and second oligonucleotide species can have a length of 300 nt or less. In certain embodiments, the stationary phase can be porous with an average pore size between about 500 angstroms and about 4000 angstroms, and the first and second oligonucleotide species can have a length of 300 nt or more.

[0013] In various embodiments of the first aspect, the first and second seed oligonucleotide species can be up to about 15,000 nt in length.

[0014] In various embodiments of the first aspect, the plurality of oligonucleotide species can be ribonucleic acid (RNA) oligonucleotides. In certain embodiments, the ribonucleic acid (RNA) oligonucleotides can include modifications. In certain embodiments, the ribonucleic acid (RNA) oligonucleotides can be produced in vitro. In certain embodiments, the ribonucleic acid (RNA) oligonucleotides can be produced in vivo. In certain embodiments, the ribonucleic acid (RNA) oligonucleotides can be encapsulated RNA oligonucleotides.

[0015] In a second aspect, a method for analytical separation of encapsulated oligonucleotides or nucleic acid biopolymers can include dissolving a sample containing multiple encapsulated oligonucleotide species. The multiple encapsulated oligonucleotide species can include a first encapsulated oligonucleotide species comprising a first oligonucleotide species and a second encapsulated oligonucleotide species comprising a second oligonucleotide species. Solubilizing the sample can include releasing the first and second oligonucleotide species. The method also includes loading the solubilized sample onto a column and injecting a mobile phase comprising a combination of the first and second solutions onto the column to elute the first and second oligonucleotide species. The first solution can include an ion-pairing reagent. The ion-pairing reagent can include a primary amine or a secondary amine. The ratio of the first and second solutions in the mobile phase can be varied over time to separate the first oligonucleotide species from the second oligonucleotide species. The method can further include analyzing at least one of the first and second oligonucleotide species using a mass spectrometer.

[0016] In various embodiments of the second aspect, the sample can include a plurality of oligonucleotide species dissolved in a first solution, a second solution, or a combination thereof.

[0017] In various embodiments of the second aspect, the second solution can include an ion-pairing reagent.

[0018] In various embodiments of the second aspect, the ion-pairing reagent can include at least four carbon atoms. In certain embodiments, the primary amine of the ion-pairing reagent can include an alkyl chain having at least four carbon atoms, such as amylamine or hexylamine. In certain embodiments, the secondary amine of the ion-pairing reagent can include two alkyl chains, at least one of which can include at least three carbon atoms (e.g., N-ethylpropylamine, dipropylamine (DPA), N-ethylbutylamine, N-propylbutylamine, dibutylamine (DBA), dipentalamine, or dihexylamine).

[0019] In various embodiments of the second aspect, the first solution can include a fluoroalcohol, such as hexafluoroisopropanol (HFIP).

[0020] In various embodiments of the second aspect, the first solution can include formic acid or a formate, acetic acid, or an acetate, or any combination thereof.

[0021] In various embodiments of the second aspect, the first solution can include a first organic solvent such as methanol, ethanol, acetone, acetonitrile, tetrahydrofuran, isopropanol, or any combination thereof.

[0022] In various embodiments of the second aspect, the second solution can include a second organic solvent, such as methanol, ethanol, acetone, acetonitrile, tetrahydrofuran, isopropanol, or any combination thereof.

[0023] In various embodiments of the second aspect, the column can include a stationary phase such as divinylbenzene or a derivative thereof. In certain embodiments, the stationary phase can be porous with an average pore size between about 50 angstroms and about 200 angstroms, and the first and second oligonucleotide species can have a size of less than 10 kDa. In certain embodiments, the stationary phase can be porous with an average pore size between about 200 angstroms and about 500 angstroms, and the first and second oligonucleotide species can have a length of 300 nt or less. In certain embodiments, the stationary phase can be porous with an average pore size between about 500 angstroms and about 4000 angstroms, and the first and second oligonucleotide species can have a length of 300 nt or more.

[0024] In various embodiments of the second aspect, the first and second species can be no more than about 15,000 nt in length.

[0025] In various embodiments of the second aspect, the first and second oligonucleotide species can be ribonucleic acid (RNA) oligonucleotides.

[0026] In various embodiments of the second aspect, the first and second oligonucleotide species can be deoxyribonucleic acid (DNA) oligonucleotides.

[0027] In various embodiments of the second aspect, the first and second oligonucleotide species may comprise a modification.

[0028] In various embodiments of the second aspect, the first and second oligonucleotide species can be produced in vitro.

[0029] In various embodiments of the second aspect, the first and second oligonucleotide species can be produced in vivo.

[0030] In a third aspect, a method for analytical separation of virus particles can include lysing a sample containing multiple virus particle species. The multiple virus particle species can include a first virus particle species containing a first oligonucleotide species and a second virus particle species containing a second oligonucleotide species. Solubilizing the sample can include releasing the first and second oligonucleotide species. The method can also include loading the solubilized sample onto a column and injecting a mobile phase consisting of a combination of the first and second solutions onto the column to elute the first and second oligonucleotide species. The first solution can include an ion-pairing reagent. The ion-pairing reagent can include a primary amine or a secondary amine. The ratio of the first and second solutions in the mobile phase can be varied over time to separate the first oligonucleotide species from the second oligonucleotide species. The method can further include analyzing at least one of the first and second oligonucleotide species using a mass spectrometer.

[0031] In various embodiments of the third aspect, the sample can include a plurality of oligonucleotide species dissolved in a first solution, a second solution, or any combination thereof.

[0032] In various embodiments of the third aspect, the second solution can include an ion-pairing reagent.

[0033] In various embodiments of the third aspect, the ion pairing reagent can include at least four carbons.

[0034] In certain embodiments, the primary amine of the ion pairing reagent may include an alkyl chain having at least four carbon atoms, such as amylamine or hexylamine.

[0035] In certain embodiments, the secondary amine of the ion pairing reagent can include two alkyl chains, and at least one of the alkyl chains can include at least three carbons (e.g., N-ethylpropylamine, dipropylamine (DPA), N-ethylbutylamine, N-propylbutylamine, dibutylamine (DBA), dipentalamine, or dihexylamine).

[0036] In various embodiments of the third aspect, the first solution can include a fluoroalcohol, such as hexafluoroisopropanol (HFIP).

[0037] In various embodiments of the third aspect, the first solution can include formic acid or a formate, acetic acid, or an acetate, or any combination thereof.

[0038] In various embodiments of the third aspect, the first solution can include a first organic solvent such as methanol, ethanol, acetone, acetonitrile, tetrahydrofuran, isopropanol, or any combination thereof.

[0039] In various embodiments of the third aspect, the second solution can include a second organic solvent, such as methanol, ethanol, acetone, acetonitrile, tetrahydrofuran, isopropanol, or any combination thereof.

[0040] In various embodiments of the third aspect, the column can include a stationary phase comprising divinylbenzene or a derivative thereof. In certain embodiments, the stationary phase can be porous with an average pore size between about 50 angstroms and about 200 angstroms, and the first and second oligonucleotide species can have a size of less than 10 kDa. In certain embodiments, the stationary phase can be porous with an average pore size between about 200 angstroms and about 500 angstroms, and the first and second oligonucleotide species can have a length of 300 nt or less. In certain embodiments, the stationary phase can be porous with an average pore size between about 500 angstroms and about 4000 angstroms, and the first and second oligonucleotide species can have a length of 300 nt or more.

[0041] In various embodiments of the third aspect, the first and second species can be no more than about 15,000 nt in length.

[0042] In various embodiments of the third aspect, the first and second oligonucleotide species can be ribonucleic acid (RNA) oligonucleotides.

[0043] In various embodiments of the third aspect, the first and second oligonucleotide species can be deoxyribonucleic acid (DNA) oligonucleotides.

[0044] In various embodiments of the third aspect, the first and second oligonucleotides can include a modification. For a more complete understanding of the principles disclosed herein and their advantages, reference is now made to the following description taken in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0045] [Figure 1] 1A, 1B, 1C, and 1D show the chemical structures of RNA nucleosides A, G, C, and U and the locations of potential hydrogen bond donors and hydrogen bond acceptors, respectively. [Figure 2]2A and 2B show the chemical structures of representative tertiary amines triethylamine and diisopropylethylamine, respectively, and the locations of potential hydrogen bond donors and hydrogen bond acceptors. [Figure 3] 3A, 3B, and 3C show the chemical structures and locations of potential hydrogen bond donors and hydrogen bond acceptors for exemplary secondary amines diethylamine, dipropylamine, and dibutylamine, respectively. [Figure 4] 4A and 4B show the chemical structures and locations of potential hydrogen bond donors and hydrogen bond acceptors for exemplary primary amines amylamine and hexylamine, respectively. [Figure 5] 5A, 5B, and 5C are flow diagrams of methods for separating and analyzing oligonucleotides according to various embodiments. [Figure 6] FIG. 6 is a block diagram of an exemplary chromatography system, according to various embodiments. [Figure 7] FIG. 7 is a block diagram of an exemplary mass spectrometry system, according to various embodiments. [Figure 8] 8 shows the results of analyzing virus particles according to various embodiments. It should be understood that the figures are not necessarily drawn to scale, and that objects within the figures are not necessarily drawn to scale in relationship to each other. The figures are intended to provide clarity and understanding of the various embodiments of the devices, systems, and methods disclosed herein. Wherever possible, the same reference numbers will be used throughout the figures to refer to the same or like parts. Furthermore, it should be understood that the figures are not intended to limit the scope of the teachings herein in any way. DETAILED DESCRIPTION OF THE INVENTION

[0046] Described herein are embodiments of methods for separating long oligonucleotides.

[0047] The section headings used herein are for organizational purposes only and are not intended to limit the scope of the described subject matter in any way.

[0048] In this detailed description of various embodiments, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the disclosed embodiments. However, those skilled in the art will appreciate that these various embodiments may be practiced without or with the specific details. In other instances, structures and devices are shown in block diagram form. Furthermore, those skilled in the art will readily appreciate that the specific order in which methods are presented and performed is illustrative, and it is contemplated that the order may be changed and still remain within the spirit and scope of the various embodiments disclosed herein.

[0049] All literature and similar materials cited in this application (including, but not limited to, patents, patent applications, papers, books, treatises, and internet web pages) are expressly incorporated by reference in their entirety for any purpose. Unless otherwise specified, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which each embodiment described herein belongs.

[0050] In the present teachings, values ​​described for temperature, concentration, time, pressure, flow rate, cross-sectional area, etc., should be understood to be preceded by an implicit "about." This allows for minor deviations that do not have a substantial effect to be considered within the scope of the present specification. In this application, the use of the singular includes the plural unless specifically stated otherwise. Similarly, the use of "comprises," "comprises," "comprising," "contain," "contains," "containing," "include," "includes," and "including" is not intended to be limiting. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present teachings.

[0051] As used herein, "a" or "an" can refer to "at least one" or "one or more." Also, the use of "or" is inclusive, so that the phrase "A or B" is true when "A" is true, when "B" is true, or when both "A" and "B" are true. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0052] A "system" refers to a set of components, whether real or abstract, in which each component interacts with or comprises a whole relative to at least one other component within the whole.

[0053] Ion-pair chromatography (IPC) is an effective reversed-phase liquid chromatography (RPLC) method for the separation of organic ions and partially ionized organic analytes. IPC techniques utilize the same types of stationary and mobile phases as RPLC. The key feature of IPC is the addition of one or more ion-pairing reagents to the mobile phase.

[0054] The purpose of adding an ion-pairing reagent to the mobile phase is usually to modify the retention time of ionic analytes. By varying the mobile phase concentration of the ion-pairing reagent, the retention factors of oppositely charged analytes can be continuously increased by 10-20 times compared to the case without the addition of the ion-pairing reagent. Correspondingly, the retention factors of like-charged analytes can be continuously decreased by 10-20 times. The retention factors of uncharged analytes are usually unaffected by the presence of the ion-pairing reagent.

[0055] IPC has applications in almost all areas of chromatographic analysis. It utilizes a water-rich mobile phase that can be combined with a variety of buffers and ionic and non-ionic additives. This method is well suited for the separation of important classes of biomolecules, particularly amino acids, peptides, proteins, and nucleic acids.

[0056] RNA (ribonucleic acid) is a nucleic acid consisting of a long chain of nucleotides, each consisting of a sugar, a phosphate group, and a base. RNA has four bases: adenine, guanine, cytosine, and uracil. Figures 1A-1D show the chemical structures of the nucleosides (ribose sugar and base, excluding the phosphate group) of adenine (Figure 1A), guanine (Figure 1B), cytosine (Figure 1C), and uracil (Figure 1D). Groups that can act as hydrogen bond donors are labeled D, and groups that can act as hydrogen bond acceptors are labeled A. Some groups can act as either donors or acceptors and are labeled both D and A. As can be seen, there are many groups that act as hydrogen bond acceptors and only a few that act as hydrogen bond donors.

[0057] Figures 2A and 2B show the structures and locations of potential hydrogen bond donors and hydrogen bond acceptors for the representative tertiary amines triethylamine (TEA) and diisopropylethylamine (DIPEA), respectively. TEA and DIPEA are commonly used ion-pairing reagents. However, as shown in Figures 2A and 2B, tertiary amines can only act as hydrogen bond acceptors.

[0058] Figures 3A, 3B, and 3C show the chemical structures and locations of potential hydrogen bond donors and hydrogen bond acceptors for exemplary secondary amines, diethylamine (DEA), dipropylamine (DPA), and dibutylamine (DBA), respectively. As can be seen in Figures 3A, 3B, and 3C, secondary amines can act as either hydrogen bond donors or hydrogen bond acceptors.

[0059] Figures 4A and 4B show the chemical structures of exemplary primary amines amylamine and hexylamine, respectively, and the locations of potential hydrogen bond donors and hydrogen bond acceptors. As can be seen in Figures 4A and 4B, primary amines can act as either hydrogen bond donors or hydrogen bond acceptors.

[0060] The use of ion-pairing reagents (which function as hydrogen bond donors) enhances interactions with nucleic acid bases, especially at the Watson-Crick interface. Hydrogen-bonding interactions with bases can enhance retention. Because the number of hydrogen-bonding acceptor sites increases with nucleic acid length, retention is dependent on the length of the nucleic acid. This can improve the separation of large oligonucleotides. Furthermore, differences in the number and placement of hydrogen-bonding acceptors for different bases can lead to differences in retention based on the sequence of the oligonucleotide.

[0061] FIG. 5A illustrates an example method 500 for analyzing a sample containing multiple oligonucleotide species. In various embodiments, the oligonucleotide species can include ribonucleic acid (RNA) oligonucleotides or deoxyribonucleic acid (DNA) oligonucleotides. The oligonucleotides can include naturally occurring or non-naturally occurring modifications. The oligonucleotides can be produced in vitro or in vivo. In various embodiments, the oligonucleotides can be encapsulated RNA oligonucleotides that are encapsulated in a structure comprising a lipid, a protein, or a combination thereof.

[0062] At 502, an ion-pairing reagent can be mixed with the sample. In various embodiments, the sample can be dissolved or resuspended in a first solution, a second solution, or any combination thereof. The ion-pairing reagent can include a primary amine or a secondary amine. In various embodiments, the ion-pairing reagent can include at least about 4 carbons, but generally no more than about 16.

[0063] The primary amine ion pairing reagent may include an alkyl chain of at least four carbons. In certain embodiments, the primary amine ion pairing reagent may include amylamine or hexylamine.

[0064] The secondary amine ion pairing reagent may contain two alkyl chains, at least one of which has 3 or more carbon atoms, but generally has less than 8 carbon atoms, such as N-ethylpropylamine, dipropylamine (DPA), N-ethylbutylamine, N-propylbutylamine, dibutylamine (DBA), dipentalamine, or dihexylamine.

[0065] At 504, the sample and ion-pairing reagent can be loaded onto a chromatography column. In various embodiments, the chromatography column can include a stationary phase comprising divinylbenzene or a derivative thereof. In various embodiments, the stationary phase can be porous, for example, having an average pore size of at least about 50 angstroms, such as at least about 200 angstroms, or even at least about 500 angstroms. The selection of the average pore size can be influenced by the size of the oligonucleotides to be separated. A stationary phase having an average pore size of about 50 angstroms to about 200 angstroms can be used to separate oligonucleotides less than 10 kDa in size. A stationary phase having an average pore size between about 200 angstroms to about 500 angstroms can be used to separate oligonucleotides of about 300 nt or less. A stationary phase having an average pore size between about 500 angstroms to about 4000 angstroms can be used to separate oligonucleotides greater than about 300 nt, but typically less than about 15,000 nt.

[0066] At 506, an elution solution can be applied to the column to elute the multiple oligonucleotide species. Generally, it is advantageous for the solution conditions of the sample applied to the column to approximate the starting conditions of the elution gradient. In various embodiments, the elution solution can include a combination of a first solution and a second solution. In certain embodiments, the ratio of the first solution to the second solution can be varied to form a gradient of solvent conditions that cause separation of various oligonucleotide species, such as the first oligonucleotide species, from the second oligonucleotide species based on differential retention within the column.

[0067] In various embodiments, the concentration of the ion-pairing reagent can be varied only in the first solution and the second solution, and the gradient of solvent conditions includes a gradient of the concentration of the ion-pairing reagent. For example, the second solution can contain more or less ion-pairing reagent than the first solution. Alternatively, only the first solution can contain ion-pairing reagent, and the second solution can contain no ion-pairing reagent. In other embodiments, the first and second solutions can contain the same concentration of ion-pairing reagent, such that the concentration of the ion-pairing reagent is substantially constant throughout the separation process. In various embodiments, the concentration of the ion-pairing reagent can be between about 1 mM and about 500 mM, e.g., between about 5 mM and about 500 mM.

[0068] In various embodiments, the first solution can include a fluoroalcohol such as hexafluoroisopropanol (HFIP). In various embodiments, the concentration of the fluoroalcohol can be about 5 mM to about 500 mM. In other embodiments, the fluoroalcohol may be absent. In various embodiments, the first solution can include formic acid or formate, acetic acid or acetate, or any combination thereof (e.g., to achieve a pH range of about 2 to about 11, such as about 4 to 10). In other embodiments, the first solution may be absent formic acid, formate, acetic acid, or acetate. In various embodiments, the first solution can include a first organic solvent such as methanol, ethanol, acetone, acetonitrile, tetrahydrofuran, isopropanol, or any combination thereof. In various embodiments, the organic solvent can be used at a concentration of about 0% to about 100%. If the concentration is less than about 100%, the remaining amount of the solution may include water.

[0069] In various embodiments, the second solution can include similar or identical components to the first solution, but at different concentrations. For example, the second solution can include fluoroalcohol, formic acid, formate salts, acetic acid, acetate salts, and / or organic solvents. In other embodiments, the second solution can include at least some components that are different from the first solution.

[0070] At 508, the oligonucleotide species can be detected using a UV / VIS detector or a mass analyzer, etc. The sample can be further analyzed based on the signal and retention time observed in the detector to identify the oligonucleotide species and / or quantify the oligonucleotide species.

[0071] 5B illustrates an example method 530 for analyzing a sample containing a plurality of encapsulated oligonucleotide species. In various embodiments, the encapsulated RNA oligonucleotides can be encapsulated in a lipid, protein, or a structure comprising either. In various embodiments, the encapsulated oligonucleotide species can include ribonucleic acid (RNA) oligonucleotides or deoxyribonucleic acid (DNA) oligonucleotides. The oligonucleotides can include naturally occurring or non-naturally occurring modifications. The oligonucleotides can be produced in vitro or in vivo.

[0072] At 532, an ion pairing reagent can be mixed with the sample. In various embodiments, the sample can be dissolved or resuspended in a first solution, a second solution, or any combination thereof. In various embodiments, dissolving or resuspending the sample can dissolve lipids and / or degrade proteins in encapsulating structures, releasing the oligonucleotides contained therein into solution.

[0073] The ion pairing reagent may include a primary amine or a secondary amine. In various embodiments, the ion pairing reagent may include at least about 4 carbons, but generally no more than about 16.

[0074] The primary amine ion pairing reagent may include an alkyl chain of at least four carbons. In certain embodiments, the primary amine ion pairing reagent may include amylamine or hexylamine.

[0075] The secondary amine ion pairing reagent may contain two alkyl chains, at least one of which has 3 or more carbon atoms, but generally has less than 8 carbon atoms, such as N-ethylpropylamine, dipropylamine (DPA), N-ethylbutylamine, N-propylbutylamine, dibutylamine (DBA), dipentalamine, or dihexylamine.

[0076] At 534, the sample and ion-pairing reagent can be loaded onto a chromatography column. In various embodiments, the chromatography column can include a stationary phase comprising divinylbenzene or a derivative thereof. In various embodiments, the stationary phase can be porous, for example, having an average pore size of at least about 50 angstroms, such as at least about 200 angstroms, or even at least about 500 angstroms. The selection of the average pore size can be influenced by the size of the oligonucleotides to be separated. A stationary phase having an average pore size of about 50 angstroms to about 200 angstroms can be used to separate oligonucleotides less than 10 kDa in size. A stationary phase having an average pore size between about 200 angstroms to about 500 angstroms can be used to separate oligonucleotides of about 300 nt or less. A stationary phase having an average pore size between about 500 angstroms to about 4000 angstroms can be used to separate oligonucleotides greater than about 300 nt, but typically less than about 15,000 nt.

[0077] At 536, an elution solution can be applied to the column to elute the multiple oligonucleotide species. Generally, it is advantageous for the solution conditions of the sample applied to the column to approximate the starting conditions of the elution gradient. In various embodiments, the elution solution can include a combination of a first solution and a second solution. In certain embodiments, the ratio of the first solution to the second solution can be varied to form a gradient of solvent conditions that cause separation of various oligonucleotide species, such as the first oligonucleotide species, from the second oligonucleotide species based on differential retention within the column.

[0078] In various embodiments, the concentrations of the ion-pairing reagent in the first and second solutions can be varied, and the gradient of solvent conditions can include a gradient of the concentration of the ion-pairing reagent. For example, the second solution can contain more or less ion-pairing reagent than the first solution. Alternatively, only the first solution can contain ion-pairing reagent, while the second solution does not. In other embodiments, the first and second solutions can contain the same concentration of ion-pairing reagent, such that the concentration of the ion-pairing reagent is substantially constant throughout the separation process. In various embodiments, the concentration of the ion-pairing reagent can be from about 5 mM to about 500 mM.

[0079] In various embodiments, the first solution can include a fluoroalcohol such as hexafluoroisopropanol (HFIP). In various embodiments, the concentration of the fluoroalcohol can be about 5 mM to about 500 mM. In various embodiments, the first solution can include formic acid or formate, acetic acid or acetate, or any combination thereof (e.g., to achieve a pH range of about 2 to about 11, such as about 4 to 10). In various embodiments, the first solution can include a first organic solvent such as methanol, ethanol, acetone, acetonitrile, tetrahydrofuran, isopropanol, or any combination thereof. In various embodiments, the organic solvent can be used at a concentration of about 0% to about 100%. If the concentration is less than about 100%, the remaining amount of the solution can include water.

[0080] In various embodiments, the second solution can include similar or identical components to the first solution, but at different concentrations. For example, the second solution can include fluoroalcohol, formic acid, formate salts, acetic acid, acetate salts, and / or organic solvents. In other embodiments, the second solution can include at least some components that are different from the first solution.

[0081] At 538, the oligonucleotide species can be detected using a UV / VIS detector or a mass analyzer, etc. The sample can then be analyzed based on the signal and retention time observed in the detector to identify the oligonucleotide species and / or quantify the oligonucleotide species.

[0082] 5C illustrates an example method 560 for analyzing a sample containing a plurality of viral particles. The viral particles may include lipid and / or protein structures encapsulating oligonucleotides. In various embodiments, the oligonucleotide species may include ribonucleic acid (RNA) oligonucleotides or deoxyribonucleic acid (DNA) oligonucleotides. The oligonucleotides may include naturally occurring or non-naturally occurring modifications. The oligonucleotides may be produced in vitro or in vivo.

[0083] At 562, an ion pairing reagent can be mixed with the sample. In various embodiments, the sample can be dissolved or resuspended in a first solution, a second solution, or any combination thereof. In various embodiments, dissolving or resuspending the sample can dissolve viral particles by dissolving lipids and / or denaturing proteins of the viral particles such that oligonucleotides contained within the viral particles are released into solution.

[0084] The ion pairing reagent may include a primary amine or a secondary amine. In various embodiments, the ion pairing reagent may include at least about 4 carbons, but generally no more than about 16.

[0085] The primary amine ion pairing reagent may include an alkyl chain of at least four carbons. In certain embodiments, the primary amine ion pairing reagent may include amylamine or hexylamine.

[0086] The secondary amine ion pairing reagent may contain two alkyl chains, at least one of which has 3 or more carbon atoms, but generally has less than 8 carbon atoms, such as N-ethylpropylamine, dipropylamine (DPA), N-ethylbutylamine, N-propylbutylamine, dibutylamine (DBA), dipentalamine, or dihexylamine.

[0087] At 564, the sample and ion-pairing reagent can be loaded onto a chromatography column. In various embodiments, the chromatography column can include a stationary phase comprising divinylbenzene or a derivative thereof. In various embodiments, the stationary phase can be porous, for example, having an average pore size of at least about 50 angstroms, such as at least about 200 angstroms, or even at least about 500 angstroms. The selection of the average pore size can be influenced by the size of the oligonucleotides to be separated. A stationary phase having an average pore size of about 50 angstroms to about 200 angstroms can be used to separate oligonucleotides less than 10 kDa in size. A stationary phase having an average pore size between about 200 angstroms to about 500 angstroms can be used to separate oligonucleotides of about 300 nt or less. A stationary phase having an average pore size between about 500 angstroms to about 4000 angstroms can be used to separate oligonucleotides greater than about 300 nt, but typically less than about 15,000 nt.

[0088] At 566, an elution solution can be applied to the column to elute the multiple oligonucleotide species. Generally, it is advantageous for the solution conditions of the sample applied to the column to approximate the starting conditions of the elution gradient. In various embodiments, the elution solution can include a combination of a first solution and a second solution. In certain embodiments, the ratio of the first solution to the second solution can be varied to form a gradient of solvent conditions that cause separation of various oligonucleotide species, such as the first oligonucleotide species, from the second oligonucleotide species based on differential retention within the column.

[0089] In various embodiments, the concentration of the ion-pairing reagent can be varied only in the first solution and the second solution, and the gradient of solvent conditions includes a gradient of the concentration of the ion-pairing reagent. For example, the second solution can contain more or less ion-pairing reagent than the first solution. Alternatively, only the first solution can contain ion-pairing reagent, and the second solution can contain no ion-pairing reagent. In other embodiments, the first and second solutions can contain the same concentration of ion-pairing reagent, such that the concentration of the ion-pairing reagent is substantially constant throughout the separation process. In various embodiments, the concentration of the ion-pairing reagent can be from about 5 mM to about 500 mM.

[0090] In various embodiments, the first solution can include a fluoroalcohol such as hexafluoroisopropanol (HFIP). In various embodiments, the concentration of the fluoroalcohol can be about 5 mM to about 500 mM. In various embodiments, the first solution can include formic acid or formate, acetic acid or acetate, or any combination thereof (e.g., to achieve a pH range of about 2 to about 10). In various embodiments, the first solution can include a first organic solvent such as methanol, ethanol, acetone, acetonitrile, tetrahydrofuran, isopropanol, or any combination thereof. In various embodiments, the organic solvent can be used at a concentration of about 0% to about 100%. If the concentration is less than about 100%, the remaining amount of the solution can include water.

[0091] In various embodiments, the second solution can include similar or identical components to the first solution, but at different concentrations. For example, the second solution can include fluoroalcohol, formic acid, formate salts, acetic acid, acetate salts, and / or organic solvents. In other embodiments, the second solution can include at least some components that are different from the first solution.

[0092] At 568, the oligonucleotide species can be detected using a UV / VIS detector or a mass analyzer, etc. The sample can then be analyzed based on the signal and retention time observed in the detector to identify the oligonucleotide species and / or quantify the oligonucleotide species.

[0093] 6 illustrates a liquid chromatography system 600 according to one embodiment of the present invention. The liquid chromatography system 600 includes an analytical pump 602 for pumping a solvent through the system 600. The system 600 includes a sample reservoir 604 containing the sample to be analyzed. The system 600 further includes a separation column 606 and a detector 608. The system 600 also includes a controller 610.

[0094] The liquid chromatography system 600 is adapted to collect a sample from a sample reservoir 604. The sample can then be introduced into the system.

[0095] The liquid chromatography system 1000 is further adapted to introduce a sample into the separation column 606 .

[0096] System 600 is also adapted to inject a sample into separation column 606 via an analytical flow. This can be accomplished by directing the sample through analytical pump 602. Separation column 606 can separate the sample into component species based on retention time within separation column 606. After separation of the sample by separation column 606, the separated components can be detected by detector 608. In some embodiments, detector 608 can be an optical detector, such as an absorbance detector, a refractive index detector, or a fluorescence detector. In other embodiments, detector 608 can be a conductivity detector or an electrochemical detector. In yet other embodiments, detector 608 can be a mass spectrometer.

[0097] In various embodiments, the separation column 606 generally consists of a tube packed with a stationary phase medium. The stationary phase medium can affect the time it takes a compound to travel through the column (retention time). This effect can be different for different compounds, so that individual components of a sample can be separated based on their individual retention times. Numerous stationary phase media exist, including porous materials, ionic materials, polar materials, non-polar materials, etc. Porous materials can affect retention time based on the size of the molecules and their ability to enter the porous material. Ionic materials can affect retention time based on charge attraction or repulsion between the ionic material and the compound. Polar and non-polar materials can affect retention time based on the hydrophobicity or hydrophilicity of the compound.

[0098] In various embodiments, nucleotides and nucleosides can be separated using reversed-phase separation, in which a hydrophobic, nonpolar stationary phase material is used with a mobile phase of varying hydrophobicity, depending on its polarity relative to the organic solvent. For example, a C18 column can be used with an ammonium acetate or ammonium formate buffer system to separate nucleosides. In certain embodiments, an aqueous mobile phase of 5 mM ammonium acetate at a pH of about 5 can be used, and a gradient of increasing concentrations of acetonitrile (up to about 40%) or methanol (up to about 50%) can be used to separate nucleosides. Suitable columns and buffer systems will be apparent to those skilled in the art and are within the scope of this disclosure.

[0099] In other embodiments, nucleotides and nucleosides can be separated using hydrophilic interaction liquid chromatography (HILIC), in which a hydrophilic stationary phase material is used with a hydrophobic mobile phase such as acetonitrile. Suitable columns and buffer systems will be apparent to those skilled in the art and are within the scope of this disclosure.

[0100] Various embodiments of mass spectrometry platform 700 can include the components shown in the block diagram of Figure 7. In various embodiments, mass spectrometry platform 700 can operate as detector 608 of system 600. In various embodiments, the elements of Figure 7 can be incorporated into mass spectrometry platform 700. According to various embodiments, mass spectrometer 700 can include an ion source 702, a mass analyzer 704, an ion detector 706, and a controller 708.

[0101] In various embodiments, the ion source 702 generates a plurality of ions from a sample. The ion source may include, but is not limited to, a matrix-assisted laser desorption / ionization (MALDI) source, an electrospray ionization (ESI) source, an atmospheric pressure chemical ionization (APCI) source, an atmospheric pressure photoionization source (APPI), an inductively coupled plasma (ICP) source, an electron ionization source, a chemical ionization source, a photoionization source, a glow discharge ionization source, a thermospray ionization source, etc.

[0102] In various embodiments, the mass analyzer 704 can separate ions based on their mass-to-charge ratio. For example, the mass analyzer 704 can include a quadrupole mass filter analyzer, a quadrupole ion trap analyzer, a time-of-flight (TOF) analyzer, an electrostatic trap (e.g., Orbitrap) mass analyzer, a Fourier transform ion cyclotron resonance (FT-ICR) mass analyzer, etc. In various embodiments, the mass analyzer 704 can also be configured to fragment ions using collision-induced dissociation (CID), electron transfer dissociation (ETD), electron capture dissociation (ECD), photo-induced dissociation (PID), surface-induced dissociation (SID), etc., and further separate the fragmented ions based on their mass-to-charge ratio.

[0103] In various embodiments, the ion detector 706 can detect ions. For example, the ion detector 706 can include an electron multiplier, a Faraday cup, etc. Ions exiting the mass analyzer can be detected by the ion detector. In various embodiments, the ion detector can be capable of quantitative detection, allowing the precise amount of ions to be determined. In various embodiments, the mass analyzer 704 and the ion detector 706 can be combined into a single device, such as an electrostatic trap (e.g., ORBITRP) mass analyzer.

[0104] In various embodiments, the controller 708 can be in communication with the ion source 702, the mass analyzer 704, and the ion detector 706. For example, the controller 708 can configure the ion source or enable / disable the ion source. Further, the controller 708 can configure the mass analyzer 704 to select a particular mass range for detection. Further, the controller 708 can adjust the sensitivity of the ion detector 706, such as by adjusting the gain. Additionally, the controller 708 can adjust the polarity of the ion detector 706 based on the polarity of the ions being detected. For example, the ion detector 706 can be configured to detect positive ions or negative ions.

[0105] While the present teachings will be described in conjunction with various embodiments, it is not intended to limit the present teachings to such embodiments. Rather, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those skilled in the art.

[0106] Furthermore, in describing various embodiments, the specification may present methods and / or processes as a particular sequence of steps. However, to the extent that the method or process does not rely on the particular order of steps described herein, the method or process should not be limited to the particular order of steps described. As one of ordinary skill in the art will understand, other orders of steps may be possible. Accordingly, the particular order of steps described herein should not be construed as a limitation on the claims. In addition, the claims directed to the method and / or process should not be limited to performing those steps in the order written; one of ordinary skill in the art will readily understand that the order may be changed and still remain within the spirit and scope of the various embodiments.

[0107] Example 1 is an analytical nucleic acid encapsulated in bacteriophage. Figure 8A is a blank injection control. Figure 8B shows the results of injecting RNA encapsulated in bacteriophage. Figure 8C shows the results of injecting purified RNA extracted from bacteriophage.

Claims

1. 1. A method for analytical separation of oligonucleotides or nucleic acid biopolymers, comprising: loading a sample containing a plurality of oligonucleotide species onto the column, wherein the plurality of oligonucleotide species comprises a first oligonucleotide species and a second oligonucleotide species; applying a mobile phase consisting of a combination of the first and second solutions to the column to elute the plurality of oligonucleotide species; the first solution contains an ion pairing reagent; the ion pairing reagent comprises a primary or secondary amine; Varying the ratio of the first solution to the second solution in the mobile phase over time to separate the first oligonucleotide species from the second oligonucleotide species; and analyzing at least one of the first oligonucleotide species and the second oligonucleotide species using a mass spectrometer.

2. 10. The method of claim 1, wherein the sample comprises a plurality of oligonucleotide species dissolved in a first solution, a second solution, or any combination thereof.

3. The method of claim 1 , wherein the second solution comprises an ion-pairing reagent.

4. 10. The method of claim 1, wherein the ion pairing reagent comprises at least four carbons.

5. 5. The method of claim 4, wherein the primary amine of the ion pairing reagent comprises an alkyl chain of at least four carbons.

6. 6. The method of claim 5, wherein the primary amine comprises amylamine or hexylamine.

7. the secondary amine of the ion pairing reagent comprises two alkyl chains; The method of claim 4, wherein at least one of the alkyl chains contains at least three carbons.

8. 8. The method of claim 7, wherein the secondary amine comprises N-ethylpropylamine, dipropylamine (DPA), N-ethylbutylamine, N-propylbutylamine, dibutylamine (DBA), dipentalamine, or dihexylamine.

9. The method of claim 1 , wherein the first solution comprises a fluoroalcohol.

10. 10. The method of claim 9, wherein the fluoroalcohol comprises hexafluoroisopropanol (HFIP).

11. 10. The method of claim 1, wherein the first solution comprises formic acid or a formate, acetic acid or an acetate, or any combination thereof.

12. The method of claim 1 , wherein the first solution comprises a first organic solvent.

13. 13. The method of claim 12, wherein the first organic solvent comprises methanol, ethanol, acetone, acetonitrile, tetrahydrofuran, isopropanol, or any combination thereof.

14. The method of claim 1 , wherein the second solution comprises a second organic solvent.

15. 15. The method of claim 14, wherein the second organic solvent comprises methanol, ethanol, acetone, acetonitrile, tetrahydrofuran, isopropanol, or any combination thereof.

16. 10. The method of claim 1, wherein the column comprises a stationary phase comprising divinylbenzene or a derivative thereof.

17. the stationary phase is porous with an average pore size of about 50 angstroms to about 200 angstroms; 17. The method of claim 16, wherein the first and second species are less than 10 kDa in size.

18. the stationary phase is porous with an average pore size of about 200 angstroms to about 500 angstroms; 17. The method of claim 16, wherein the first and second species are about 300 nt or less in length.

19. the stationary phase is porous with an average pore size of about 500 angstroms to about 4000 angstroms; 17. The method of claim 16, wherein the first and second species are greater than 300 nt in length.

20. 2. The method of claim 1, wherein the first and second species are less than or equal to about 15,000 nt in length.

21. 10. The method of claim 1, wherein the plurality of oligonucleotide species are ribonucleic acid (RNA) oligonucleotides.

22. 22. The method of claim 21, wherein the ribonucleic acid (RNA) oligonucleotide comprises a modification.

23. 22. The method of claim 21, wherein the ribonucleic acid (RNA) oligonucleotide is generated in vitro.

24. 22. The method of claim 21, wherein the ribonucleic acid (RNA) oligonucleotide is generated in vivo.

25. 22. The method of claim 21, wherein the ribonucleic acid (RNA) oligonucleotide is an encapsulated RNA oligonucleotide.

26. 1. A method for analytical separation of encapsulated oligonucleotides or nucleic acid biopolymers, comprising: solubilizing a sample containing a plurality of encapsulated oligonucleotide species, wherein the plurality of encapsulated oligonucleotide species includes a first encapsulated oligonucleotide species comprising a first oligonucleotide species and a second encapsulated oligonucleotide species comprising a second oligonucleotide species; solubilizing the sample includes releasing the first oligonucleotide species and the second oligonucleotide species; loading the solubilized sample onto a column; applying a mobile phase consisting of a combination of the first and second solutions to the column to elute the first and second oligonucleotide species; the first solution contains an ion pairing reagent; the ion pairing reagent comprises a primary or secondary amine; Varying the ratio of the first solution to the second solution in the mobile phase over time to separate the first oligonucleotide species from the second oligonucleotide species; and analyzing at least one of the first oligonucleotide species and the second oligonucleotide species using a mass spectrometer.

27. 27. The method of claim 26, wherein the sample comprises a plurality of oligonucleotide species dissolved in a first solution, a second solution, or any combination thereof.

28. 27. The method of claim 26, wherein the second solution comprises an ion-pairing reagent.

29. 27. The method of claim 26, wherein the ion pairing reagent comprises at least four carbons.

30. 30. The method of claim 29, wherein the primary amine of the ion pairing reagent comprises an alkyl chain of at least four carbons.

31. 31. The method of claim 30, wherein the primary amine comprises amylamine or hexylamine.

32. the secondary amine of the ion pairing reagent comprises two alkyl chains; 30. The method of claim 29, wherein at least one of the alkyl chains comprises at least 3 carbons.

33. 33. The method of claim 32, wherein the secondary amine comprises N-ethylpropylamine, dipropylamine (DPA), N-ethylbutylamine, N-propylbutylamine, dibutylamine (DBA), dipentalamine, or dihexylamine.

34. 27. The method of claim 26, wherein the first solution comprises a fluoroalcohol.

35. 35. The method of claim 34, wherein the fluoroalcohol comprises hexafluoroisopropanol (HFIP).

36. 27. The method of claim 26, wherein the first solution comprises formic acid or a formate, acetic acid or an acetate, or any combination thereof.

37. 27. The method of claim 26, wherein the first solution comprises a first organic solvent.

38. 38. The method of claim 37, wherein the first organic solvent comprises methanol, ethanol, acetone, acetonitrile, tetrahydrofuran, isopropanol, or any combination thereof.

39. 27. The method of claim 26, wherein the second solution comprises a second organic solvent.

40. 40. The method of claim 39, wherein the second organic solvent comprises methanol, ethanol, acetone, acetonitrile, tetrahydrofuran, isopropanol, or any combination thereof.

41. 27. The method of claim 26, wherein the column comprises a stationary phase comprising divinylbenzene or a derivative thereof.

42. the stationary phase is porous with an average pore size of about 50 angstroms to about 200 angstroms; 42. The method of claim 41, wherein the first and second species are less than 10 kDa in size.

43. the stationary phase is porous with an average pore size of about 200 angstroms to about 500 angstroms; 42. The method of claim 41, wherein the first and second species are about 300 nt or less in length.

44. the stationary phase is porous with an average pore size of about 500 angstroms to about 4000 angstroms; 42. The method of claim 41, wherein the first and second species are greater than 300 nt in length.

45. 27. The method of claim 26, wherein the first and second species are about 15,000 nt or less in length.

46. 27. The method of claim 26, wherein the first and second oligonucleotide species are ribonucleic acid (RNA) oligonucleotides.

47. 27. The method of claim 26, wherein the first and second oligonucleotide species are deoxyribonucleic acid (DNA) oligonucleotides.

48. 27. The method of claim 26, wherein the first and second oligonucleotide species comprise a modification.

49. 27. The method of claim 26, wherein the first and second oligonucleotide species are generated in vitro.

50. 27. The method of claim 26, wherein the first and second oligonucleotide species are generated in vivo.

51. 1. A method for the analytical separation of viral particles, comprising: solubilizing a sample containing multiple virus particle species, wherein: the plurality of virus particle species includes a first virus particle species comprising a first oligonucleotide species and a second virus particle species comprising a second oligonucleotide species; solubilizing the sample includes releasing the first oligonucleotide species and the second oligonucleotide species; loading the solubilized sample onto a column; applying a mobile phase consisting of a combination of the first and second solutions to the column to elute the first and second oligonucleotide species; the first solution contains an ion pairing reagent; the ion pairing reagent comprises a primary or secondary amine; Varying the ratio of the first solution to the second solution in the mobile phase over time to separate the first oligonucleotide species from the second oligonucleotide species; and analyzing at least one of the first oligonucleotide species and the second oligonucleotide species using a mass spectrometer.

52. 52. The method of claim 51, wherein the sample comprises a plurality of oligonucleotide species dissolved in a first solution, a second solution, or any combination thereof.

53. 52. The method of claim 51, wherein the second solution comprises an ion-pairing reagent.

54. 52. The method of claim 51, wherein the ion pairing reagent comprises at least four carbons.

55. 55. The method of claim 54, wherein the primary amine of the ion pairing reagent comprises an alkyl chain of at least four carbons.

56. 56. The method of claim 55, wherein the primary amine comprises amylamine or hexylamine.

57. the secondary amine of the ion pairing reagent comprises two alkyl chains; 55. The method of claim 54, wherein at least one of the alkyl chains comprises at least 3 carbons.

58. 58. The method of claim 57, wherein the secondary amine comprises N-ethylpropylamine, dipropylamine (DPA), N-ethylbutylamine, N-propylbutylamine, dibutylamine (DBA), dipentalamine, or dihexylamine.

59. 52. The method of claim 51, wherein the first solution comprises a fluoroalcohol.

60. 60. The method of claim 59, wherein the fluoroalcohol comprises hexafluoroisopropanol (HFIP).

61. 52. The method of claim 51, wherein the first solution comprises formic acid or a formate, acetic acid or an acetate, or any combination thereof.

62. 52. The method of claim 51, wherein the first solution comprises a first organic solvent.

63. 63. The method of claim 62, wherein the first organic solvent comprises methanol, ethanol, acetone, acetonitrile, tetrahydrofuran, isopropanol, or any combination thereof.

64. 52. The method of claim 51, wherein the second solution comprises a second organic solvent.

65. 65. The method of claim 64, wherein the second organic solvent comprises methanol, ethanol, acetone, acetonitrile, tetrahydrofuran, isopropanol, or any combination thereof.

66. 52. The method of claim 51, wherein the column comprises a stationary phase comprising divinylbenzene or a derivative thereof.

67. the stationary phase is porous with an average pore size of about 50 angstroms to about 200 angstroms; 67. The method of claim 66, wherein the size of the first and second species is less than 10 kDa.

68. the stationary phase is porous with an average pore size of about 200 angstroms to about 500 angstroms; 67. The method of claim 66, wherein the first and second species are about 300 nt or less in length.

69. the stationary phase is porous with an average pore size of about 500 angstroms to about 4000 angstroms; 67. The method of claim 66, wherein the first and second species are greater than 300 nt in length.

70. 52. The method of claim 51, wherein the first and second species are about 15,000 nt or less in length.

71. 52. The method of claim 51, wherein the first and second oligonucleotide species are ribonucleic acid (RNA) oligonucleotides.

72. 52. The method of claim 51, wherein the first and second oligonucleotide species are deoxyribonucleic acid (DNA) oligonucleotides.

73. 52. The method of claim 51 , wherein the first and second oligonucleotide species comprise a modification.

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