A pre-treatment method for resolving interacting hydrophobic oligonucleotides in liquid chromatography

Abstract

The present disclosure is directed to methods of assaying mixtures or pools of oligonucleotides where at least two oligonucleotides within the pool of oligonucleotides interact, associate, or bind to one another such that their identities cannot be resolved chromatographically.

Description

[0001] A PRE-TREATMENT METHOD FOR RESOLVING INTERACTING HYDROPHOBIC OLIGONUCLEOTIDES IN LIQUID CHROMATOGRAPHY FIELD OF THE INVENTION

[0002] The present disclosure relates to methods of preparing or separating at least two different oligonucleotides prior to downstream utilization.

[0003] BACKGROUND OF THE DISCLOSURE

[0004] An oligonucleotide is a short sequence of nucleotides (e.g., between 2-25, 5-25, or 8-25 bases), which are used in various biological and medical research applications. These sequences can be designed to hybridize (bind) to specific complementary sequences of nucleic acids (deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)), making them valuable tools in techniques such as polymerase chain reaction (PCR), DNA sequencing, gene synthesis, and as probes or primers for targeted molecule detection. Oligonucleotides can be chemically synthesized and customized based on the requirements of the specific application. Oligonucleotides find utility in a number of biological applications. Oligonucleotide sequences form duplexes with complementary oligonucleotide targets and can be used as molecular biological probes in genome research and in clinical diagnostic applications involving disease causing genes.

[0005] Oligonucleotides also find utility as primers for various polymerase applications such as the polymerase chain reaction ("PCR"). In PCR, an oligonucleotide primer is added to a sample containing single stranded template nucleic acid fragments in the presence of an enzyme and mononucleotides. Starting at the primer, the enzyme builds a nucleic acid strand which is complementary to the template nucleic acid. The reaction is carried out several times in succession where in each cycle the newly built strand is amplified.

[0006] Oligonucleotides have also been used as a discovery tool to design oligonucleotide sequences that act as competitive inhibitors of certain disease causing and unwanted proteins. In this application, also known as the "aptamer approach," the high affinity of a particular protein of interest with a specific oligonucleotide sequence present in a random pool of millions of randomly synthesized oligonucleotide sequences is determined. This technique has been instrumental in developing novel oligonucleotide inhibitors of various proteins such as thrombin, small interfering RNA (siRNA), antisense oligonucleotides, guide RNA in CRISPR for gene silencing at DNA and mRNA levels, etc.

[0007] It is often advantageous to be able to store oligonucleotides for a long duration (e.g., greater than two months). It would also be advantageous to be able to accurately assay stored oligonucleotides for quality control purposes.BRIEF SUMMARY OF THE DISCLOSURE

[0008] Methods of assaying mixtures or pools of oligonucleotides are provided herein, including those oligonucleotides that include one or more hydrophobic groups which may cause two or more different oligonucleotides in the mixture or pool of oligonucleotides to be indiscernible (e.g., by appearing as a single broad peak instead of two discrete peaks) by a chromatography method. It has been found that chemically and / or thermally disrupting this interaction resolves the nonspecific hydrophobic interaction of modified oligonucleotides during storage.

[0009] A first aspect of the present disclosure is a method of assaying a pool of oligonucleotides including at least two or more different oligonucleotides comprising: (a) obtaining the pool of oligonucleotides comprising the two or more different oligonucleotides; (b) chemically disrupting hydrophobic interactions between the two or more different oligonucleotides in the pool of oligonucleotides; and (c) eluting the two or more different oligonucleotides from a solid phase support.

[0010] In some embodiments, at least one of the oligonucleotides of the two or more different oligonucleotides includes a hydrophobic group or moiety. In some embodiments, the at least one of the two or more different oligonucleotides that includes the hydrophobic group or moiety non-specifically interacts with the other of the two or more different oligonucleotides. In some embodiments, both of the two or more oligonucleotides include a hydrophobic group or moiety. In some embodiments, the two or more different oligonucleotides in the pool of oligonucleotides each include one or more labels. In some embodiments, the one or more labels are fluorescent labels. In some embodiments, the fluorescent labels are hydrophobic. In some embodiments, the fluorescent labels include one or more hydrophobic groups. In some embodiments, at least one of the two or more different oligonucleotides include one or more modified nucleotides, wherein the one or more modified nucleotides include a hydrophobic moiety. In some embodiments, the hydrophobic moiety is an alkynyl group. In some embodiments, the hydrophobic moiety is a propynyl group. In some embodiments, the label includes a heavy metal (e.g., a ferrocene label). In some embodiments, at least two of the two or more different oligonucleotides in the sample non-specifically interact to each other. In some embodiments, at least two of the two or more oligonucleotides in the sample cannot be resolved by chromatography, e.g., liquid chromatography.

[0011] In some embodiments, the chemically disrupting of the hydrophobic interactions comprises contacting the obtained pool of oligonucleotides with at least one surfactant. In some embodiments, the surfactant is an anionic surfactant. In some embodiments, the surfactant is a cationic surfactant. In some embodiments, the surfactant is a non-ionic surfactant.In some embodiments, the chemically disrupting of the hydrophobic interactions comprises contacting the obtained pool of oligonucleotides with at least one denaturing agent. In some embodiments, the denaturing agent is formamide. In some embodiments, the denaturing agent is selected from the group consisting of guanidine, sodium salicylate, dimethyl sulfoxide (DMSO), propylene glycol, and urea. In some embodiments, the two or more oligonucleotides are contacted with both a surfactant and a denaturing agent.

[0012] A second aspect of the present disclosure is a method of assaying a pool of oligonucleotides including two or more different oligonucleotides comprising: (a) obtaining the pool of oligonucleotides comprising the two or more different oligonucleotides; (b) heating the pool of oligonucleotides to a first predetermined temperature for a first predetermined amount of time; (c) flash cooling the pool of oligonucleotides to a second predetermined temperature for a second predetermined amount of time, wherein second predetermined temperature is 10°C or less; and (d) eluting the pool of oligonucleotides flash cooled to the second predetermined temperature from a solid phase support. In some embodiments, no additional reagents are introduced during any of steps (a) - (d) (e.g., no denaturing agents or surfactants are added during these steps).

[0013] In some embodiments, the first predetermined temperature is at least about 90°C. In some embodiments, the first predetermined temperature ranges from between about 90°C to about 120°C. In some embodiments, the first predetermined temperature ranges from between about 100°C to about 110°C. In some embodiments, the predetermined time is at least about 2 minutes. In some embodiments, the predetermined time is at least about 4 minutes. In some embodiments, the predetermined time ranges from between about 2 minutes to about 12 minutes. In some embodiments, the predetermined time ranges from between about 4 minutes to about 10 minutes. In some embodiments, the second predetermined temperature ranges from between about 0°C to about 10°C. In some embodiments, the second predetermined temperature ranges from between about 0°C to about 5°C.

[0014] In some embodiments, at least one of the oligonucleotides of the two or more different oligonucleotides includes a hydrophobic group or moiety. In some embodiments, the at least one of the two or more different oligonucleotides that includes the hydrophobic group or moiety non-specifically interacts with the other of the two or more different oligonucleotides. In some embodiments, both of the two or more oligonucleotides include a hydrophobic group or moiety. In some embodiments, the two or more different oligonucleotides in the pool of oligonucleotides each include one or more labels. In some embodiments, the one or more labels are fluorescent labels. In some embodiments, the fluorescent labels are hydrophobic. In some embodiments, thefluorescent labels include one or more hydrophobic groups. In some embodiments, at least one of the two or more different oligonucleotides include one or more modified nucleotides, wherein the one or more modified nucleotides include a hydrophobic moiety. In some embodiments, the hydrophobic moiety is an alkynyl group. In some embodiments, the hydrophobic moiety is a propynyl group. In some embodiments, the label includes a heavy metal (e.g., a ferrocene label). In some embodiments, at least two of the two or more different oligonucleotides in the sample non-specifically interact with each other. In some embodiments, at least two of the two or more oligonucleotides in the sample cannot be resolved by chromatography, e.g., liquid chromatography.

[0015] In some embodiments, the solid phase support is a chromatography column. In some embodiments, the chromatography column is a reverse phase chromatography column. In some embodiments, the pool of oligonucleotides is eluted from the solid phase support using a solvent selected from acetonitrile, methanol, isopropanol, ethanol, tetrahydrofuran, or a combination thereof.

[0016] A third aspect of the present disclosure method of assaying at least two different oligonucleotides comprising: (a) obtaining a sample comprising the at least two different oligonucleotides; (b) heating the sample comprising the at least two different at a first predetermined temperature for a first predetermined amount of time; (c) flash cooling the at least two different oligonucleotides to a second predetermined temperature for a second predetermined amount of time, wherein second predetermined temperature is 10°C or less.

[0017] In some embodiments, the method further comprises introducing the at least two different oligonucleotides flash cooled to the second predetermined temperature to a liquid chromatography instrument.

[0018] In some embodiments, the liquid chromatography instrument is a high-performance liquid chromatography instrument. In some embodiments, the liquid chromatography instrument is an ultra-performance liquid chromatography instrument.

[0019] In some embodiments, the method further comprises introducing the at least two different oligonucleotides flash cooled to the second predetermined temperature to a chromatography column. In some embodiments, the column is a reverse phase chromatography column. In some embodiments, the chromatography column has a temperature ranging from between about 0°C to about 60°C.

[0020] In some embodiments, each of the at least two different oligonucleotides include one or more labels. In some embodiments, the one or more labels are fluorescent labels. In some embodiments, the fluorescent labels are hydrophobic. In some embodiments, the fluorescent labels include one or more hydrophobic groups. In some embodiments, the at least two different oligonucleotidesinclude one or more modified nucleotides, wherein the one or more modified nucleotides include a hydrophobic moiety. In some embodiments, the hydrophobic moiety is an alkynyl group. In some embodiments, the hydrophobic moiety is a propynyl group. In some embodiments, the hydrophobic moiety is a group including a metal, such as a heavy metal. In some embodiments, the at least two different oligonucleotides are labeled with ferrocene.

[0021] In some embodiments, the at least two of the two or more oligonucleotides in the sample non-specifically bind to each other.

[0022] In some embodiments, the first predetermined temperature is at least about 90°C. In some embodiments, the first predetermined temperature ranges from between about 80°C to about 140°C. In some embodiments, the first predetermined temperature ranges from between about 90°C to about 120°C. In some embodiments, the predetermined time is at least about 2 minutes. In some embodiments, the predetermined time is at least about 4 minutes. In some embodiments, the predetermined time ranges from between about 2 minutes to about 12 minutes. In some embodiments, the predetermined time ranges from between about 4 minutes to about 10 minutes. In some embodiments, the second predetermined temperature ranges from between about 0°C to about 10°C. In some embodiments, the second predetermined temperature ranges from between about 0°C to about 5°C.

[0023] In some embodiments, the method further comprises quantifying an amount of the at least two oligonucleotides in the sample. In some embodiments, the method further comprises determining the relative concentration of two or more interacting oligonucleotides in a formulation including a plurality of oligonucleotides; and / or determining the relative concentration of two or more interacting oligonucleotides in a formulation including multiple oligonucleotides and other components, such as the components present in PCR master mixes.

[0024] BRIEF DESCRIPTION OF THE FIGURES

[0025] For a general understanding of the features of the disclosure, reference is made to the drawings. In the drawings, like reference numerals have been used throughout to identify identical elements. FIG. 1 depicts a method of preparing or separating two or more different oligonucleotides in accordance with one embodiment of the present disclosure, whereby any interactions between the two or more different oligonucleotides are chemically disrupted.

[0026] FIG. 2 depicts a method of preparing or separating two or more different oligonucleotides in accordance with one embodiment of the present disclosure, whereby any interactions between the two or more different oligonucleotides are thermally disrupted.FIG. 3 is a chromatogram showing the merged peaks of two different oligonucleotides (labeled "3" and "4"), where the two different oligonucleotides were stored, such as for a period of two or more months. In some embodiments, the oligonucleotides labeled "3" and "4" were stored with yet other oligonucleotides in a pool of oligonucleotides.

[0027] FIG. 4 illustrates how the two different oligonucleotides of FIG. 3 (labeled "3" and "4") should appear in a standard operating procedure (SOP) chromatogram. Said another way, the figure illustrates how two different oligonucleotides (labeled "3 "and "4") should appear in a freshly prepared oligonucleotide pool prior to storage.

[0028] FIG. 5 is a chromatogram showing the peaks of the two different oligonucleotides (labeled "B3" and "B4") of FIG. 3 after thermal disruption of hydrophobic interactions between the two different oligonucleotides.

[0029] FIG. 6 is a chromatogram showing the merged peaks of two different oligonucleotides (labeled "2" and "3"), where the two different oligonucleotides were stored, such as for a period of 2 or more months. In some embodiments, the oligonucleotides labeled "2" and "3" were stored with yet other oligonucleotides in a pool of oligonucleotides.

[0030] FIG. 7 is a chromatogram showing the peaks of the two different oligonucleotides (labeled "Peak 2" and "Peak 3") of FIG. 6 after thermal disruption of hydrophobic interactions between the two different oligonucleotides.

[0031] FIG. 8 illustrates how the two different oligonucleotides of FIG. 6 (labeled "2" and "3") should appear in a SOP chromatogram.

[0032] DETAILED DESCRIPTION

[0033] The present disclosure is directed to methods of preparing or separating two or more different oligonucleotides present, such as two or more different oligonucleotides present in a pool of oligonucleotides (such as a pool including 2 or more different oligonucleotides, such as 3 or more different oligonucleotides, such as 4 or more different oligonucleotides, such as 5 or more different oligonucleotides, such as 8 or more different oligonucleotides, such as 10 or more different oligonucleotides, such as 15 or more different oligonucleotides, such as 20 or more different oligonucleotides, such as 25 or more different oligonucleotides, such as 30 or more different oligonucleotides, such as 35 or more different oligonucleotides, such as 40 or more different oligonucleotides, etc.). In some embodiments, the two or more different oligonucleotides are prepared or separated by: (i) chemically or thermally disrupting interactions (defined herein) between the two or more different oligonucleotides; and (ii) chromatographically resolving the two or more different oligonucleotides. In some embodiments, the thermal disruption does notrequire the use of any additives, reagents, etc. In some embodiments, the chromatographic resolution comprises reverse phase chromatography, ionic phase chromatography, size exclusion chromatography, affinity chromatography, chiral chromatography, etc. In some embodiments, the two or more different oligonucleotides are introduced, following chemical or thermal disruption of any interactions, to an ultra-performance liquid chromatography (UPLC) instrument or a high-performance liquid chromatography (HPLC) instrument.

[0034] Definitions

[0035] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0036] As used herein, the singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "includes" is defined inclusively, such that "includes A or B" means including A, B, or A and B.

[0037] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of' or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0038] The terms "comprising," "including," "having," and the like are used interchangeably and have the same meaning. Similarly, "comprises," "includes," "has," and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of "comprising" and is therefore interpreted to be an open term meaning "at least the following," and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, "a device having components a, b, and c" means that the device includes at least components a, b, and c. Similarly, the phrase: "a method involving steps a, b, and c" means that the method includes at least steps a, b, and c. Moreover, while thesteps and processes may be outlined herein in a particular order, the skilled artisan will recognize that the ordering steps and processes may vary.

[0039] As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and / or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0040] Use of the term "about" is intended to describe values either above or below the stated value in a range of approx. + / — 10%; in other forms the values may range in value either above or below the stated value in a range of approx. + / -5%; in other forms the values may range in value either above or below the stated value in a range of approx. + / -2%; in other forms the values may range in value either above or below the stated value in a range of approx. + / -1%. The preceding ranges are intended to be made clear by context, and no further limitation is implied.

[0041] The terms "reverse phase" and "reverse phase support" as used herein refer to any stationary support, including the base material and any chemically bonded phase, for use in liquid chromatography, e.g., high-performance liquid chromatography (HPLC) or ultra-performance liquid chromatography, which is less polar (e.g., more hydrophobic) than the starting mobile phase. The term is intended to encompass a porous and / or a non-porous support.

[0042] Overview

[0043] The present disclosure provides methods of assaying a pool of oligonucleotides including at least two different oligonucleotides. In some embodiments, the at least two different oligonucleotides non-specifically interact or bind with each other. In some embodiments, the at least two different oligonucleotides cannot be resolved or resolved well through a chromatography technique, such as high-performance liquid chromatography (HPLC) or ultra-performance liquid chromatography(UPLC). For instance, the peaks of two interacting oligonucleotides in a chromatogram may appear as a single broad peak instead of two discrete, resolvable peaks.

[0044] In some embodiments, the present disclosure provides methods of preparing or separating at least two different oligonucleotides from each other, or from other oligonucleotides present in a pool of oligonucleotides. In some embodiments, the present disclosure provides methods of preparing or separating at least two different oligonucleotides which non-specifically interact, associate, or bind (hereinafter collectively referred to "interact" or "interaction" or the like) with each other for assaying. In some embodiments, the at least two different oligonucleotides non-specifically bind to each other through hydrophobic interactions. In some embodiments, any interactions, associations, or bonds between the at least two different oligonucleotides are disrupted chemically or thermally.

[0045] In some embodiments, the at least two different oligonucleotides are modified oligonucleotides. In some embodiments, the at least two different oligonucleotides are modified to include one or more labels or other chemical groups. In some embodiments, the one or more labels or other chemical groups are hydrophobic groups. In some embodiments, the hydrophobic group is or includes an alkynyl bond. "Alkynyl" refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one triple bond, optionally having an indicated number of carbons, e.g., having from two to twelve carbon atoms, or two to eight carbon atoms, or two to six carbon atoms, or two to four carbon atoms, and which is attached to the rest of the molecule by a single bond, e.g., ethenyl, prop-l-enyl, but-l-enyl, pent-1-enyl, penta- 1,4-dienyl, and the like. Non-limiting examples of alkynyl radicals include C2-C8 hydrocarbon moieties such as, but not limited to: acetylenic ( — C=CH) (abbreviated herein as "C2H) and propargyl ( — CH2OCH). An alkynyl group can be substituted or unsubstituted. In some embodiments, the hydrophobic moiety is a propynyl group.

[0046] In some embodiments, the one or more labels are fluorescent labels. In other embodiments, the one or more fluorescent labels are hydrophobic. In some embodiments, the one or more fluorescent labels are selected from acridine, am Alexa dye (e.g., Alexa 350, 488, 594, 610, 633, 700, etc.) AMCA, an ATTO dye (e.g., ATTO 390, 425, 465, 488, 495, 590, 610, 633, 647, 700), Bodipy FL, Bodipy TMT, Bodipy TR, Cascade Blue, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Edans, Eosin, Erythrosin, 6-FAM, 6-TET, 6-HEX, JOE, LightCycler 640, LightCycler 705, Lissamine, NBD, Rhodamine 6G, Rhoadamine Green, Rhoadamine Red, TAMRA< ROX, Texas Red, NED, and VIC.

[0047] In some embodiments, the at least two different oligonucleotides are modified to include group such as DBCO, TCO, ferrocene, bromohexyl, coumarin343-threioninol, etc. In otherembodiments, the at least two different oligonucleotides include modified sugars, such as oligonucleotides including LNA or L-DNA. For instance, the at least two different oligonucleotides may include one or more of D-LNA-C, D-LNA-A, D-LNA-T, L-DNA-A, L-DNA-C, L-DNA-G, or L-DNA-T. In other embodiments, the at least two different oligonucleotides may include 2'0-methyl nucleotides or other nucleobase modifications (e.g., 2'-thiouridine, cytosine analogs, etc.). In yet other embodiments, the at least two different oligonucleotides are modified to include any of: N6-Benzyl-dA, 7-deaza-dG; BHQ-2 DMT amidite,Cy5.25B-Threioninol, D-LNA-5Me Cytosine, DBCO-Threioninol, Threo-FAM, Threo-HEX, JA270-Threo, 3'-Phosphate, 3'-Space C3, GalNAc (N-acetylgalactosamine) modifications, cholesterol modifications, phosphorothioate bonds, etc.

[0048] Chemical Disruption of Interactions Between Oligonucleotides

[0049] In some embodiments, interactions between the at least two different oligonucleotides are chemically disrupted by contacting the sample (e.g., a pool of at least two different oligonucleotides) including the at least two different oligonucleotides with a composition including a denaturing agent and / or a surfactant, such as an anionic surfactant, a cationic surfactant ionic surfactant, a non-ionic surfactant, or a mixture thereof. In some embodiments, and with reference to FIG. 1, a pool of oligonucleotides including two or more different oligonucleotides is obtained (step 1). In some embodiments, the pool of oligonucleotides includes at least 2, such as at least 3, such as at least 4, such as at least 6, such as at least 8, such as at least 10, etc. oligonucleotides. In some embodiments, the two or more different oligonucleotides may include one or more groups or moieties that confer hydrophobicity to at least one of the two or more different oligonucleotides, causing them to non-specifically interact with each other. In some embodiments, the two or more different oligonucleotides are contacted with a composition so as to chemically disrupt any interactions between the two or more different oligonucleotides (step 2). In some embodiments, the method further comprises introducing the introducing the two or more different oligonucleotides, following chemical treatment, to a column (step 3). In some embodiments, the method further comprises eluting the two or more different oligonucleotides from the column (in accordance with methods known to those of ordinary skill in the art, such as by introducing one or more solvents and / or buffers to the column).

[0050] In some embodiments, the at least two different oligonucleotides are incubated with the composition for a time period ranging from about 1 minute to about 60 minutes, such as about 2 minutes to about 50 minutes, such as about from 3 minutes to about 40 minutes, such as about 5 minutes to about 30 minutes. In some embodiments, the at least two different oligonucleotides areincubated with the composition for about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, etc.

[0051] Anionic Surfactants

[0052] Anionic surfactants are generally based upon sulfates, sulfonates, phosphates, or carboxylates and contain a water-soluble cation. A representative formula of a sulfonate is R — SO3M, where R is a hydrocarbon group of from about 5 to 22 carbon atoms which may be linked through an alkoxy or oxyalkoxy to the sulfonate functionality and M is a water-soluble cation such as an alkali metal. Anionic surfactants include alkyl ether sulfates, alkyl sulfates and sulfonates, alkyl carboxylates, alkyl phenyl ether sulfates, sodium salts of alkyl poly(oxyethylene) sulfonates, sodium salts of alkyl benzyl sulfonate, such as sodium salts of dodecylbenzyl sulfonate and sodium lauryl ether sulfate. Anionic surfactants also include anionic phosphate esters.

[0053] In some embodiments, the surfactants include, but are not limited to polyoxyethylene alkyl ether, wherein the alkyl is (CH2)M and the oxyethylene is (C2H4O)N, wherein M is an integer from 5 to 16, from 8 to 14, or from 10 to 12 and N is an integer from 10 to 40, from 15 to 30, or from 20 to 28. In one embodiment, the surfactant is polyoxyethylene lauryl ether having a formula (C2H4O)23Ci2H25OH. In another embodiment, the surfactant is a polyoxyethylene (20) sorbitan monoalkylate, the monoalkylate comprising between 8 and 14 carbons. In another embodiment, the surfactant is a linear secondary alcohol polyoxyethylene having a formula C12-14H25-29O(CH2CH2O]X, wherein x equals an integer between 2 and 12. In yet another embodiment, the surfactant is a polyoxyethylene octyl phenyl ether. Exemplary surfactants are sold under the names: Brij® 35, TWEEN®, Tergitol™, Triton™, Ecosurf™, Dowfax™, polysorbate 80™, BigCHAP, Deoxy BigCHAP, IGEPAL®, Saponin, Thesit®, Nonidet®, Pluronic F-68, digitonin, deoxycholate, and the like. Particular disclosed working embodiments concern using surfactants selected from Brij® 35, TWEEN®, Tergitol™, Triton™.

[0054] Cationic Surfactants

[0055] Suitable cationic surfactants for use with the methods of the present disclosure contain amino or quaternary ammonium moieties. Cationic surfactants among those useful herein are disclosed in the following documents: M.C. Publishing Co., McCutcheon's, Detergents & Emulsifiers, (North American edition 1979); Schwartz, et al.; Surface Active Agents, Their Chemistry and Technology, New York: Interscience Publishers, 1949; U.S. Pat. No. 3,155,591, Hilfer, issued Nov. 3, 1964; U.S. Pat. No. 3,929,678, Laughlin et al., issued Dec. 30, 1975; U.S. Pat. No.3,959,461, Bailey et al., issued May 25, 1976; and U.S. Pat. No. 4,387,090, Bolich, Jr., issued Jun.

[0056] 7, 1983.

[0057] Among the quaternary ammonium-containing cationic surfactants include those having the general formula:

[0058]

[0059] wherein R1-R4 are independently an aliphatic group of from about 1 to about 22 carbon atoms or an aromatic, alkoxy, polyoxyalkylene, alkylamido, hydroxyalkyl, aryl or alkylaryl group having from about 1 to about 22 carbon atoms; and X is a salt-forming anion such as those selected from halogen, (e.g. chloride, bromide), acetate, citrate, lactate, glycolate, phosphate nitrate, sulfate, and alkylsulfate radicals. The aliphatic groups may contain, in addition to carbon and hydrogen atoms, ether linkages, and other groups such as amino groups. The longer chain aliphatic groups, e.g., those of about 12 carbons, or higher, can be saturated or unsaturated. Especially preferred are mono-long chain (e.g., mono C12 to C22, preferably C12 to Cis, more preferably Ci6, aliphatic, preferably alkyl), di-short chain (e.g., Ci to C3 alkyl, preferably Ci to C2 alkyl) quaternary ammonium salts.

[0060] Salts of primary, secondary, and tertiary fatty amines are also suitable cationic surfactant materials. In some embodiments, the alkyl groups of such amines preferably have from about 12 to about 22 carbon atoms and may be substituted or unsubstituted. Such amines, useful herein, include stearamido propyl dimethyl amine, diethyl amino ethyl stearamide, dimethyl stearamine, dimethyl soyamine, soyamine, myristyl amine, tridecyl amine, ethyl stearylamine, N-tallowpropane diamine, ethoxylated (with 5 moles of ethylene oxide) stearylamine, dihydroxy ethyl stearylamine, and arachidylbehenylamine. Suitable amine salts include the halogen, acetate, phosphate, nitrate, citrate, lactate, and alkyl sulfate salts. Such salts include stearylamine hydrochloride, soyamine chloride, stearylamine formate, N-tallowpropane diamine dichloride, stearamidopropyl dimethylamine citrate, cetyl trimethyl ammonium chloride and dicetyl diammonium chloride. Preferred for use in the compositions herein are cetyl trimethyl ammonium chloride, stearyl trimethyl ammonium chloride, tetradecyltrimethly ammonium chloride, dicetyldimethyl ammonium chloride, dicocodimethyl ammonium chloride and mixtures thereof. More preferred is cetyl trimethyl ammonium chloride.Non-ionic Surfactants

[0061] Suitable nonionic surfactants include condensation products of -C30 alcohols with sugar or starch polymers. These compounds can be represented by the formula (S)n— O — R, wherein S is a sugar moiety such as glucose, fructose, mannose, and galactose; n is an integer of from about 1 to about 1000, and R is C8-C30 alkyl. Examples of suitable C8-C30 alcohols from which the R group may be derived include decyl alcohol, cetyl alcohol, stearyl alcohol, lauryl alcohol, myristyl alcohol, oleyl alcohol, and the like. Specific examples of these surfactants include decyl polyglucoside and lauryl polyglucoside.

[0062] Other suitable nonionic surfactants include the condensation products of alkylene oxides with fatty acids (i.e., alkylene oxide esters of fatty acids). These materials have the general formula RCO(X)nOH, wherein R is a C10-C30 alkyl, X is — OCH2CH2 — (derived from ethylene oxide) or — OCH2CHCH3 — (derived from propylene oxide), and n is an integer from about 1 to about 200. Yet other suitable nonionic surfactants are the condensation products of alkylene oxides with fatty acids (i.e., alkylene oxide diesters of fatty acids) having the formula RCO(X)nOOCR, wherein R is a C10-C30 alkyl, X is — OCH2CH2 — (derived from ethylene oxide) or — OCH2CHCH3 — (derived from propylene oxide), and n is an integer from about 1 to about 200. Yet other nonionic surfactants are the condensation products of alkylene oxides with fatty alcohols (i.e., alkylene oxide ethers of fatty alcohols) having the general formula R(X)nOR', wherein R is C10-C30 alkyl, n is an integer from about 1 to about 200, and R' is H or a C10-C30 alkyl.

[0063] Yet other nonionic surfactants are the compounds having the formula RCO(X)nOR' wherein R and R' are C10-C30 alkyl, X is — OCH2CH2 — (derived from ethylene oxide) or — OCH2CHCH3 — (derived from propylene oxide), and n is an integer from about 1 to about 200. Examples of alkylene oxide-derived nonionic surfactants include ceteth-1, ceteth-2, ceteth-6, ceteth-10, ceteth- 12, ceteraeth-2, ceteareth6, ceteareth-10, ceteareth-12, steareth-1, steareth-2, stearteth-6, steareth- 10, steareth-12, PEG-2 stearate, PEG4 stearate, PEG6 stearate, PEG- 10 stearate, PEG- 12 stearate, PEG-20 glyceryl stearate, PEG-80 glyceryl tallowate, PPG- 10 glyceryl stearate, PEG-30 glyceryl cocoate, PEG-80 glyceryl cocoate, PEG-200 glyceryl tallowate, PEG-8 dilaurate, PEG- 10 distearate, and mixtures thereof. Still other useful nonionic surfactants include polyhydroxy fatty acid amides disclosed, for example, in U.S. Pat. Nos. 2,965,576, 2,703,798, and 1,985,424, which are incorporated herein by reference.

[0064] Exemplary surfactants include sodium dodecyl sulfate, Tomadol 1200 (Air Products), Tomadol 900 (Air Products), Tomadol 91-8 (Air Products), Tomadol 1-9 (Air Products), Tergitol 15-S-9 (Sigma), Tergitol 15-S-12 (Sigma), Masurf NRW-N (Pilot Chemical), Bio-Soft N91-6 (Stepan), and Brij-35 (Polyethylene glycol dodecyl ether) (Sigma).Denaturing Agents

[0065] In some embodiments, interactions between the at least two different oligonucleotides in the pool of oligonucleotides are chemically disrupted by contacting the sample including the at least two different oligonucleotides with a denaturing agent. In some embodiments, the denaturing agent is formamide, guanidine, sodium salicylate, dimethyl sulfoxide (DMSO), propylene glycol, or urea. In some embodiments, the denaturing agent is a base. In some embodiments, the base has a pH ranging from between about 10.5 to about 12.5. In other embodiments, the base has a pH ranging from between about 11 to about 12. In some embodiments, the base is NaOH.

[0066] Thermal Disruption of Interactions

[0067] In some embodiments, interactions between the at least two different oligonucleotides are thermally disrupted, such as by sequentially heating and cooling (especially flash cooling) the at least two different oligonucleotides. In particular, the specific combination of heating and flash cooling (specifically flash cooling to 10°C or below) provides for a surprising technical effect compared to standard slow cooling. Herein, it has been found that slow cooling allows the hydrophobic interactions to re-form, whereas flash cooling "freezes" the oligonucleotides in a single-stranded conformation. In some embodiments, and with reference to FIG. 2, a pool of oligonucleotides including two or more different oligonucleotides is obtained (step 4). In some embodiments, the two or more different oligonucleotides may include one or more groups or moieties that confer hydrophobicity to at least one of the two or more different oligonucleotides, causing them to non-specifically interact with each other. In some embodiments, the two or more different oligonucleotides are sequentially heated and cooled (such as by flash cooling) so as to disrupt any interactions between the two or more different oligonucleotides (step 5). In some embodiments, no reagents are added to the at least two different oligonucleotides during the thermal disruption (step 5). For instance, no chemicals (e.g., surfactants, denaturing agents, etc.) are added to the at least two different oligonucleotides during the thermal disruption (step 5). In some embodiments, the method further comprises introducing the introducing the two or more different oligonucleotides, following thermal treatment, to a column (step 6). In some embodiments, the method further comprises eluting the two or more different oligonucleotides from the column (in accordance with methods known to those of ordinary skill in the art, such as by introducing one or more solvents and / or buffers to the column).

[0068] In some embodiments, interactions between the at least two different oligonucleotides are thermally disrupted by heating the at least two different oligonucleotides to a first predetermined temperature and such as for a first predetermined amount of time. In some embodiments, the firstpredetermined temperature is at least about 40°C. In other embodiments, the first predetermined temperature is at least about 50°C. In other embodiments, the first predetermined temperature is at least about 60°C. In other embodiments, the first predetermined temperature is at least about 70°C. In other embodiments, the first predetermined temperature is at least about 80°C. In other embodiments, the first predetermined temperature is at least about 90°C. In other embodiments, the first predetermined temperature is at least about 100°C. In other embodiments, the first predetermined temperature is at least about 110°C. In other embodiments, the first predetermined temperature is at least about 120°C.

[0069] In other embodiments, the first predetermined temperature ranges from between about 80°C to about 150°C. In other embodiments, the first predetermined temperature ranges from between about 80°C to about 140°C. In other embodiments, the first predetermined temperature ranges from between about 85°C to about 130°C. In other embodiments, the first predetermined temperature ranges from between about 90°C to about 120°C. In other embodiments, the first predetermined temperature ranges from between about 95°C to about 115°C. In other embodiments, the first predetermined temperature ranges from between about 100°C to about 110°C.

[0070] In some embodiments, the first predetermined amount of time is at least about 2 minutes, such as at least about 3 minutes, such as at least about 4 minutes, such as at least about 5 minutes, such as at least about 6 minutes, such as at least about 7 minutes, such as at least about 8 minutes, such as at least about 9 minutes, such as at least about 10 minutes, such as at least about 15 minutes, such as at least about 20 minutes, etc.

[0071] Following heating of the at least two different oligonucleotides, the at least two different oligonucleotides are cooled, such as to a second predetermined temperature and such as for a second predetermined amount of time. In some embodiments, the at least two oligonucleotides are abruptly cooled, such as "flash cooled”. In some embodiments, the second predetermined temperature is about 15°C or less. In other embodiments, the second predetermined temperature is about 10°C or less. In other embodiments, the second predetermined temperature is about 8°C or less. In other embodiments, the second predetermined temperature is about 6°C or less. In other embodiments, the second predetermined temperature is about 5°C or less. In other embodiments, the second predetermined temperature ranges from between about 0°C to about 10°C. In other embodiments, the second predetermined temperature ranges from between about 0°C to about 8°C. In other embodiments, the second predetermined temperature ranges from between about 0°C to about 6°C. In other embodiments, the second predetermined temperature ranges from between about 0°C to about 5°C.In some embodiments, the second predetermined amount of time is at least about 2 minutes, such as at least about 3 minutes, such as at least about 4 minutes, such as at least about 5 minutes, such as at least about 6 minutes, such as at least about 7 minutes, such as at least about 8 minutes, such as at least about 9 minutes, such as at least about 10 minutes, such as at least about 15 minutes, such as at least about 20 minutes, etc.

[0072] EXAMPLES

[0073] Example 1

[0074] An oligonucleotide pool including two oligonucleotides was thawed and introduced to a column of a ultra-performance liquid chromatography (UPLC). Each oligonucleotide included a FAM probe. One of the oligonucleotides included a stretch of propynyl groups. The pool was maintained frozen at -20°C for a time period greater than 2 months.

[0075] The UPLC trace indicates that the peaks associated with the two oligonucleotides appeared as a blob (FIG. 3) instead of appearing as two individual peaks (FIG. 4, a standard-operating-procedure ("SOP") chromatogram). It was believed that the two probes, given the hydrophobic groups included in each, non-specifically bound to each other (hence, the blob as depicted in FIG. 3). Specifically, it was hypothesized that both FAM probes, with oligo 4 being rich in Propynyl groups can non-specifically bind to each other through hydrophobic interactions, particularly during a prolonged storage of the pools in the freezer.

[0076] A novel, yet simple, method was developed to overcome this problem and break down the hydrophobic interactions between the two different oligonucleotides in the pool. Samples were heated to denature and separate the oligos and flash cooled (flash chilled) immediately on ice to freeze the individual oligos in single stranded conformation prior to injection into the UPLC. This treatment of the Pool B was able to break down the binding mediated by hydrophobic interactions between the two different oligonucleotides.

[0077] The two different oligonucleotides ("the pool") was diluted 4x as described in the OTD / SOP. 10 pL of the pool was diluted to 40 pL with Milli Q water. This diluted pool in a 0.2 mL microfuge tube was placed in a boiling (95°C) water bath for 4 minutes. After 4 minutes, the tube containing the Pool B sample was rapidly chilled (flash cooled) on a crushed ice bath. The sample was kept on an ice bath for at least 30 minutes (if the sample is in a small 0.2 to 2 mL tube). The sample can be on ice up to 2 hours before it is injected into the UPLC. The critical part is that after heating the sample in boiling water bath it has to be chilled on ice immediately (i.e. flash cooled). The sample was analyzed by UPLC as usual as described in SOP. This treatment prior to injection into the UPLC was able to resolve the peaks between the two different oligonucleotides to the base lineresolution (see FIG. 4). It is clear that this treatment prior to the UPLC analysis helps to achieve sharp separation of these merged peaks.

[0078] The method of heating and flash cooling (flash chilling) the oligonucleotide pool samples prior to UPLC injection is believed to help break down the hydrophobic non-specific interactions of merged adjacently eluting oligos with very close retention times resulting in baseline resolution.

[0079] Example 2

[0080] The procedure of Example 1 applied to a second pool of oligonucleotides, each again including two hydrophobic groups (FAM groups, propynyl groups) (see FIG. 6 showing the merged peaks). The same method of heating and flash cooling (flash chilling) prior to UPLC analysis was carried out and, after the treatment, the peaks 2 and 3 were well resolved even better than in the SOP standard chromatogram (FIG. 7 shows the result after thermal disruption of the interactions; while FIG. 8 provides the SOP chromatogram). In this example, the resolution of peaks 2 and 3 is even better than in SOP where we were able to only see the apex of the peaks 2 and 3. So, can we state that this method can adapted as a general method to get a better separation of the oligonucleotides. The method of heating and flash cooling (flash chilling) the oligonucleotide pool samples prior to UPLC injection is believed to help break down the hydrophobic non-specific interactions of merged adjacently eluting oligos with very close retention times resulting in baseline resolution.

[0081] Although the present disclosure has been described with reference to several illustrative embodiments, it should be understood that numerous other modifications and embodiments can be devised by those skilled in the art that will fall within the spirit and scope of the principles of this disclosure. More particularly, reasonable variations and modifications are possible in the component parts and / or arrangements of the subject combination arrangement within the scope of the foregoing disclosure, the drawings, and the appended claims without departing from the spirit of the disclosure. In addition to variations and modifications in the component parts and / or arrangements, alternative uses will also be apparent to those skilled in the art.

Claims

CLAIMS1. A method of assaying a pool of oligonucleotides comprising at least two or more different oligonucleotides comprising:(a) obtaining the pool of oligonucleotides comprising the at least two or more different oligonucleotides;(b) chemically disrupting hydrophobic interactions between the at least two or more different oligonucleotides in the pool of oligonucleotides; and(c) eluting the at least two or more different oligonucleotides from a solid phase support.

2. The method of claim 1, wherein the chemically disrupting of the hydrophobic interactions comprises contacting the obtained pool of oligonucleotides with a surfactant.

3. The method of claim 2, wherein the surfactant is an anionic surfactant.

4. The method of claim 2, wherein the surfactant is a cationic surfactant.

5. The method of claim 2, wherein the surfactant is a non-ionic surfactant.

6. The method of any one of claims 1 to 5, wherein the chemically disrupting of the hydrophobic interactions comprises contacting the obtained pool of oligonucleotides with a denaturing agent.

7. The method of claim 6, wherein the denaturing agent is formamide.

8. The method of claim 6, wherein the denaturing agent is selected from the group consisting of guanidine, sodium salicylate, dimethyl sulfoxide (DMSO), propylene glycol, and urea.

9. A method of assaying a pool of oligonucleotides including two or more different oligonucleotides comprising:(a) obtaining the pool of oligonucleotides comprising the two or more different oligonucleotides;(b) heating the pool of oligonucleotides to a first predetermined temperature for a first predetermined amount of time;(c) flash cooling the pool of oligonucleotides to a second predetermined temperature for a second predetermined amount of time, wherein second predetermined temperature is 10°C or less; and(d) eluting the pool of oligonucleotides cooled to the second predetermined temperature from a solid phase support.

10. The method of claim 9, wherein the first predetermined temperature is at least about 90°C.

11. The method of claim 9, wherein the first predetermined temperature ranges from between about 90°C to about 120°C.

12. The method of claim 9, wherein the first predetermined temperature ranges from between about 100°C to about 110°C.

13. The method of any one of claims 9 to 12, wherein the predetermined time is at least about 2 minutes.

14. The method of any one of claims 9 to 12, wherein the predetermined time is at least about 4 minutes.

15. The method of any one of claims 9 to 12, wherein the predetermined time ranges from between about 2 minutes to about 12 minutes.

16. The method of any one of claims 9 to 12, wherein the predetermined time ranges from between about 4 minutes to about 10 minutes.

17. The method of any one of claims 9 to 16, wherein the second predetermined temperature ranges from between about 0°C to about 10°C.

18. The method of any one of claims 9 to 16, wherein the second predetermined temperature ranges from between about 0°C to about 5°C.

19. The method of any one of claims 9 to 18, wherein the two or more different oligonucleotides in the pool of oligonucleotides each include one or more labels.

20. The method of claim 19, wherein the one or more labels are fluorescent labels.

21. The method of claim 20, wherein the fluorescent labels are hydrophobic.

22. The method of claim 20, wherein the fluorescent labels include one or more hydrophobic groups.

23. The method of any one of claims 9 to 22, wherein at least one of the two or more different oligonucleotides include one or more modified nucleotides, wherein the one or more modified nucleotides include a hydrophobic moiety.

24. The method of claim 23, wherein the hydrophobic moiety is an alkynyl group.

25. The method of claim 23, wherein the hydrophobic moiety is a heavy metal label.

26. The method of any one of claims 9 to 25, wherein at least two of the two or more different oligonucleotides in the sample non-specifically interact to each other.

27. The method of any one of claims 9 to 26, wherein at least two of the two or more oligonucleotides in the sample cannot be resolved by liquid chromatography.

28. The method of any one of claims 9 to 27, wherein the solid phase support is a chromatography column.

29. The method of claim 28, wherein the chromatography column is a reverse phase chromatography column.

30. The method of any one of claims 9 to 29, wherein the pool of oligonucleotides is eluted from the solid phase support using a solvent selected from acetonitrile, methanol, isopropanol, ethanol, tetrahydrofuran, or a combination thereof.

31. A method of assaying at least two different oligonucleotides comprising:(a) obtaining a sample comprising the at least two different oligonucleotides;(b) heating the sample comprising the at least two different at a first predetermined temperature for a first predetermined amount of time; and(c) flash cooling the at least two different oligonucleotides to a second predetermined temperature for a second predetermined amount of time, wherein second predetermined temperature is 10°C or less.

32. The method of claim 31 further comprising introducing the at least two different oligonucleotides flash cooled to the second predetermined temperature to a liquid chromatography instrument.

33. The method of claim 32, wherein the liquid chromatography instrument is a high- performance liquid chromatography instrument.

34. The method of claim 32, wherein the liquid chromatography instrument is an ultraperformance liquid chromatography instrument.

35. The method of any one of claims 31 to 34 further comprising introducing the at least two different oligonucleotides flash cooled to the second predetermined temperature to a chromatography column.

36. The method of claim 35, wherein the column is a reverse phase chromatography column.

37. The method of any one of claims 35 to 36, wherein the chromatography column has a temperature ranging from between about 0°C to about 60°C.

38. The method of any one of claims 31 to 37, wherein each of the at least two different oligonucleotides include one or more labels.

39. The method of claim 38, wherein the one or more labels are fluorescent labels.

40. The method of claim 39, wherein the fluorescent labels are hydrophobic.

41. The method of claim 39, wherein the fluorescent labels include one or more hydrophobic groups.

42. The method of any one of claims 31 to 41 , wherein the at least two different oligonucleotides include one or more modified nucleotides, wherein the one or more modified nucleotides include a hydrophobic moiety.

43. The method of claim 42, wherein the hydrophobic moiety is an alkynyl group.

44. The method of claim 42, wherein the hydrophobic moiety is a propynyl group.

45. The method of any one of claims 31 to 44, wherein at least two of the two or more oligonucleotides in the sample non-specifically bind to each other.

46. The method of any one of claims 31 to 45, wherein the first predetermined temperature is at least about 90°C.

47. The method of any one of claims 31 to 45, wherein the first predetermined temperature ranges from between about 80°C to about 140°C.

48. The method of any one of claims 31 to 45, wherein the first predetermined temperature ranges from between about 90°C to about 120°C.

49. The method of any one of claims 31 to 48, wherein the predetermined time is at least about 2 minutes.

50. The method of any one of claims 31 to 48, wherein the predetermined time is at least about 4 minutes.

51. The method of any one of claims 31 to 48, wherein the predetermined time ranges from between about 2 minutes to about 12 minutes.

52. The method of any one of claims 31 to 48, wherein the predetermined time ranges from between about 4 minutes to about 10 minutes.

53. The method of any one of claims 31 to 52, wherein the second predetermined temperature ranges from between about 0°C to about 10°C.

54. The method of any one of claims 31 to 52, wherein the second predetermined temperature ranges from between about 0°C to about 5°C.

55. The method of any one of claims 31 to 54 further comprising the quantifying an amount of the at least two oligonucleotides in the sample.

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