Non-ionic surfactants and methods of using the same

Abstract

Disclosed herein are compounds that can be used as surfactants for various applications. The disclosed compounds are biodegradable and do not form toxic by-products when degraded. As such, the disclosed compounds can be used as replacements for current surfactants, such as current octylphenol ethoxylates (OPEs) and nonylphenol ethoxylates (NPEs) that have toxic effects when degraded. Also disclosed are methods of making and using the disclosed compounds.

Description

NON-IONIC SURFACTANTS ANDMETHODS OF USING THE SAMECROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 739,959 filed on December 30, 2024, the contents of which are hereby expressly incorporated herein by reference in their entirety as though fully set forth herein.FIELD

[0002] The present disclosure is directed to new biodegradable surfactants and methods of making and using the same.BACKGROUND

[0003] Surfactants play an important role in life science technology and other various industries. With respect to their roles in life sciences, they are often important components used in both nucleic acid purification and direct PCR processes. For example, in direct PCR, surfactants are needed to enhance the efficiency of the reaction by improving the accessibility of the DNA template. They also can be used to aid in cell, vesicle, and virus lysis, viral inactivation, enhancing DNA accessibility, reducing non-specific binding, and ultimately contributing to the success of obtaining pure nucleic acids and amplifying specific DNA targets. Other processes and / or applications that rely on surfactants are discussed herein and can include, but are not limited to, hybridization, multiple arrays, kits for diagnosing diseases, nucleic acid-based and polypeptide-based sample preparation workflows, and the like.

[0004] Current non-ionic surfactants, such as Triton™ X-100 and Tergitol™ NP-40 or the like, that are available as surfactants for myriad biological and / or other industrial uses have a trade-off associated with good performance: they produce degradation by-products that exhibit endocrine disruption effects that interfere with the hormonal system of numerous organisms, particularly aquatic species. These surfactants are often used in sample preparation products, including nucleic acid and protein extraction buffers, wash buffers, nucleic acid and protein interaction kits, and nucleic acid and protein purification kits.

[0005] To date, no alternative surfactants are available as direct replacements for Triton™ X-100 and / or Tergitol™ NP-40, particularly those that are suitable for life science applications.12 / 22 / 25 TP391474W01Existing, more environmentally friendly alternatives, including polysorbate detergents such as Tween-20 and alcohol ethoxylates such as Ecosurf™ EH-9, fail to provide the stability, robustness, and workflow consistency required for magnetic bead-based sample preparation and immunoassay systems. Tween-20 is prone to autoxidation under commonly encountered storage and handling conditions, such as exposure to light, heat, dissolved oxygen, and trace metal ions, leading to formation of peroxides, aldehydes, and acidic species that cause pH drift, visible discoloration and subsequent customer concerns, and detrimental changes to surfactant properties, as documented in the scientific literature (Kishore et al., Pharm. Res. 28, 1194-1210 (2011), DOI: 10.1007 / sl 1095-011-0385-x; Aryal et al., Pharm. Res. 41, 1217-1232 (2024), DOI: 10.1007 / S11095-024-03700-7) and supplier technical reports (MilliporeSigma, polysorbate degradation overview: https: / / www.siqmaaldrich.com). Another commonly used alternative to Triton X-100, Ecosurf EH-9, although initially presenting acceptable internal results, can exhibit workflow-dependent unreliability, including significantly reduced nucleic acid recovery in external molecular diagnostics testing environments, and appears sensitive to differences in precipitation chemistries such as ethanol versus isopropanol.SUMMARY

[0006] Provided herein are methods for lysing cells, vesicles and viruses, isolating target molecules from samples, and kits and compositions comprising the disclosed surfactants for such methods. In one aspect, teachings herein include a method for preparing a sample containing nucleic acids, e.g., for downstream analysis. In some aspects, the method includes contacting the sample containing nucleic acids with a lysis buffer to produce a lysis mixture, and incubating the lysis mixture and for a period of time.

[0007] Provided is a method for sequential isolating target molecules such as polypeptides, DNA, and RNA from a single biological sample. This method allows for the sequential isolation of different types of target molecules from a single sample, ensuring that each type can be selectively bound to a specific solid support and separated accordingly. The method for isolating polypeptides, DNA (e.g., gDNA, cfDNA, viral DNA and the like), and / or RNA from a biological sample involves lysing the sample with a lysis solution comprising the disclosed surfactants.

[0008] Also provided herein are methods for preparing nucleic acids such as plasmid DNA (pDNA) from samples, and kits and compositions for such methods. In some aspects, methodsdescribed herein are based, in part, upon the surprising discovery of buffers comprising surfactants disclosed herein, and systems for nucleic acid (e.g. pDNA) preparations that are eco-friendly (e.g., are REACH compliant), produce pDNA suitable for therapeutic applications (e.g., are endotoxin-free), and at the same time are readily amenable to automation (e.g., can be used in workflows that eliminate the need for centrifugation).

[0009] Other technical features may be readily apparent to one skilled in the art from the following figures, descriptions, and claims.

[0010] Disclosed herein is a compound having a structure according to Formula I,Formula Iwherein:R1is a hydrophilic group;R2is a lipophilic group;X is selected from oxygen, sulfur, or N(R5);R3is selected from heteroaliphatic, aliphatic, or aryl;each R4independently is aliphatic;Y is selected from oxygen, sulfur, or N(R5);the linker, when present, is aliphatic or has a formula -C(=Z)-W-C(=Z)-X’, wherein each Z independently is oxygen, sulfur, or NR5, W is an aliphatic or heteroaliphatic group, and X’ is oxygen, sulfur, or NR5;n is an integer selected from 0 or 1 ;m is an integer selected from 0 to 6, provided that, if n is 0, then m is 1;p is an integer selected from 0 to 2; andeach R5group independently is selected from hydrogen, aliphatic, aromatic, or heteroaliphatic.

[0011] In one aspect the disclosure provides a composition, comprising:(a) one or more surfactant, wherein the surfactant is a compound having a structure according to Formula I:Formula Iwherein:R1is a hydrophilic group;R2is a lipophilic group;X is selected from oxygen, sulfur, or N(R5);R3is selected from heteroaliphatic, aliphatic, or aryl;each R4independently is aliphatic;Y is selected from oxygen, sulfur, or N(R5);the linker, when present, is aliphatic or has a formula -C(=Z)-W-C(=Z)-X’ , wherein each Z independently is oxygen, sulfur, or NR5, W is an aliphatic or heteroaliphatic group, and X’ is oxygen, sulfur, or NR5;n is an integer selected from 0 or 1 ;m is an integer selected from 0 to 6, provided that, if n is 0, then m is 1;p is an integer selected from 0 to 2; andeach R5group independently is selected from hydrogen, aliphatic, aromatic, or heteroaliphatic;(b) at least one polar protic solvent;(c) optionally, one or more inorganic salt;(d) optionally one or more buffering agent; and(e) optionally one or more chaotropic agents.

[0012] In one aspect the disclosure provides a composition, comprising a surfactant: (a) wherein the surfactant is a compound having a structure according to Formulas IA, IB, IC, ID, IE, or IF:12 / 22 / 25 TP391474W01wherein:R1is a hydrophilic group;R2is a lipophilic group;X is selected from oxygen, sulfur, or N(R5);R3is selected from heteroaliphatic, aliphatic, or aryl;each R4independently is aliphatic;Y is selected from oxygen, sulfur, or N(R5);the linker, when present, is aliphatic or has a formula -C(=Z)-W-C(=Z)-X’, wherein each Z independently is oxygen, sulfur, or NR5, W is an aliphatic or heteroaliphatic group, and X’ is oxygen, sulfur, or NR5;n is an integer selected from 0 or 1 ;m is an integer selected from 0 to 6, provided that, if n is 0, then m is 1;p is an integer selected from 0 to 2; andeach R5group independently is selected from hydrogen, aliphatic, aromatic, or heteroaliphatic;(b) at least one polar protic solvent;(c) optionally, one or more inorganic salt;(d) optionally one or more buffering agent; and(e) optionally one or more chaotropic agents.

[0013] Also disclosed is a method, comprising exposing a sample to a compound according to the present disclosure.

[0014] Also disclosed is a method of lysing a cell, comprising contacting the cell with an amount of a compound according to the present disclosure sufficient to facilitate cell lysis.

[0015] Also disclosed is a kit, comprising a compound according to the present disclosure; and a container.

[0016] In one aspect the disclosure provides a method for lysing cells, vesicles, or viruses, if present, in a sample, comprising, contacting the sample with the aforementioned composition; thereby generating a lysed sample.

[0017] In one aspect the disclosure further provides a method for isolating nucleic acids from a sample, comprising, generating a lysed sample by performing the aforementioned method andisolating nucleic acids from the lysed sample.

[0018] In one aspect the disclosure further provides a method for isolating polypeptides and nucleic acids from a sample containing nucleic acids and polypeptides, the method comprising:generating a lysed sample by performing the aforementioned method and isolating polypeptides and nucleic acids from the lysed sample.

[0019] In one aspect the disclosure provides a method for preparing nucleic acids from a sample containing nucleic acids, comprising, generating a lysed sample by performing the aforementioned method; and incubating the lysed sample at an incubation temperature and for a time to produce a treated cell lysate, wherein the composition comprises an RNase inhibitor, wherein the cell lysate is compatible with in situ polymerase or reverse transcription reactions.

[0020] In one aspect the disclosure provides a method; wherein the method further comprises: (a) contacting the lysed sample with a first solid support in the presence of a binding buffer to bind the polypeptides to the solid support; (b) wherein the binding buffer is a composition having a concentration of the surfactant according to Formula I effective to allow binding of the polypeptides to the first solid support.

[0021] In one aspect the disclosure provides a method wherein the method further comprises: (a) contacting the lysed sample with a second solid support in the presence of a12 / 22 / 25 TP391474W01binding buffer to bind the nucleic acids to the second solid support, (b) wherein the binding buffer is a composition having a concentration of the surfactant according to Formula I effective to allow binding of the nucleic acids to the second solid support.

[0022] The foregoing and other objects, features, and advantages of the present disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 shows the comparative genomic DNA (gDNA) yield and purity obtained from 100 pL whole blood using compounds 1 (SY-2), 3 (SY-207), and 2 (SY-210) as the surfactant incorporated into a commercially available lysis / binding buffer at 10% and 20% concentrations, compared with EcoSurf™ EH-9 and Triton™ X-100 controls and a no-surfactant condition.

[0024] FIG. 2 shows the comparative genomic DNA (gDNA) yield and purity obtained from 100 pL whole blood using compounds 1 (SY-2), 3 (SY-207), and 2 (SY-210) as the surfactants incorporated into a commercially available lysis / binding buffer at 2% and 5% concentrations, compared with EcoSurf™ EH-9 and Triton™ X-100 controls and a no-surfactant condition.

[0025] FIG. 3 shows the comparative genomic DNA (gDNA) yield and purity obtained from 200 pL whole blood using compounds of this disclosure as surfactants containing poly(ethylene glycol) linkers (Compound 7 (SY-DMB-dPEG9-OMe), Compound 6 (SY-iPe-dPEG9-OMe), Compound 7 (SY-No-dPEG9-OMe), Compound 4 (SY-Oc-dPEG9-OMe), and Compound 5 (SY-Pe-dPEG9-OMe)) which were incorporated into a commercially available lysis / binding buffer at 20% concentration.

[0026] FIG. 4 shows the quantitative PCR (qPCR) performance of genomic DNA (gDNA) extracted from 200 pL whole blood using compounds of this disclosure as surfactants containing polyethylene glycol) linkers (Compound 7 (SY-DMB-dPEG9-OMe), Compound 6 (SY-iPe-dPEG9-OMe), Compound 1 (SY-No-dPEG9-OMe), Compound 4 (SY-Oc-dPEG9-OMe), and Compound 5 (SY-Pe-dPEG9-OMe)) incorporated into a commercially available lysis / binding buffer at 20% concentration.

[0027] FIG. 5 demonstrates that compounds of this disclosure are effective at lysing human cell cultures at 0.1% and 1% concentration of the compound as a surfactant.

[0028] FIG. 6 demonstrates that compounds of this disclosure are compatible in RT-qPCR workflow at 0.1% and 1% concentration of the compound as a surfactant.12 / 22 / 25 TP391474W01

[0001] FIG. 7 shows the Western blot results for lysates prepared using Pierce™ IP Lysis Buffer, formulated using 0.5%, 1%, and 2% surfactants or no surfactant.

[0029] FIG. 8 shows the Western blot results for E. coli DH1a lysates prepared using B-PER™ buffers, formulated using 2% surfactants or no surfactant.

[0030] FIG. 9 shows the Western blot results for yeast lysates prepared using B-PER™ buffers, formulated using 2% surfactants or no surfactant.

[0031] FIG. 10 shows the Western blot results for lysates prepared using N-PER™ buffers, formulated using 1% and 2% surfactants or no surfactant.

[0032] FIG. 11 shows the Western blot results for lysates prepared using RIPA buffers, formulated using 1% surfactants or no surfactant.

[0033] FIG. 12 shows IP-Western blot results for antibody denaturation assay.

[0034] FIG. 13 shows signals in the AT and GC channels for hybridization buffer formulations prepared with either control Tween-20 or compounds of this disclosure and stored at -20 °C (storage temperature) or subjected to accelerated aging at 37 °C.

[0035] FIG. 14 shows the quality control performance of microarray hybridization buffers incorporating a compound of this disclosure or Tween 20 and stored at -20 °C or accelerated aged at 37 °C.

[0036] FIG. 15 shows effect of surfactant type and concentration on direct PCR amplification efficiency for DNA and RNA targets.

[0037] FIG. 16 shows multicomponent no-template control (NTC) fluorescence profiles for the FAM channel in the presence of surfactants.

[0038] FIG. 17 shows multicomponent no-template control (NTC) fluorescence profiles for the VIC channel in the presence of surfactants.DETAILED DESCRIPTIONOverview of Terms

[0039] The following explanations of terms are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. As used herein, “comprising” means “including” and the singular forms “a” or “an” or “the” include plural references unless the context clearly dictates otherwise. The term “or” refers to a single element of stated alternative elements or a combination of two or more elements unless the context clearly indicates otherwise.

[0040] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

[0041] Although the steps of some of the disclosed methods are described in a particular, sequential order for convenient presentation, it should be understood that this manner of description encompasses rearrangement, unless a particular ordering is required by specific language set forth below. For example, steps described sequentially may in some cases be rearranged or performed concurrently. Additionally, the description sometimes uses terms like “produce” or “provide” to describe the disclosed methods. These terms are high-level abstractions of the actual steps that are performed. The actual steps that correspond to these terms will vary depending on the particular implementation and are readily discernible by one of ordinary skill in the art.

[0042] Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting, unless otherwise indicated. Other features of the disclosure are apparent from the following detailed description and the claims.

[0043] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, percentages, temperatures, times, and so forth, as used in the specification or claims are to be understood as being modified by the term “about.” Accordingly, unless otherwise indicated, implicitly or explicitly, the numerical parameters set forth are approximations that can depend on the desired properties sought and / or limits of detection under standard test conditions / methods and in some aspects encompasses a range up to ± 15% of that numerical value, unless the context clearly dictates otherwise.

[0044] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not intended to limit the scope of the current teachings. In this application, the use of the singular includes the plural unless specifically stated otherwise. The use of "comprise", “contain”, and "include", or modifications ofthose root words, for example but not limited to, “comprises”, “contained”, and “including”, are not intended to be limiting. Use of “or” means “and / or” unless stated otherwise. The term “and / or” means that the terms before and after can be taken together or separately. For illustration purposes, but not as a limitation, “X and / or Y” can mean “X” or “Y” or “X and Y”.

[0045] Whenever a range of values is provided herein, the range is meant to include the starting value and the ending value and a value or value range there between unless otherwise specifically stated. For example, “from 0.2 to 0.5” means 0.2, 0.3, 0.4, 0.5; ranges there between such as 0.2-0.3, 0.3 - 0.4, 0.2 - 0.4; increments there between such as 0.25, 0.35, 0.225, 0.335, 0.49; increment ranges there between such as 0.26 - 0.39; and the like.

[0046] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way. All literature and similar materials cited in this application including, but not limited to, patents, patent applications, articles, books, treatises, and internet web pages, regardless of the format of such literature and similar materials, are expressly incorporated by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials defines or uses a term in such a way that it contradicts that term’s definition in this application, this application controls. While the present teachings are described in conjunction with various aspects, it is not intended that the present teachings be limited to such aspects. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.

[0047] The term “or combinations thereof” as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof” is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0048] Certain functional group terms used herein include a symbolwhich is used to show how the defined functional group attaches to, or within, the compound to which it is bound. Also, a dashed bond (i.e.,as used in certain formulas described herein indicates an “optional” bond to a substituent or atom of the formula other than hydrogen in the sense that thebond (and in some embodiments, the substituent) may or may not be present. In any formulas comprising a dashed bond, if the optional bond and / or any corresponding substituent is not present, then the valency requirements of any atom(s) bound thereto is completed by a bond to a hydrogen atom.

[0049] The symbol js used to indicate a bond disconnection in abbreviated structures / formulas provided herein. A person of ordinary skill in the art recognizes that the definitions provided below and the compounds and formulas included herein are not intended to include impermissible substitution patterns (e.g., methyl substituted with 5 different groups, and the like). Such impermissible substitution patterns are easily recognized by a person of ordinary skill in the art. In formulas and compounds disclosed herein, a hydrogen atom is present and completes any formal valency requirements (but may not necessarily be illustrated) wherever afunctional group or other atom is not illustrated. For example, a phenyl ring that is drawn ascomprises a hydrogen atom attached to each carbon atom of the phenyl ring other than the “a” carbon, even though such hydrogen atoms are not illustrated. Any functional group disclosed herein and / or defined above can be substituted or unsubstituted, unless otherwise indicated herein.

[0050] If a group R is depicted as “floating” on a ring system, as for example in the group:then, unless otherwise defined, a substituent R can reside on any atom of the fused bicyclic ring system, excluding the atom carrying the bond with the symbol, so long as a stable structure is formed. In the example depicted, the R group can reside on an atom in either the left side or the right side ring of the naphthylene ring system.

[0051] When there are more than one such depicted “floating” groups, as for example in the formula:where there are two groups, namely, the R and the bond indicating attachment to a parent structure; then, unless otherwise defined, the “floating” groups can reside on any atoms of the ring system, again assuming each replaces a depicted, implied, or expressly defined hydrogen on the ring system and a chemically stable compound would be formed by such an arrangement.

[0052] To facilitate review of the the disclosure, the following explanations of specific terms are provided.

[0053] Aldehyde: -C(O)H.

[0054] Aliphatic: A hydrocarbon group having at least one carbon atom to 50 carbon atoms (C1-50), such as one to 25 carbon atoms (C1-25), or one to ten carbon atoms (C1.10), and which includes alkanes (or alkyl), alkenes (or alkenyl), alkynes (or alkynyl), including cyclic versions thereof, and further including straight- and branched-chain arrangements, and all stereo and position isomers as well. Aliphatic groups may be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0055] Alkenyl: An unsaturated monovalent hydrocarbon having at least two carbon atoms to 50 carbon atoms (C2-50), such as two to 25 carbon atoms (C2-25), or two to ten carbon atoms (C2-10), and at least one carbon-carbon double bond, wherein the unsaturated monovalent hydrocarbon can be derived from removing one hydrogen atom from one carbon atom of a parent alkene. An alkenyl group can be branched, straight-chain, cyclic, cis, or trans (e.g., E orZ).

[0056] Alkoxy: -O-aliphatic, such as -O-alkyl, -O-alkenyl, -O-alkynyl; with exemplary embodiments including, but not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, f-butoxy, sec-butoxy, n-pentoxy (wherein any of the aliphatic components of such groups can comprise no double or triple bonds, or can comprise one or more double and / or triple bonds). Alkoxy groups may be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0057] Alkyl: A saturated monovalent hydrocarbon having at least one carbon atom to 50 carbon atoms (C1.50), such as one to 25 carbon atoms (C1-25), or one to ten carbon atoms (Ci-10), wherein the saturated monovalent hydrocarbon can be derived from removing one hydrogen atom from one carbon atom of a parent compound (e.g., alkane). An alkyl group can be branched, straight-chain, or cyclic.

[0058] Alkynyl: An unsaturated monovalent hydrocarbon having at least two carbon atoms to 50 carbon atoms (C2-50), such as two to 25 carbon atoms (C2-25), or two to ten carbon atoms (C2-10), and at least one carbon-carbon triple bond, wherein the unsaturated monovalent hydrocarbon can be derived from removing one hydrogen atom from one carbon atom of a parent alkyne. An alkynyl group can be branched, straight-chain, or cyclic.

[0059] Amide: -C(O)NRbRcor -NRbC(O)Rcwherein each of Rband Rcindependently is selected from hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group and can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0060] Amino: -NRbRc, wherein each of Rband Rcindependently is selected from hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group, and can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0061] Aromatic: A cyclic, conjugated group or moiety of, unless specified otherwise, from 5 to 15 ring atoms having a single ring (e.g., phenyl) or multiple condensed rings in which at least one ring is aromatic (e.g., naphthyl, indolyl, or pyrazolopyridinyl); that is, at least one ring, and optionally multiple condensed rings, have a continuous, delocalized ir-electron system. Typically, the number of out of plane rr-electrons corresponds to the Huckel rule (4n + 2). The point of attachment to the parent structure typically is through an aromatic portion of thecondensed ring system. For example,. However, in certain examples, context or express disclosure may indicate that the point of attachment is through a non-aromatic portion ofthe condensed ring system. For example,. An aromatic group or moiety may comprise only carbon atoms in the ring, such as in an aryl group or moiety, or it may comprise one or more ring carbon atoms and one or more ring heteroatoms comprising a lone pair of electrons (e.g. S, O, N, P, or Si), such as in a heteroaryl group or moiety. Aromatic groups may be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0062] Aroxy: -O-aromatic. Aroxy groups may be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0063] Aryl: An aromatic carbocyclic group comprising at least five carbon atoms to 15 carbon atoms (C5-15), such as five to ten carbon atoms (C5-10), having a single ring or multiple condensed rings, which condensed rings can or may not be aromatic provided that the point of attachment to a remaining position of the compounds disclosed herein is through an atom of the aromatic carbocyclic group. Aryl groups may be substituted with one or more groups other than hydrogen, such as an aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0064] Azo: -N=NRawherein Rais hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Azo groups may be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0065] Carbamate: -OC(O)NRbRc, wherein each of Rband Rcindependently is selected from hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Carbamate groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0066] Carbonate: -OC(O)ORa, wherein Rais selected from aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Carbonate groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. In independent embodiments, Racan be hydrogen.

[0067] Carboxyl: -C(O)OH.

[0068] Carboxylate: -C(O)O' or salts thereof, wherein the negative charge of the carboxylate group may be balanced with an M+counterion, wherein M+may be an alkali ion, such as K+, Na+, Li+; an ammonium ion, such as+N(Rb)4where Rbis H, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, or aromatic; or an alkaline earth ion, such as [Ca2+]o.5, [Mg2+]o.s, or [Ba2+]0.5.

[0069] Cyano: -CN.

[0070] Degree of Polymerization: The number of monomer units in a polymer. In the context of the present disclosure, when discussing, for example, polyalkene oxide and / or polyalkylene amine polymers comprising repeat units of monomers, the degree of polymerization is typically defined by a mass average molecular weight.

[0071] Disulfide: -SSRa, wherein Rais selected from hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Disulfide groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0072] Dithiocarboxylic: -C(S)SRawherein Rais selected from hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Dithiocarboxylic groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0073] Ester: -C(O)ORaor -OC(O)Ra, wherein Rais selected from aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Ester groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0074] Ether: -aliphatic-O-aliphatic, -aliphatic-O-aromatic, -aromatic-O-aliphatic, or -aromatic-O-aromatic, including any polymers thereof having repeats of any such groups (e.g., polyalkene oxide compounds). Ether groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0075] Halo (or halide or halogen): Fluoro, chloro, bromo, or iodo. In some embodiments, halo can also include astatine.

[0076] Haloaliphatic: An aliphatic group wherein one or more hydrogen atoms, such as one to 10 hydrogen atoms, independently is replaced with a halogen atom, such as fluoro, bromo, chloro, or iodo. Haloaliphatic groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0077] Haloheteroaliphatic: A heteroaliphatic group wherein one or more hydrogen atoms, such as one to 10 hydrogen atoms, independently is replaced with a halogen atom, such as fluoro, bromo, chloro, or iodo. Haloheteroaliphatic groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.12 / 22 / 25 TP391474W01

[0078] Heteroatom: An atom other than carbon or hydrogen, such as (but not limited to) oxygen, nitrogen, sulfur, silicon, boron, selenium, or phosphorous. A heteroatom does not include a halogen atom.

[0079] Heteroaliphatic: An aliphatic group comprising at least one heteroatom to 20 heteroatoms, such as one to 15 heteroatoms, or one to 5 heteroatoms, which can be selected from, but not limited to oxygen, nitrogen, sulfur, silicon, boron, selenium, phosphorous, and oxidized forms thereof within the group. Alkoxy, ether, amino (excluding NH2), disulfide (wherein Rais other than H), peroxy (wherein Rais other than H), and thioether groups are exemplary (but non-limiting) examples of heteroaliphatic. Heteroaliphatic groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0080] Hydrophilic: A hydrophilic group according to the present disclosure is a functional group or other chemical group that exhibits affinity for water. In some aspects, a hydrophilic group can further comprise a lipophilic portion; however, in such instances, the hydrophilic group is predominately hydrophilic and the lipophilic portion makes up a minor (e.g., less than 50%) of the hydrophilic group. Solely by way of example, a hydrophilic group can comprise a combination of a heteroaliphatic group and an aliphatic group and still be hydrophilic because the heteroaliphatic group makes up a greater proportion of the molecular weight of the total hydrophilic group than does the aliphatic group.

[0081] Hydrophilic-Lipophilic Balance (HLB): A numerical value that represents the balance of the size and strength of the hydrophilic and lipophilic moieties of a surfactant compound. The HLB scale ranges from 0 to 20.

[0082] Hydroxyl: -OH

[0083] Ketone: -C(O)Ra, wherein Rais selected from aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Ketone groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0084] Lipophilic: A lipophilic group according to the present disclosure is a functional group or other chemical group that lacks affinity for water.

[0085] Tergitol™ NP-40: A compound having

[0086] Organic Functional Group: A functional group that may be provided by any combination of aliphatic, heteroaliphatic, aromatic, haloaliphatic, and / or haloheteroaliphatic groups, or that may be selected from, but not limited to, aldehyde; aroxy; acyl halide; halogen; nitro; cyano; azide; carboxyl (or carboxylate); amide; ketone; carbonate; imine; azo; carbamate; hydroxyl; thiol; sulfonyl (or sulfonate); oxime; ester; thiocyanate; thioketone; thiocarboxylic acid; thioester; dithiocarboxylic; phosphonate; phosphate; silyl ether; sulfinyl; sulfonamide; thial; or combinations thereof. Organic functional groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0087] Oxime: -CRa=NOH, wherein Rais hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Oxime groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0088] Peroxy: -O-ORawherein Rais hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Peroxy groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0089] Phosphate: -O-P(O)(ORa)2, wherein each Raindependently is hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group; or wherein one or more Ragroups are not present and the phosphate group therefore has at least one negative charge, which can be balanced by a counterion, M+, wherein each M+independently can be an alkali ion, such as K+, Na+, Li+; an ammonium ion, such as+N(Rb)4 where Rbis H, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, or aromatic; or an alkaline earth ion, such as [Ca2+]0.5, [Mg2+]05, or [Ba2+]o.s. The Ragroups of the phosphate can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0090] Phosphonate: -P(O)(ORa)2, wherein each Raindependently is hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group; or wherein one or more Ragroups are not present and the phosphate group therefore has at least one negative charge, which can be balanced by a counterion, M+, wherein each M+independently can be an alkali ion, such as K+, Na+, Li+; an ammonium ion, such as+N(Rb)4where Rbis H, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, or aromatic; or an alkaline12 / 22 / 25 TP391474W01earth ion, such as [Ca2+]0.5, [Mg2+]o.5, or [Ba2+]0.5. The Ragroups of the phosphonate group can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0091] Silyl Ether: -OSiRaRbRc, wherein each of Ra, Rband Rcindependently is selected from hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Silyl ether groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0092] Sulfinyl: -S(O)Ra, wherein Rais selected from hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Sulfinyl groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0093] Sulfonyl: -SO2Ra, wherein Rais selected from hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Sulfonyl groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0094] Sulfonamide: -SO2NRbRcor -N(Rb)SC>2Rc, wherein each of Rband Rcindependently is selected from hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Sulfonamide groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0095] Sulfonate: -SO3; wherein the negative charge of the sulfonate group may be balanced with an M+counter ion, wherein M+may be an alkali ion, such as K+, Na+, Li+; an ammonium ion, such as+N(Rb)4where Rbis H, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, or aromatic; or an alkaline earth ion, such as [Ca2+]0.5, [Mg2+]o.s, or [Ba2+]o.s.

[0096] Surfactant: A compound that lowers surface tension between two fluids (liquids and / or gases) between a solid and a fluid. Surfactants can exhibit properties the allow their use as surfactants, dispersants, emulsifiers, foaming agents, wetting agents, lubricants, or a combination thereof.

[0097] Terminating Group: A functional group that is used to terminate an R1group of the formulas according to the present disclosure.12 / 22 / 25 TP391474W01

[0098] Thial: -C(S)H.

[0099] Thiocarboxylic acid: -C(O)SH, or-C(S)OH.

[0100] Thiocyanate: -S-CN or -N=C=S.

[0101] Thioester: -C(O)SRaor -C(S)ORawherein Rais selected from hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Thioester groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0102] Thioether: -S-aliphatic or -S-aromatic, such as -S-alkyl, -S-alkenyl, -S-alkynyl, -S-aryl, or -S-heteroaryl; or -aliphatic-S-aliphatic, -aliphatic-S-aromatic, -aromatic-S-aliphatic, or -aromatic-S-aromatic. Thioether groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0103] Thioketone: -C(S)Rawherein Rais selected from hydrogen, aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group. Thioketone groups can be substituted with one or more groups other than hydrogen, such as aliphatic, heteroaliphatic, haloaliphatic, haloheteroaliphatic, aromatic, or an organic functional group.

[0104] Triton™ X-100: A compound having a structure

[0105] Solid support: The solid support may comprise beads, for example monodisperse beads. The (optionally monodisperse) beads may be magnetic. In some instances, the solid support may be a component of a robotic liquid handling platform. The solid support may be a membrane, a resin or a bead. Advantageously, a membrane or resin is provided in a tube or column that allows easy contacting the support with the lysed biological sample in a corresponding binding buffer, while solid supports provided as beads are simply added to the corresponding binding buffer or the lysed biological sample or the corresponding binding buffer and the lysed biological sample are mixed prior to adding the beads.

[0106] The solid support may be a magnetic bead, a paramagnetic bead or a superparamagnetic bead. Suitable beads or membranes are known in the art and include but not limited to, for e.g. MagAttract® silica-coated beads (Qiagen), Sera-Mag® Carboxyl Magnetic Beads (Cytiva), Dynabeads® MyOne™ Streptavidin C1 (Life Technologies), ag ReSy n® Protein A agarose-coated beads (ReSyn Biosciences), BioMag® polystyrene beads (Qiagen), Amersham Hybond-P PVDF membranes (Cytiva), Optitran® BA-S 85 nitrocellulose membranes (GE Healthcare), and Stericup® PES membranes (MilliporeSigma).

[0107] Magnetic: The term “magnetic” means responds to a magnetic field. For example, magnetic beads respond to a magnetic field. Magnetic materials (such as magnetic beads) may be paramagnetic or superparamagnetic. When the magnetic material is paramagnetic, the magnetic properties are switched off when the magnetic field is removed. When the magnetic material is superparamagnetic, the magnetic material becomes saturated at relatively low magnetic fields and switching off of the magnetic properties with removal of the magnetic field is very rapid / instant. Some magnetic material, e.g. iron oxides, form superparamagnetic crystals when the size of the crystals is sufficiently small (e.g. below about 15 nm scale for iron oxides).

[0108] “Nucleic acid” refers to a polynucleotide molecule. The polynucleotide may be a naturally occurring polynucleotide or a synthetic polynucleotide. A nucleic acid may be a DNA, RNA or mixture of DNA and RNA nucleotides. Typically, a nucleic acid contains from 20 to 10,000 nucleotides or more, such as from 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, or 5000 nucleotides to 10,000 nucleotides. The term “nucleic acid” means, unless otherwise stated, a polynucleotide molecule made up of ribonucleotides and / or deoxyribonucleotides as well as synthetic nucleotide residues that are capable of participating in Watson-Crick type or analogous base pair interactions, i.e. "hybridization" or the formation of a "duplex". Thus, the nucleic acid may be DNA or RNA or any modification thereof, including conformationally restricted or nucleobase analogue-bearing oligomers such as “locked-nucleic acids” (LNA) or “peptide nucleic acids” (PNA) or other derivatives containing non-nucleotide backbones. The nucleic acid may be a naturally occurring molecule, i.e. DNA or RNA but also include DNA / RNA hybrids where the DNA is in separate strands or in the same strand) in which the 3' position of the pentose of one nucleotide is joined by a phosphodiester linkage to the 5' position of the pentose of the next nucleotide. Nucleic acids used in various aspects may comprise chemically, enzymatically, or metabolically modified forms of nucleotides or combinations thereof,12 / 22 / 25 TP391474W01such as primers, probes, oligonucleotides or aptamers. Exemplary types of DNA include synthetic DNA, plasmid DNA, genomic DNA, viral DNA (e.g. dsDNA or ssDNA), cDNA or cfDNA. Exemplary types of RNA include mRNA, siRNA, microRNA, tRNA, cfRNA, rRNA, or viral RNA (e.g. dsRNA or ssRNA).

[0109] “Polypeptide,” “Protein,” and “Peptide:” are used interchangeably herein and refer to an amino acid polymer or a set of two or more interacting or bound amino acid polymers. Polypeptides sometimes have a secondary, tertiary and / or quaternary structure that is typically is using non-covalent bonds, such as hydrogen bonds, ionic bonds, hydrophobic interactions, and / or van der Walls interactions, and / or covalent bonds, for example, disulfide bonds, such as between the thiol groups of cysteine residues.

[0110] The terms “isolation”, “separation” and “purification” are used synonymously and are interchangeable. The terms describe processes involved in obtaining target molecules such as the polypeptides or nucleic acids from a complex mixture, which include extracting the target molecules from their original source, and removing impurities to achieve a form that is suitable for further use or analysis. These processes collectively ensure that the target molecules are obtained in a higher concentration and purity compared to their initial state.

[0111] The term “monodisperse” means that for a plurality of particles or beads (e.g. at least 100, more preferably at least 1,000) the particles or beads have a coefficient of variation (CV) or % polydispersity of their diameters of less than 20%, for example less than 15%, typically of less than 10% and optionally of less than 8%, e.g. less than 5%. The term monodisperse is used herein to characterize a population of particles or beads with low heterogeneity and a homogenous size distribution. The size distribution of a particle or bead may be defined by the percentage CV (coefficient of variation) which may be determined on a CPS disc centrifuge as described e.g. in the Analytical Methods section of WO2017211913A1 which is incorporated by reference herein. CV is defined as 100 times (standard deviation) divided by average where “average” is mean particle or bead diameter 10 and standard deviation is standard deviation in particle size. The CV for a plurality of particles may for example be within a range of 50 to 100%. For example, a monodisperse particle or beads population may have more than 90%, preferably more than 95% of the particles or beads with sizes within their mean diameter of ± 5 %.

[0112] A Polypeptide having Protease Activity: In certain aspects herein, the lysis solution comprises a polypeptide having protease activity such as for example, proteinase K. In lieu of, or in addition to, proteinase K, the lysis solution can comprise a serine protease such astrypsin, chymotrypsin, elastase, subtilisin, streptogrisin, thermitase, aqualysin, plasmin, cucumisin, or carboxypeptidase A, D, C, or Y; a cysteine protease such as papain, calpain, or clostripain; an acid protease such as pepsin, chymosin, or cathepsin; or a metalloprotease such as pronase, thermolysin, collagenase, dispase, an aminopeptidase or carboxypeptidase A, B, E / H, M, T, or U.

[0113] A polypeptide having deoxyribonuclease activity: A polypeptide having deoxyribonuclease activity is present in certain lysis mixtures as set forth in aspects herein where RNA is to be detected. The polypeptide having deoxyribonuclease activity is dependent upon cations such as Ca++or Mg++for stability and activity. In the case where a polypeptide having deoxyribonuclease activity is obtained with a cation already present, which is commonly the case, additional cations are not needed in the lysis mixture. In the case where a polypeptide having deoxyribonuclease activity is obtained lacking cations, exogenous cations are added to the lysis mixture. A polypeptide having deoxyribonuclease activity can be DNase I or Nuclease BAL-31, both of which are Ca++- and Mg++-dependent; or exonuclease I, exonuclease III, Lambda exonuclease, CviKI-1 endonuclease, or McrBC endonuclease, all of which are Mg++-dependent, or an enzymatically active mutant or variant thereof. A polypeptide having deoxyribonuclease activity can be present in the lysis mixture from 100 U / ml to 600 U / ml in some aspects and, for other aspects, about 200 U / ml, about 300 U / ml, about 400 U / ml, about 500 U / ml or any range of concentrations therebetween. In certain aspects, the volume of deoxyribonuclease added is less than about 1% of the volume of the final lysis reaction. Preferably, the DNase is a heat-labile double-strand specific DNase (HL-dsDNase). As the HL-dsDNase is double-strand specific, it would not interfere with cDNA synthesis, e.g., in a downstream reverse transcriptase reaction. Advantageously, the HL-dsDNase is heat inactivated at 55 °C. Accordingly, in RT-qPCR reactions, which generally include inactivation of reverse transcriptase, the HL-dsDNase would be simultaneously inactivated.

[0114] A cation chelator effective to inactivate the polypeptide having deoxyribonuclease activity of the stop mixture: For aspects where the polypeptide having deoxyribonuclease activity is dependent upon calcium ions for stability and activity, the cation chelator comprises a calcium chelator such as EGTA or EDTA, for example. For aspects where the polypeptide having deoxyribonuclease activity is dependent upon magnesium ions for stability and activity, the cation chelator comprises a magnesium chelator such as EDTA, for example. Of course, divalent cation chelators bind a variety of divalent cations and overlap in specificity for divalent cations isexpected. Cation chelators include EGTA, ethylenediamine tetraacetic acid (EDTA), cation exchange beads such as SP SEPHAROSE™ beads (GE Healthcare), 1,10-phenanthroline, tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), or a combination thereof. EGTA inhibits DNase I at u 4 mM and is compatible with RT-PCR.

[0115] An inhibitor of a polypeptide having protease activity: The stop mixture comprises a chemical or molecular inhibitor of the polypeptide having protease activity. In addition, the chemical inhibitor has essentially no inhibitory effect on reverse transcriptase or DNA polymerase. For aspects where the polypeptide having protease activity is proteinase K, the chemical inhibitor can be a methoxysuccinyl-Ala-Ala-Pro / Leu-Ala / Val-chloroalkyl ketone where the alkyl is Ci to C3 or active derivatives or analogs thereof. Methoxysuccinyl-AAPV-chloromethyl ketone (for example, in DMSO, Sigma-Aldrich, St. Louis, MO) at a concentration as low as 0.75 mM was demonstrated to have inhibitory activity for 100 pg / ml PK and is compatible with both one-step and two-step RT-PCR reactions. Other inhibitors of proteinase K include carbobenzoxy-Ala-Ala-COCH2CI, carbobenzoxy-Ala-Ala-Phe-COCH2CI or carbobenzoxy-Phe-Pro-Arg-COCH2CI as described by Wolf et al. (JBC 266:26, 17695, 1991), and phenylmethylsulfonyl fluoride (PMSF) at a concentration of up to 2 mM in the stopped mixture (up to 400 mM in an RT reaction). Further protease / protease inhibitor pairs include leupeptin as an inhibitor for serine and cysteine proteases such as plasmin, trypsin, papain, kallikrein and cathepsin B; 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF) as an inhibitor for serine proteases such as chymotrypsin, kallikrein, plasmin, thrombin, and trypsin; aprotinin as an inhibitor of serine proteases such as trypsin, chymotrypsin, plasmin and kallikrein; benzamidine as an inhibitor of trypsin; N-acetyl eglin-C as an inhibitor of chymotrypsin, subtilisin, leukocyte elastase and cathepsin G; and antipain or plasmin for inhibition of a serine or cysteine such as papain and trypsin. Further protease inhibitors include aptamers, or polyclonal or monoclonal antibodies having binding affinity and binding specificity for the polypeptide having protease activity of the lysis mixture.

[0116] Enzymatically Active Mutants or Variants Thereof: The term “enzymatically active mutants or variants thereof” when used in reference herein to an enzyme such as a protease, deoxyribonuclease, a polymerase or the like, refers to a polypeptide derived from the corresponding enzyme that retains at least some of the desired enzymatic activity. Enzymatically active mutants or variants include, for example, fragments, recombinantly expressed fragments, naturally-occurring mutants, mutants generated using mutagens, genetically engineered mutants,mutants due to amino acid insertions or deletions or due to nucleic acid nonsense, missense, or frameshift mutations, reversibly modified enzymes, splice variants, polypeptides having modifications such as altered glycosylation, disulfide bonds, hydroxyl side chains, and phosphate side chains, or crosslinking, and the like. Protocols for measuring enzymatic activity using an appropriate assay are known to one of ordinary skill in the art.

[0117] Temperature: The sample preparation processes of teachings herein include a contacting step to produce a lysate and an admixing step where the lysate is mixed with a stop mixture where the steps are carried out at substantially the same temperature. “Substantially the same temperature” generally refers to an isothermal process of holding the temperature relatively constant during the contacting and admixing steps and, for certain aspects described herein, means ambient temperature which temperature may change during the day or from lab to lab. In general, the contacting and admixing steps are carried out at substantially the same temperature, which temperature is about 15 °C to 40 °C, or about 16 °C to 28 °C or about 19 °C to 26 °C, or about 19 °C to 25 °C, or about 22 °C to 25 °C, or at ambient temperature, or about 15 °C, 16 °C, 17 °C, 18 °C, 19 °C, 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, or 40 °C. An isothermal process is particularly amenable for high throughput analyses.

[0118] Sample: The term “sample,” as used herein, refers to any material, substance, or composition of matter, whether biological, environmental, synthetic, or otherwise derived, that contains or is suspected of containing nucleic acids, polypeptides, cells, cellular components, vesicles, viruses, genetic material, or other analytes of interest. In certain aspects, the genetic material of the sample comprises RNA. In other aspects, the genetic material of the sample is DNA, or both RNA and DNA. In certain aspects, a tissue sample includes a cell isolated from a subject. A subject includes any organism from which a sample can be isolated. Non-limiting examples of organisms include prokaryotes, eukaryotes or archaebacteria, including bacteria, fungi, animals, plants, or protists. The animal, for example, can be a mammal or a non-mammal. The mammal can be, for example, a rabbit, dog, pig, cow, horse, human, or a rodent such as a mouse or rat. In particular aspects, the tissue sample is a human tissue sample. The tissue sample can be, for example, a blood sample. The blood sample can be whole blood or a blood product (e.g., red blood cells, white blood cells, platelets, plasma, serum). The sample, in other non-limiting aspects, can be saliva, a cheek, throat, or nasal swab, a fine needle aspirate, a tissue print, cerebral spinal fluid, mucus, lymph, feces, urine, skin, spinal fluid, peritoneal fluid, lymphaticfluid, aqueous or vitreous humor, synovial fluid, tears, semen, seminal fluid, vaginal fluids, pulmonary effusion, serosal fluid, organs, bronchio-alveolar lavage, tumors, and constituents and components of in vitro cell cultures. In other aspects, the tissue sample is a solid tissue sample. In still further aspects, the sample comprises a virus, bacteria, or fungus. The sample can be an ex vivo tissue or sample. The sample can be a fixed sample, including as set forth by U.S. Published Patent Application No. 2003 / 0170617 filed January 28, 2003. Sample preparation processes provided by teachings herein are for from one cell up to about 105cells per sample or any range therebetween. For example, a patient needle biopsy often consists of thousands of cells. A biopsy could be prepared using methods herein, PCR amplified and analyzed by measuring the expression of certain genes, for example. The sample may comprise or be a pretreated or untreated biological sample, clinical or environmental sample, or an enzymatic reaction mixture.

[0119] The sample may be or comprise a pre-treated or untreated biological sample. The biological sample may be in a physiological buffer or transport medium. The biological sample may be a harvested or biopsied sample or a cultured sample. The sample may be or comprise an environmental sample. The sample may be or comprise an enzymatic reaction mixture.

[0120] The biological sample may be any suitable biological sample. Exemplary biological samples may comprise but are not limited to one or more of blood, blood stain, cord blood, blood components (e.g., platelet concentrates), blood cultures, peripheral blood mononuclear cells, peripheral blood leukocytes, plasma lysates, leukocyte lysates, buffy coat leukocytes, serum, plasma, saliva, saliva stain, buccal cells, buccal swab, semen, semen stain, urine, fecal matter, fecal stain, cigarette butt, chewing gum, formalin-fixed paraffin-embedded (FFPE) sample, biopsy (e.g. tumor biopsy) sample, bone marrow or other tissue sample, plant sample, cell lysate, bacterial or yeast culture, sputum, tear, throat swabs, oral rinses, nasopharyngeal swabs, nasopharyngeal aspirates, exhalates, nasal swabs, nasal washes, mucus, bronchial aspirations, bronchoalveolar lavage fluid, pleural fluid, endotracheal aspirates, cerebrospinal fluid, anal swabs, rectal swabs, vaginal swabs, endocervical swabs, vitreous fluid, amniotic fluid, breast milk, exosomes, circulating tumor cells, tissue lysates, bacterial lysates, yeast lysates, and plant lysates. The biological sample may comprise exosomes. The biological sample may be a fluid biological sample. In some instances, the biological sample may be a clinical sample. In some instances, the sample may be a cell-free sample.12 / 22 / 25 TP391474W01

[0121] The environmental sample may comprise a water sample (such as a wastewater sample, swimming pool water sample, ocean water sample), a soil sample, a sediment sample, a surface swab, an air derived sample (such as an air filter residue), a cosmetic, a food ingredient or a food sample, or a combination thereof.

[0122] The enzymatic reaction mixture could be any such mixture that may contain nucleic acid sequences. For example, the enzymatic reaction mixture may comprise in vitro transcription reaction mixture, a reverse transcription reaction mixture, a second strand synthesis reaction mixture, a nucleic acid assembly reaction mixture, an amplification reaction mixture, a library preparation reaction mixture, a restriction reaction mixture, a nucleic acid assembly reaction mixture, ora barcoding reaction mixture.

[0123] Lysis Mixture: A “lysis mixture,” as used herein, refers to the combination of a sample with a lysis buffer, wherein the lysis buffer includes components for lysing cells, viruses, vesicles, or the like present in the sample. The lysis buffer and lysis mixture lack components that can interfere with downstream processing of target molecules, such as nucleic acids, e.g., reverse transcription and / or amplification reactions. Preferably, the lysis buffer and lysis mixture also lack components that could interfere with methods of detecting nucleic acids using emission detection at wavelengths of 300 nm to 750 nm. The cell, virus or vesicle lysates (e.g., produced by incubating the lysis mixtures as described herein) described herein may or may not require further processing (e.g., inactivation of enzymes), prior to use in downstream analyses such as reverse transcription and / or amplification reactions. For some compositions and methods provided herein, the compositions and methods may be faster and simpler when compared to traditional sample preparation processes (e.g., including proteinase K digestion or lysis with harsh chemicals that must be removed prior to use of the nucleic acids in downstream reactions), making the methods provided herein suitable for automation, and high throughput applications.

[0124] The lysis mixtures described herein may be incubated for a period of time, which can range from less than 1 minute to several hours, depending upon the incubation temperature. For example, the lysis mixtures can be incubated for a period of time between 1 minute and 2 hours, at about 16 °C to 28 °C. For example, lysis mixtures can be incubated at 16 °C to 28 °C for 2 minutes to about 60 minutes, about 2 minutes to about 20 minutes, about 3 minutes to about 15 minutes, about 4 minutes to about 10 minutes or about 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, 31 minutes, 32 minutes, 33 minutes 34 minutes 35 minutes 36 minutes, 37 minutes, 38 minutes, 39 minutes, 40 minutes, 41 minutes 42 minutes 43 minutes, 44 minutes 45 minutes, 46 minutes 47 minutes, 48 minutes 49 minutes, 50 minutes, 51 minutes, 52 minutes 53 minutes 54 minutes 55 minutes 56 minutes, 57 minutes, 58 minutes, 59 minutes, 60 minutes, or longer, or any time in between. Alternatively, lysis mixtures can be held on ice, or incubated at 4 °C for 15 minutes to 12 hours or longer, e.g., 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, or longer, or any time in between.

[0125] Temperature: The methods provided herein may include incubation of the lysis mixture at a temperature, which is any temperature appropriate for the desired process. In some aspects, the temperature is preferably between about 15 °C to 40 °C, or about 16 °C to 28 °C or about 19 °C to 26 °C, or about 19 °C to 25 °C, or about 22 °C to 25 °C, or at ambient temperature, or about 15 °C, 16 °C, 17 °C, 18 °C, 19 °C, 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, or 40 °C. In some aspects, the lysis mixture may remain at substantially the same temperature during the incubation time. “Substantially the same temperature” generally refers to an isothermal process of holding the temperature relatively constant during the incubation time, for certain aspects described herein, means ambient temperature which temperature may change during the day or from lab to lab. An isothermal process is particularly amenable for high throughput analyses. In some aspects, wherein the lysis buffer includes a HL-dsDNase, or wherein a HL-dsDNase is added to the sample or lysis mixture, the incubation temperature is such that the HL-dsDNase is not inactivated (e.g., below 50 °C).

[0126] Lysis Buffer: The term “lysis buffer” means, unless the context requires otherwise, to any solution formulated to disrupt, break open, or permeabilize biological cells, viral particles, vesicles, or subcellular structures to release intracellular components, including but not limited to nucleic acids, proteins, lipids, or organelles. Exemplary lysis buffers provided herein are aqueous buffers comprising a polar protic solvent and optionally a compound of this disclosure. In some aspects, the polar protic solvent may be selected from a C1-C5 alcohol, C2-C6 polyol, polyethylene glycol, water, and mixtures thereof. In some aspects, the polar protic solvent is selected from ethanol, isopropyl alcohol, 2-methyl-1,3-propanediol (MPD), isoamyl alcohol, water, and mixtures thereof. In some aspects, the polar protic solvent is a polyethylene glycol (PEG) which may be selected from the group including, but not limited to, PEG1500, PEG8000 PEG6000, PEG2000,PEG 1000, PEG600, and mixtures thereof. Lysis buffers provided herein optionally include an anionic oligomer having RNase inhibitory activity, and a compound of this disclosure. Lysis buffers provided herein can optionally include one or more inorganic salts, which may include any inorganic salt disclosed herein or any other suitable inorganic salt. Lysis buffers provided herein can optionally include one or more chaotropic agents, which may include any chaotropic agent disclosed herein or any other suitable agent. Lysis buffers provided herein can optionally include one or more chelating agents, which may include any chelating agent disclosed herein or any other suitable agent. Lysis buffers provided herein can optionally include one or more buffering agents, which may include any buffering agent disclosed herein or any other suitable agent. The lysis buffers provided herein can optionally include a buffer, such as Tris or Tris base, HEPES, CHAPS, or the like, at pH 6.0 to 9.0 for a range of temperatures such as 5 °C to 40 °C. The lysis buffers can optionally include a chelator (e.g., EDTA, EGTA or the like) or can optionally be substantially free of a chelator.

[0127] “Inorganic Salt:” The term “inorganic salt” means, unless the context requires otherwise, any inorganic salt, including but not limited to an alkali or alkaline earth metal salt, LiCI, NaCI, KCI, MgCh, BaCh, and CaCh, and corresponding citrates, acetates, any combination thereof, ammonium chloride, sodium acetate, potassium acetate, ammonium acetate, sodium citrate, potassium citrate, sodium phosphate, magnesium phosphate, tetramethylammonium chloride, tetraethylammonium chloride, tetrapropylammonium chloride, tetrabutylammonium chloride, guanidinium thiocyanate, sodium thiocyanate, or potassium thiocyanate, and any other appropriate inorganic salt.

[0128] “Chaotropic Agent:” The term “chaotropic agent” means, unless the context requires otherwise, any inorganic chaotropic agent, including but not limited to lithium perchlorate, lithium acetate, sodium dodecyl sulfate, thiourea, urea, guanidinium chloride, guanidinium thiocyanate, a combination thereof, and any other suitable agent.

[0129] “Chelating Agent:” The term “chelating agent” means, unless the context requires otherwise, any chelating agent, including but not limited to EDTA, EGTA, DTPA, and the like, and any other suitable agent

[0130] “Buffering Agent:” The term “buffering agent” means, unless the context requires otherwise, a compound or mixture that helps maintain or stabilize a pH in a solution within a desired range. A buffering agent may include, but is not limited to any of MOPS (3-(N-morpholino)propanesulfonic acid), citrate buffers (saline-sodium citrate (SSC)), phosphate buffers(phosphate buffered saline (PBS)), tris (tris(hydroxymethyl)aminomethane) or (2-amino-2-(hydroxymethyl)propane-1,3-diol)-based buffers, TAPS ([tris(hydroxymethyl)methylamino]propanesulfonic acid)-based buffers, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)-based buffers, Bicine (2-(bis(2-hydroxyethyl)amino)acetic acid)-based buffers, tricine (N-[tris(hydroxymethyl)methyl]glycine)-based buffers, TAPSO (3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid)-based buffers, TES (2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid)-based buffers, MES (2-(N-morpholino)ethanesulfonic acid)-based buffers, PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid))-based buffers, Cacodylate (dimethylarsenic acid)-based buffers, or any combination thereof, or any other suitable buffering agent.

[0131] Binding Buffer: The term “binding buffer” means, unless the context requires otherwise, a buffer that is useful for precipitating a target molecule, such as a nucleic acid or polypeptide, from solution to a solid support. Nucleic acids are often solvated in aqueous solutions, therefore typical binding buffers are miscible in aqueous solution, e.g. a binding buffer may be provided as an aqueous buffer. Exemplary binding buffers of the present disclosure are aqueous buffers comprising a polar protic solvent or polar aprotic solvent and optionally a compound of this disclosure. Binding buffers provided herein can optionally include one or more chelating agents, or buffering agents.

[0132] Wash Buffer: The terms “wash buffer” or “washing buffer” mean, unless the context requires otherwise, a buffer that is useful for washing a nucleic acid or polypeptide bound to a substrate, solid support, biological sample, reagent mixture, or reaction environment. Binding buffers, in view of their function to precipitate nucleic acid from solution, may also be useful as wash buffers, as they should provide minimal loss of the target molecule from the solid support during washing. Binding conditions, which precipitate the nucleic acid onto the solid support, may be harsher than washing conditions, which need to prevent the nucleic acid eluting from the solid support. In view of this, the wash buffer may represent an aqueous dilution of the binding buffer, such as a 1.1 to 5 times dilution of a corresponding binding buffer. Exemplary wash buffers of the present disclosure are aqueous buffers comprising a polar protic solvent or polar aprotic solvent and optionally a compound of this disclosure. Wash buffers provided herein can optionally include one or more buffering agents.

[0133] Targeted protein isolation is specific to a particular protein, or class of protein. An example of targeted protein isolation is immunoprecipitation in which an antibody with a specific12 / 22 / 25 TP391474W01affinity for a protein of interest immobilized to a solid support (e.g. a bead) is contacted with the sample causing that protein to bind to the support (e.g. the bead).

[0134] Non-targeted protein isolation is not specific to a particular type of protein and aims to isolate a certain subgroup of proteins (e.g. all lipophilic proteins) or all proteins from a sample. An example of non-targeted protein isolation is contacting the sample with a solid support comprising silica in which the surface silanol groups or bound to alkyl chains of a certain length, e.g. C4, Cs, or Cis. In this example, the solid support may be the stationary phase of a column, e.g. a reverse phase column. The terms “protein extraction,” “protein isolation,” “protein separation” “polypeptide extraction,” “polypeptide isolation,” and “polypeptide separation” are used synonymously.

[0135] Anionic oligomers having RNase inhibitory activity: Several anionic oligomers having RNase inhibitory activity are known in the art, and are useful in the aspects provided herein. Non-limiting examples of anionic oligomers having RNase inhibitory activity useful in the aspects described herein include Poly(vinylphosphonic acid), Heparin, Sulfated cellulose, Sulfated nitro-carboxymethyl cellulose, Sulfated amylose, Sulfated amylopectin, Sulfated pectic acid, Sulfated nitrochitin, Sulfated nitrochitosan, Sulfated polyvinyl alcohol, Poly-p,p-dioxy-dibenzyl phosphate, Poly-p,p-dioxydiphenyldimethyl metaphosphate, Polyaspartic acid, Polyglutamic acid, Polyacrylic acid, Poly(methacrylic acid), Poly(maleic acid), Pentosan polysulfate, Chondroitin sulfate, polyglycerol sulfate, Polyethylene sulfonate, Poly(4-styrenesulfonic acid-co-maleic acid), Poly(vinyl sulfonic acid) (PVSA), Poly(4-styrenesulfonic acid), Dextran sulfate, or any combination thereof.

[0136] Anionic oligomers can be present in the lysis mixture in amounts ranging from about 0 ug / mL to about 300 ug / mL. In some aspects, the anionic oligomer is present in the lysis mixture in amounts ranging from about 20 ug / mL to about 280 ug / mL. In some aspects, the anionic oligomer is present in the lysis mixture in amounts ranging from about 40 ug / mL to about 250 ug / mL. In some aspects, the anionic oligomer is present in the lysis mixture in amounts ranging from about 50 ug / mL to about 200 ug / mL. In some aspects, the anionic oligomer is present in the lysis mixture in amounts ranging from about 50 ug / mLto about 150 ug / mL. In some aspects, the anionic oligomer is present in the lysis mixture in amounts ranging from about 50 ug / mL to about 125 ug / mL. In some aspects, the anionic oligomer is present in the lysis mixture in amounts ranging from about 60 ug / mL to about 100 ug / mL. In some aspects, the anionicoligomer is present in the lysis mixture in amounts ranging from about 70 ug / mL to about 90 ug / mL.

[0137] In some aspects, the anionic oligomer having RNase inhibitory activity is selected from the group consisting of: Poly(vinyl sulfonic acid), Poly(4-styrenesulfonic acid), and Dextran sulfate, for example. In certain aspects, the anionic oligomer is present at about 20 ug / mL, 25 ug / mL, 30 ug / mL 35 ug / mL 40 ug / mL, 45 ug / mL 50 ug / mL, 55 ug / mL, 60 ug / mL, 65 ug / mL, 70 ug / mL, 75 ug / mL, 80 ug / mL, 85 ug / mL, 87.5 ug / mL, 90 ug / mL, 95 ug / mL, 100 ug / mL, 105 ug / mL, 110 ug / mL, 115 ug / mL, 120 ug / mL, 125 ug / mL, 130 ug / mL, 135 ug / mL, 140 ug / mL, 145 ug / mL, 150 ug / mL, 155 ug / mL, 160 ug / mL, 165 ug / mL, 170 ug / mL, 175 ug / mL, 180 ug / mL, 185 ug / mL, 190 ug / mL, 195 ug / mL, 200 ug / mL, 205 ug / mL, 210 ug / mL, 215 ug / mL, 220 ug / mL, 225 ug / mL, 230 ug / mL, 235 ug / mL, 240 ug / mL, 245 ug / mL, 250 ug / mL, 255 ug / mL, 260 ug / mL, 265 ug / mL, 270 ug / mL, 275 ug / mL, 280 ug / mL, 285 ug / mL, 290 ug / mL, 295 ug / mL, 300 ug / mL, or any amounts therebetween.

[0138] The lysis mixtures and cell, vesicle or virus lysates produced therefrom provided herein can be used in any number of downstream reactions and processes. By way of example only, the cell, vesicle or virus lysates can be used in sequencing workflows, RT-qPCR reactions, single-cell analysis reactions (e.g., RNA-seq and the like), next-generation sequencing (NGS) reactions, amplification workflows including multiplex amplification reactions (e.g., AMPLISEQ®), microarrays, Northern Blotting, in vitro transcription, or the like. The skilled artisan will readily appreciate that the compounds according to the disclosure are useful in a variety of downstream reactions and processes, including those that do not require downstream polymerization or amplification reactions. By way of example only, the compounds of the present disclosure can be used in branched DNA-based assays to detect target nucleic acids present in a sample lysate, e.g., as described in US Patent No. 8426578. Non-limiting examples of downstream reactions and processes in which the cell, vesicle or virus lysates provided herein can be used are discussed in further detail below.

[0139] Detection of RNA or DNA or a surrogate thereof: Aspects of detecting RNA or DNA or, a surrogate thereof, in a cell lysate as provided herein includes detection means using emission by an emitter that is representative of the RNA or DNA.

[0140] In some aspects, RNA present in a cell lysate produced by the methods described herein is detected in situ by adding or mixing at least a portion of the lysis mixture with a composition comprising reverse transcriptase to produce an RT product, e.g., that comprises12 / 22 / 25 TP391474W01cDNA. The RT product provides a surrogate of the RNA that can be detectable. Any reverse transcriptase known to those of ordinary skill in the art can be used such as, for example, MMLV-RT (murine maloney leukemia virus-reverse transcriptase), avian myelogenous virus reverse transcriptase (AMV-RT), human immunodeficiency virus (HIV)-RT and the Tth DNA polymerase which has reverse transcriptase activity if Mn++ is provided. In some aspects, the HL-dsDNase is heat inactivated during the downstream processes of the RT protocol. Optionally, a positive control RNA can be added to the lysis buffer or the cell lysate.

[0141] Amplification: As used herein, "amplification” or “amplify” and the like refers to a process that results in an increase in the copy number of a molecule or set of related molecules. As the term applies to a lysis mixture herein, amplification means the production of multiple copies of the target nucleic acid, a surrogate of a target nucleic acid, or a portion thereof. Amplification can encompass a variety of chemical and enzymatic processes such as a polymerase chain reaction (PCR), a strand displacement amplification reaction, a transcription mediated amplification reaction, an isothermal amplification reaction, or a nucleic acid sequence-based amplification reaction, for example. Following at least one amplification cycle, the amplification products can be detected or can be separated from at least one other component of the amplification mixture based on their molecular weight or length or mobility prior to detection.

[0142] Polymerase Chain Reaction: PCR includes introducing a molar excess of two or more extendable oligonucleotide primers to a reaction mixture comprising the lysis mixture where the primers hybridize to opposite strands of a DNA, RNA, or RNA surrogate. The reaction mixture is subjected to a program of thermal cycling in the presence of a DNA polymerase, resulting in the amplification of the DNA or RNA surrogate sequence flanked by the primers. Reverse transcriptase PCR is a PCR reaction that uses an RNA template and a reverse transcriptase, or a polypeptide having reverse transcriptase activity, to first generate a single stranded DNA molecule prior to the multiple cycles of DNA-dependent DNA polymerase primer elongation as cited above. Methods for a wide variety of PCR applications are widely known in the art, and described in many sources, for example, Ausubel et al. (eds.), Current Protocols in Molecular Biology, Section 15, John Wiley & Sons, Inc., New York (1994).

[0143] Hybridization: Hybridization is a process in which single-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules anneal to complementary nucleic acids. When placed in appropriate surroundings, complementary single-stranded nucleic acid molecules will anneal or “hybridize” to each other, forming a stable duplex whose presencecan be detected, measured, or manipulated. The compositions and methods of the present disclosure can be used fully or partly in all types of hybridization techniques known in the art and used in nucleic acid amplifications and sequencing workflows. Such techniques include, for example, nucleic acid or polypeptide microarrays, in situ hybridization (ISH) (for example, fluorescent in situ hybridization (FISH; including multi-color FISH and Fiber-FISH), chromogenic in situ hybridization (CISH), silver in situ hybridization (SISH)), Southern blot assays, Northern blot assays, dot blot assays and arrays. For example, a variety of different methods use hybridization to either pinpoint the origin of a nucleic acid (NA) sample or identify short NA sequences. Hybridizations using the compositions of the disclosure may be performed using the same methodology as for any of the many hybridizations protocols known in the art.

[0144] Master Mix: As used herein, the term “master mix” means, unless the context requires otherwise, an aqueous solution comprising some or all reagents used to amplify a target nucleic acid.

[0145] Array: As used herein, the term “array” means, unless the context requires otherwise, any spatially addressable collection of discrete probes, capture molecules, or binding sites arranged in a defined pattern on a solid substrate. An array may include probes bound to glass, silica, plastic, polymeric films, membranes, beads, or other solid-phase materials. The array elements may be arranged in a regular grid, patterned features, or other ordered configuration, and may consist of DNA, RNA, modified nucleic acids, peptides, proteins, or combinations thereof. The term “array” encompasses macroarrays, microarrays, high-density arrays, beadbased arrays, microfluidic arrays, and any functionally equivalent arrangement in which analyte binding occurs at discrete, addressable locations. Hybridization buffers are widely used in arraybased techniques to regulate ionic strength, pH, temperature, and stringency, as well as to reduce nonspecific interactions and enhance the specificity of probe-target hybridization. Virtually all nucleic-acid-based arrays employ a hybridization buffer in order to achieve reliable and interpretable binding patterns.

[0146] Hybridization buffer: As used herein, the term “hybridization buffer” means, unless the context requires otherwise, a solution comprising one or more components that facilitate the annealing or binding of a nucleic acid probe to a complementary nucleic acid target under certain conditions. Exemplary hybridization buffers of the present disclosure are aqueous buffers comprising a polar protic solvent or polar aprotic solvent and optionally a compound of this disclosure. Hybridization buffer buffers provided herein can optionally include one or moresalts, denaturants, surfactants, polymers, blocking agents, crowding agents, hybridization enhancers, accelerating agents, chelating agents, stabilizers, and buffering agents. A hybridization buffer may modulate ionic strength, pH, stringency, viscosity, or nucleic-acid-solvent interactions to promote, maintain, or control hybridization specificity and efficiency. A hybridization buffer may be supplied in concentrated or ready-to-use form and may, for example, be used in solution-phase, membrane-based, or solid-support-based hybridization assays.

[0147] Labeling Reagent: As used herein, the term “labeling reagent,” means, unless the context requires otherwise, any chemical, biochemical, macromolecular, or physical entities capable of imparting a detectable signal to a nucleic acid probe, target, or hybridization product. Labeling reagents of the present disclosure include, but are not limited to, isotopic labels, reactive chemical groups that permit covalent or non-covalent attachment of such labels, chromogenic substrates, radiolabels, organic fluorophores, fluorescent proteins, quantum dots, chromophores, enzymatic labels, chemiluminescent labels, bioluminescent labels, metal-based labels, oligonucleotide-based labels, affinity tags, haptens, detectable substrates, colorimetric proteins, peptide detection dyes, or any combination thereof. Exemplary detectable labels include, but are not limited to ligands, radionuclides, fluorescent dyes, chemiluminescent agents, microparticles, nanoparticles (e.g., a gold nanoparticle), enzymes, colorimetric labels, magnetic labels, small molecules (e.g., biotin), streptavidin, haptens, allophycocyanin (APC), oligonucleotides, and peptide tags (e.g., Myc tag, His tag, or FLAG tag). Chromophores include 3,3'-diaminobenzidine, 4-chloro-2-methylbenzenediazonium (Fast Red), 3,3'-dimethoxybiphenyl-4,4'-di(diazonium) or zinc chloride (Fast Blue). Haptens include fluorescein, biotin, nitroaryls, dinitrophenol (DNP), digoxigenin, oxazole, pyrazole, thiazole, benzofuran, urea, thiourea, rotenoid, coumarin, or cyclolignan. Enzymatic labels include peroxidase, a phosphatase, a glycosidase, or an oxidase. Detectable substrates include a substrate for a peroxidase, a substrate for horseradish peroxidase, a tyramide, or a tyramide-like molecule. Colorimetric protein or peptide detection dye include bicinchoninicacid (BCA), bathocuprione, bathocuprionedisulphonicacid, tartarate, copper sulphate, acetonitrile, salts thereof, derivatives thereof, and combinations thereof. Labels may be molecules (either attached to analyte binding agents or not) to aid in the detection of a biomolecule such as a protein, antibody, or amino acid. Fluorescent labels are also referred to as fluorophores, fluorescent tags, fluorescent dyes, or fluorescent probes. A fluorescent label may be a naturally occurring fluorescent protein (e.g., phycoerythrin, PE), a derivative thereof (e.g., PE-Cy7), a tandem dye, a polymer dye, a single molecule dye, an organic dye, a fluorescent nucleic acid, a12 / 22 / 25 TP391474W01fold-back oligonucleotide probe with a complementary 3' end for fluorescent dye incorporation, or a scaffold-based fluorescent label, for example a nucleic acid nanostructure including fluorescent DNA nanostructures such as PHTION nucleic acid nanostructures, including NOVAFLUOR dyes (Thermo Fisher Scientific, Waltham, MA). Other fluorophore labels that may be used include xanthenes, fluoresceins, rhodamines, rhodols, roseamines, carbopyranonse, indoles, indacenes, borapolyazaindacenes, furans, benzofurans, cyanines, benzocyanines, benzopyriliums, pyrenes, coumarins, carbostyryls, styryls, squarines, resorufins, anthraquinones, acridines, benzophenoxazines, cyanine-based tandem dyes, phycoerythrin-dye conjugates, allophycocyanins, allophycocyanin-dye conjugates, nanocrystals, Pdots, fluorescent conjugated polymers, and oligonucleotide-based fluorescent dyes. Labeling reagents may be incorporated directly into nucleic acids, conjugated to probes, or introduced via enzymatic or chemical labeling reactions. Labeling reagents may provide direct detection or allow indirect detection via binding partners, substrates, or amplification chemistries.

[0148] Probe: As used herein, the term “probe” when used in reference to hybridization means, unless the context requires otherwise, nucleic acid molecule capable of hybridizing, under specific conditions, to a complementary nucleic acid sequence in a sample. Probes of the present disclosure include, but are not limited to, DNA, RNA, a chimeric DNA / RNA molecule, or an analog thereof (including, without limitation, PNA, LNA, morpholino, 2'-modified nucleotides, or other synthetic nucleic acid mimetics). The probe may be labeled for detecting genomic or transcriptomic targets on a membrane substrate, or reagents for target nucleic acid immobilization and post-hybridization washing.

[0149] Accelerating Agent: As used herein, the term “accelerating agent,” when used in reference to a hybridization buffer, means, unless the context requires otherwise, an agent that is capable of accelerating hybridization rates of nucleic acids. Acceleration agents of the present disclosure include, but are not limited to, a polymer (such as ficoll, polyvinylpyrolidone (PVP), heparin, or dextran sulfate), a protein (such as bovine serum albumin), a glycol (such as ethylene glycol, glycerol, 1,3 propanediol, propylene glycol, or diethylene glycol), or an organic solvents (such as formamide, dimethylformamide, or dimethylsulfoxide).

[0150] Blocking agent: As used herein, the term “blocking agent,” when used in reference to a hybridization buffer, means, unless the context requires otherwise, an agent that blocks binding. Blocking agents of the present disclosure include, but are not limited to, yeast tRA, homopolymer DNA, denatured salmon sperm DNA, herring sperm DNA, total human DNA, and COTI DNA.

[0151] Hybridization Enhancer: As used herein, the term “hybridization enhancer” when used in reference to a hybridization buffer, means, unless the context requires otherwise, any component that increases the efficiency, sensitivity, or specificity of nucleic acid hybridization. Hybridization enhancers of the present disclosure include, but are not limited to, DMSO, formamide, dextran sulfate, PEG, or chaotropic agents such as guanidinium thiocyanate.

[0152] Accelerating Agent: As used herein, the term “accelerating agent,” when used in reference to a hybridization buffer, means, unless the context requires otherwise, an agent that is capable of accelerating hybridization rates of nucleic acids. Acceleration agents of the present disclosure include, but are not limited to, a polymer (such as ficoll, polyvinylpyrolidone (PVP), heparin, or dextran sulfate), a protein (such as bovine serum albumin), a glycol (such as ethylene glycol, glycerol, 1,3 propanediol, propylene glycol, or diethylene glycol), or an organic solvents (such as formamide, dimethylformamide, or dimethylsulfoxide).Introduction

[0153] Surfactants play an important role in life science technology and other various industries. With respect to their roles in the life sciences, they are often important components used in both nucleic acid purification and direct PCR processes. For example, in direct PCR, surfactants are needed to enhance the efficiency of the reaction by improving the accessibility of the DNA template. They also can be used to aid in cell lysis, viral inactivation, enhancing DNA accessibility, reducing non-specific binding, and ultimately contributing to the success of obtaining pure nucleic acids and amplifying specific DNA targets. Other processes and / or applications that rely on surfactants are discussed herein and can include, but are not limited to, DNA hybridization, kits for diagnosing allergy-, asthma-, and autoimmune-related diseases, nucleic acid-based sample preparation workflows, and the like.

[0154] Current non-ionic surfactants, such as Triton™ X-100, Tergitol™ NP-40, and / or EH9, that are available as surfactants for myriad biological and / or other industrial uses have a trade-off associated with good performance: they produce degradation by-products that exhibit endocrine disruption effects that interfere with the hormonal system of numerous organisms, particularly aquatic species. These surfactants are often used in sample preparation products,12 / 22 / 25 TP391474W01including protein extraction buffers, wash buffers, protein interaction kits, and protein purification kits. To date, no alternative surfactants are available as direct replacements for Triton™ X-100 and / or Tergitol™ NP-40, particularly those that are suitable for life science applications.

[0155] While less hazardous surfactants are available, they do not meet quality or performance standards across a broad range of products or applications, including sensitive biological assays typically used in life science technologies.

[0156] The present disclosure is directed to new compounds that can be used to replace current surfactants, like Triton™ X-100 and / or Tergitol™ NP-40. The disclosed compounds are less hazardous than Triton™ X-100 and / or Tergitol™ NP-40, but do not sacrifice performance as they work as effectively as, or better than, Triton™ X-100 and / or Tergitol™ NP-40. The disclosed compounds can be prepared using cost-effective methods and are biodegradable without producing toxic by-products like the endocrine disrupting by-products that are produced from degradation of Triton™ X-100 and / or Tergitol™ NP-40. The disclosed compounds are suitable for use as surfactants in myriad applications / industries.

[0157] Throughout the specification these abbreviations have the following meanings:ACTB Actin BetacDNA complementary DNAcfDNA cell-free DNAcfRNA cell-free RNACHAPS (3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate) CHAPSO 3-([3-Cholamidopropyl]dimethylammonio)-2-hydroxy-1- propanesulfonateCHES 2-(Cyclohexylamino)ethanesulfonic acidCISH Chromogenic In Situ HybridizationCTCs circulating tumor cellsctDNA circulating tumor DNActRNA circulating tumor RNADCC N,N’-DicyclohexylcarbodiimideDISH Dual In Situ HybridizationDNA deoxyribonucleic aciddPCR digital polymerase chain reactiondsDNA double Stranded DNAdsRNA double Stranded RNAEDTA ethylenediaminetetraacetic acidEDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide EGTA ethyleneglycol- b / s(P-aminoethyl)-N,N,N',N'-tetraacetic Acid FISH Fluorescence In Situ HybridizationGAPDH Glyceraldehyde-3-phosphate dehydrogenase gDNA Genomic DNAHEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid ISH In Situ HybridizationLPA linear polyacrylamideMOPS 3-morpholin-4-ylpropane-1-sulfonic acidMPD 2-methyl-1 ,3-propanediolmRNA messenger RNANHS N-hydroxysuccinamidePCR polymerase chain reactionPEG polyethylene glycolqPCR quantitative polymerase chain reactionRNA ribonucleic acidrRNA ribosomal RNARTqPCR quantitative reverse transcription polymerase chain reaction SDS sodium dodecyl sulfatesiRNA mall interfering RNAsnRNA small nuclear RNAssRNA single-stranded RNATAPS N-[T ris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid TEG tetraethylene glycolTLC Thin Layer ChromatographytRNA transfer RNACompounds

[0158] Disclosed herein are aspects of a compound that exhibits surfactant properties. Compounds of the present disclosure can have a structure according to Formula I.Formula I

[0159] With reference to Formula I, R1is selected to be predominantly hydrophilic, such as comprising a heteroaliphatic group or a combination of a heteroaliphatic group and an aliphatic group; R2and each R4independently is a lipophilic group or a hydrophilic group, provided that if one of R2or (one or more) R4is a hydrophilic group, then R1and the other of R2or (either) R4is selected to provide the compound with a hydrophilic-lipophilic balance (“HLB”) (calculated using Griffin’s Method) value ranging from 10 to 18 (or, if both R2and one or more R4are hydrophilic groups, then R1is selected to provide the compound with such an HLB value); X is selected from oxygen, sulfur, or N(R5); R3is selected from heteroaliphatic, aliphatic, or aryl; each R4independently is aliphatic; Y is selected from oxygen, sulfur, or N(R5); the linker, when present (as indicated with subscript “1” in Formula I), is aliphatic or has a formula -C(=Z)-W-C(=Z)-X’ , wherein each Z independently is oxygen, sulfur, or NR5, W is an aliphatic or heteroaliphatic group (e.g., a N-containing heteroaliphatic group, an O-containing heteroaliphatic group, or an S-containing heteroaliphatic group), and X’ is oxygen, sulfur, or N(R5); n is an integer selected from 0 or 1; m is an integer selected from 0 to 6, such as 0, 1, 2, 3, 4, 5, or 6, provided that if n is 0, then m is 1; and p is an integer selected from 0 to 2, such as 0, 1, or 2. With reference to R5groups disclosed above, each R5group independently is selected from hydrogen, aliphatic, aromatic, or heteroaliphatic.

[0160] In any of the aspects in the present disclosure, the hydrophilic and lipophilic groups for R1, R2, and / or (one or both of) R4are selected to provide a hydrophilic-lipophilic balance (“HLB”) value ranging from 10 to 18, such as 10 to 12, or 10 to 13, or 11 to 13, or 11 to 14, or 1212 / 22 / 25 TP391474W01to 14, or 12 to 15, or 13 to 15, or 13 to 16, or 14 to 16, or 11 to 18, or 12 to 18, or 13 to 18, or 14 to 18, or 15 to 18, or 16 to 18, or 17 to 18.

[0161] In some aspects, R1comprises a heteroaliphatic group, such as a linear heteroaliphatic group or a cyclic heteroaliphatic group (e.g., a five-membered or 6-membered heterocyclic group). In some aspects, R1can include an aliphatic group in combination with the heteroaliphatic group. In some aspects, R1is a polyalkylene oxide group (e.g., a polyethylene glycol (PEG), a polypropylene glycol, ora combination thereof), a polyalkylene amine group (e.g., a polyethylene amine (PEI), a polypropylene amine, or a combination thereof), or comprises a cyclic C^sheteroaliphatic group, such as a sugar molecule. In some aspects, R1comprises a combination of (i) a polyalkylene oxide group or a polyalkylene amine group or a cyclic C4-sheteroaliphatic group, and (ii) an aliphatic group. In aspects comprising an R1group that is a polyalkylene oxide or a polyalkylene amine, the polyalkylene oxide / amine can be terminated with a hydrogen atom or a terminating group, wherein the terminating group can be an aliphatic group, an aromatic group, or the like. In particular aspects, R1is a PEG or PEI group having a mass average molecular weight ranging from 500 g / mol to 800 g / mol, such as 525 g / mol to 800 g / mol, or 550 g / mol to 800 g / mol, or 575 g / mol to 800 g / mol, or 600 g / mol to 800 g / mol, or 625 g / mol to 800 g / mol, or 650 g / mol to 800 g / mol, or 675 g / mol to 800 g / mol, or 700 g / mol to 800 g / mol, or 725 g / mol to 800 g / mol, or 750 g / mol to 800 g / mol, or 775 g / mol to 800 g / mol. In particular aspects, the PEG group has a mass average molecular weight ranging from 525 g / mol to 575 g / mol, or 725 g / mol to 775 g / mol. In representative aspects, the PEG group has a mass average molecular weight of 550 g / mol or 750 g / mol. In any such aspects comprising a PEG group, the average molecular weights do not include the weight of any terminating group. In aspects comprising a PEG group, the PEG has a formula of -[CH2CH2O , where r is an integer selected to provide a PEG group having a mass average molecular weight as described above. In aspects comprising a PEI group, the PEI has a formula of -[CF^CFkNH],-, where r is an integer selected to provide a PEI group having a mass average molecular weight as described above. In aspects where R1comprises a cyclic C^sheteroaliphatic group, the cyclic C4-sheteroaliphatic group can be bound to X of Formula I directly through a bond formed between X and a carbon atom of the cyclic C4.5heteroaliphatic group; or it can be bound to X of Formula I through an external carbonyl group (i.e., -C(=O)-) of the cyclic C4-5heteroaliphatic group. In some such aspects, the cyclic C4-sheteroaliphatic group can be a sugar molecule, such as glucuronic acid, trehalose, glucopyranoside, and other sugars, including combinations of such sugars wherein more thanone sugar molecule is present (e.g., two to four sugar molecules). In some aspects, R1comprises 1 to 12 PEG groups, or a combination of 1 to 12 PEG groups and an alkyl group (e.g., C1.5 alkyl).

[0163] In any of the aspects in the present disclosure, each of R2or (one or both of) R4independently can be an aliphatic group, an aryl group, a polyalkylene oxide, or a polyalkylene amine. In some other aspects, such as when R1comprises a combination of a heteroaliphatic group and an aliphatic group, one of R2or (one or both) R4comprises a PEG group. In particular aspects, R2is Ci-2salkyl (e.g., C2-2oalkyl, or C2-isalkyl, or Ci-galkyl), phenyl, or naphthyl. In particular aspects, each R4independently is a Ci-ioalkyl group, such as Ci-9alkyl (e.g., methyl or ethyl).

[0164] In some aspects, R3is independently is -O-Ci-ealkyl, -O-Cs-ecycloalkyl,

[0165] In one aspect, SS is a solid-support, such as polystyrene (PS) beads, agarose beads, pegylated-polystyrene beads, controlled-pore glass beads, functionalized silica gel, sepharose, alumina supports, magnetic silica or polymer-coated magnetic beads, cellulose and modified cellulose resins, chitosan beads, dextran-based supports such as sephadex, and prefunctionalized systems including NHS-activated beads, maleimide-activated beads, and epoxyactivated supports, or the like.12 / 22 / 25 TP391474W01

[0166] In some aspects, the solid support (SS) may be a resin, a bead, a particle, magnetic bead, a non-magnetic bead, an ion exchange matrix, an affinity chromatography matrix, a size exclusion chromatography matrix, a hydrophobic interaction chromatography matrix, an immobilized metal affinity chromatography, a reverse phase chromatography matrix, immunoaffinity chromatography matrix, or a mixed mode chromatography matrix, or any of the solid supports disclosed herein.

[0168] In some aspects, R3is independently is -O-Ci-ealkyl, -O-Cs-ecycloalkyl,

[0169] In any of the aspects in the present disclosure, R3is selected from an alkoxy group or an aliphatic group (e.g., an alkyl group, an alkenyl group, or an alkynyl group). In some aspects, p is 0, in which case no R3group is present. In other aspects, p is 1 or 2.

[0170] In any of the aspects in the present disclosure, n is 1 and thus the C=Y group is present. In such aspects, Y typically is oxygen or sulfur. In some other aspects, n is 0 and thus the C=Y group is not present and the group bearing X is directly attached to the aryl ring of Formula I.

[0171] In some aspects, X is oxygen or NH. In particular aspects, X is oxygen.

[0172] In any of the aspects in the present disclosure, the linker is not present and thus X is bound directly to R1. In some other aspects, the linker is present. In such aspects, the linker can be a Ci-2saliphatic group (e.g., Ci-2salkyl) or has a structure according to the formula -C(=Z)-W-C(=Z)-X’-, wherein each Z independently can be selected from O, S, or NR5; W can be Ci- 2salkyl, Ci-25alkenyl, Ci-25alkynyl, ether, thioether, or amine (e.g., NH, Ci-25alkyl-N(H)-Ci-25alkyl); and X’ is O, S, or NR5. In particular aspects, the linker, if present, is -(CH2)q-, -C(=O)(CH2)qC(=O)-O-, or -C(=S)(CH2)qC(=S)-O-, -C(=O)N(H)(CH2)qN(H)C(=O)-O-, wherein each q independently is an integer ranging from 1 to 10.

[0173] In any of the aspects in the present disclosure, m is 1, 2, 3, 4, 5, or 6. In particular aspects, m is 1. In any of the foregoing aspects, p is 0, 1 , or 2. In particular aspects, p is 0.

[0174] In some aspects, the compound can have a structure according to any one of Formulas IA, IB, IC, ID, IE, or IF, illustrated below.Formula IC Formula IDFormula IE Formula IF

[0175] With respect to any of Formulas IA-IF above, each of R1, R2, R3, R4, Y, the linker group, n, m, and p can be as described above for Formula I.

[0176] In some aspects, the compound has a structure according to Formulas IG, IH, I J, and IK, shown below.Formula IK

[0177] With reference to Formulas IG, IH, IJ, and IK, the aliphatic group is linear, branched, cyclic, or a combination thereof; and the TG group, if present, is an aliphatic group. In particular aspects, the aliphatic group is a linear C2-2oalkyl group, such as a linear C2-1 salkyl group, or a linear C2-ioalkyl group; a branched C2-2oalkyl group, such as a branched C2-isalkyl group, or a branched C2- alkyl group; a cyclic Cs-zoalkyl group, such as a cyclic Cs-isalkyl group, or a cyclic C3-ioalkyl group; or a combination of any such linear, branched, and / or cyclic groups. In particular aspects, the aliphatic group is selected from isopentyl, 2-methylpentyl, heptyl, 2,2-dimethylbutyl, pentyl, octyl, nonyl, and the like. The terminating group (or “TG” group) of Formulas IG or IH can be a Ci- alkyl group, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, or decyl (with any such groups being linear, branched, cyclic, or any combination thereof). In particular aspects, the TG group is methyl. Also with reference to Formulas IG and IH, r is an integer selected to provide a PEG group having a mass average molecular weight as described above for Formula I. In some exemplary aspects, r is an integer selected from 2 to 20, such as 2 to 18, or 2 to 16, or 2 to 14, or 2 to 12, or 2 to 10. In particular aspects, r is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0178] Representative compound examples are provided in Table 1, below, wherein the “m-PEG” notations are used to indicate the average molecular weight of the PEG group (e.g., “m-PEG 550” is used to indicate a number of PEG repeats that provide a mass average molecule weight of 550 g / mol).- 47 -Methods

[0180] Disclosed herein is a method for making compounds according to the present disclosure. In some aspects, the method comprises performing chemical reactions to add the lipophilic and hydrophilic groups to a core compound, such as that shown in Scheme 1. In someaspects, the method may comprise one or more synthesis steps to add the lipophilic group. In some aspects, the method may comprise one or more, typically two or more, synthesis steps to add the hydrophilic group. In some aspects, the lipophilic group is added by exposing a starting material 100 (Scheme 1) to a base and an R2-group-bearing electrophile, such as an R2-group-bearing halide compound. In some such aspects, the R2-group-bearing halide compound can be a halide-bearing aliphatic compound, such as Br-aliphatic, l-aliphatic, or Cl-aliphatic. Starting material 100 is then converted to an R2-bearing intermediate, such as intermediate 102 (Scheme 1). Intermediate 102 can then be converted to a compound according to the disclosure that includes the R1hydrophilic group using further synthesis steps.Scheme 1

[0181] In some aspects, intermediate 102 is converted to a compound 104 using a sequence of synthesis steps wherein the aldehyde of 102 is reduced to a hydroxyl group, which is then converted to a suitable leaving group (e.g., a triflate). In such aspects, an R1group can then be added to displace the leaving group, thus providing a compound according to compound 104, wherein n is 0, m is 1 , and wherein the linker is not present. Reagents and conditions used in such aspects are recognizable to those in the art, particularly with the benefit of the present disclosure. For example, in some aspects, a suitable reducing agent (e.g., sodium borohydride) can be used to convert the aldehyde to a hydroxyl group. Then a suitable base (e.g., an amine base) and a suitable leaving group reagent (e.g., methanesulfonyl chloride) can be used to provide the leaving group-containing compound that is reacted with the R1group.

[0182] In yet other aspects intermediate 102 can be converted to a compound 104 by following a similar procedure described above wherein the aldehyde of 102 is reduced to a hydroxyl group. The hydroxyl-containing compound can then be combined with a suitable base and a suitable halide-containing reagent to add the R1group. For example, in some aspects, the hydroxyl-containing compound can be deprotonated with a suitable base and then allowed to react with an R1-halide reagent, such as a halide-bearing sugar molecule, to provide compound12 / 22 / 25 TP391474W01104, wherein n is 0, m is 1, and the linker is not present. In some such aspects, the halide-bearing sugar molecule can be 2-bromo-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol.

[0183] In yet other aspects, intermediate 102 can be converted to a compound 104 by following a similar procedure described above wherein the aldehyde of 102 is reduced to a hydroxyl group. The hydroxyl-containing compound can then be combined with a suitable base and a suitable linker (or linker precursor) reagent so as to attach a linker group to the hydroxyl group. In some such aspects, the linker can be an aliphatic halide, an anhydride (e.g., succinic anhydride), or other suitable linker groups. Once added, the linker-containing compound can then be reacted under suitable reaction conditions with a suitable R1-containing group to provide compound 104, wherein n is 0, m is 1, and the linker is present.

[0184] In yet other aspects, the aldehyde of compound 100 can be maintained and a suitable X-[Linker]0 oriR1group can be added to the aldehyde using suitable reaction conditions that would be recognized by those in the art with the benefit of the present disclosure. The resulting intermediate could then be oxidized using suitable reagents and reaction conditions to provide a compound 104 wherein N is 1, m is 0 and wherein the linker is or is not present.

[0185] With respect to solid-support (SS) bound surfactants, appropriately functionalized hydroxy-substituted syringic acid derivatives, carboxylic acid substituted syringic acid or hydroxymethyl-substituted syringic acid derivatives are converted to PEG-linked constructs bearing terminal amine or hydroxyl groups. These pegylated intermediates are subsequently coupled to functionalized bead surfaces (for example, carboxyl- or epoxy-activated agarose or polystyrene beads or the like) to generate bead-immobilized non-ionic surfactant analogs.

[0186] Alternatively, polystyrene beads are modified with poly(ethylene glycol) linkers bearing terminal hydroxyl groups (PS-PEG-OH). Syringic acid derivatives are subsequently coupled to the PEG terminus via ester or carbonate linkages to yield PS-PEG-syringic acid derivatives as solid-support bound surfactants. Polystyrene beads functionalized with carboxylic O PS J °Hacid groups ( ) or hydroxy methyl groups) can be used to react with the PEG-alcohols to obtain polystyrene beads with PEG linkers bearing terminal carboxylic acid groups or terminal hydroxy groups.12 / 22 / 25 TP391474W01Applications

[0187] The disclosed compounds are useful as surfactants and can be used in many applications where a non-ionic surfactant is suggested or preferred. Applications include, but are not limited to, lysing cells, permeabilizing membranes, inactivating viruses, separating hydrophilic proteins from membranes, reducing surface tension, or decellularizing tissue. Additionally, the disclosed compound may be useful as an excipient or adjuvant in a vaccine, a cleaning agent, a component in buffers particularly biological buffers, a wetting agent, an emulsifier, a surfactant, or as a surface treatment for metals. In some aspects, the disclosed compound can be used for DNA or nucleic acid extraction from blood or pathogens applications, protein expression and purification, ELISA’s, Western blotting and RT_PCR applications.

[0188] In one aspect, the disclosed compounds are useful in methods comprising cell lysis. In some aspects, the method comprises treating the cell with the disclosed compound(s) to facilitate cell lysis.

[0189] In one aspect, the disclosed compounds are useful in methods for virus inactivation, particularly inactivating a virus having a lipid envelope, or a virus that may develop a lipid envelope. In some aspects, a method for inactivating a virus comprises combining the disclosed compound with a liquid comprising the virus.

[0190] Also disclosed herein are aspects of a kit comprising a compound disclosed herein.

[0191] In some aspects, compounds of this disclosure are incorporated into compositions which comprise a compound of this disclosure and a polar protic solvent. In some aspects, the disclosed compositions optionally includes one or more inorganic salts, buffering agents, chaotropic agents, and chelators. In some aspects, the disclosed compositions may include one or more inorganic salts, which optionally may be selected from the inorganic salts disclosed herein. In some aspects, the disclosed compositions may include one or more buffering agents, which optionally may be selected from the buffering agents disclosed herein. In some aspects, the disclosed compositions may include one or more chaotropic agents, which optionally may be selected from the chaotropic agents disclosed herein. In some aspects, the disclosed compositions may include one or more chelating agents, which optionally may be selected from the chelating agents disclosed herein. In some aspects, the compounds of this disclosure may be present in the disclosed compositions at a concentration (v / v) of any of 0.001% to about 25%, 0.05% to about 25%, 0.05% to about 15%, 3.5% to about 15%, 1.0% to about 10%, 0.02%, 0.05%,0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, and any percentage in between. In some aspects, the disclosed compositions may include inorganic salt(s) at a concentration of about 0.25 mM to about 1000 mM, and any concentration in between. In some aspects, the disclosed compositions may include buffering agent(s) at a concentration of about 5 mM to about 500 mM, and any concentration in between. In some aspects, the disclosed compositions may include chaotropic agent(s) at a concentration of about 0.5 M to about 8 M, and any concentration in between. In some aspects, the disclosed compositions may include chelating agent(s) at a concentration of about 5 M to about 100 M, and any concentration in between. In some aspects, the disclosed compositions may include the polar protic solvent at a concentration (v / v) of about 5% to about 25%, about 2% to about 50%, about 10%-95%, and any percentage in between. In some aspects, the disclosed composition may have a pH in the range of about 5.5 to about 10.0, or about 3.0 to about 12.0, and any pH in between. In some aspects, compounds of this disclosure may enhance activity of enzymes, including enzymatic proteases, cellulase, lysozymes, and carbohydrate-degrading enzymes, which can be beneficial in certain applications, for example, lysing certain hard to lyse cells or viruses such as plant cells or on-enveloped viruses. This can also be beneficial whenever improved protein unfolding, accessibility, or solubilization is needed to allow the protease to act more efficiently on its substrates. This is especially useful when digesting membrane proteins, aggregated or denatured proteins, highly structured polypeptides, or complex biological samples such as lysates, serum, tissues, or exosomes, where hydrophobic regions or lipid components can limit cleavage. Buffers containing surfactants that enhance enzyme help expose cleavage sites, prevent aggregation, increase substrate mobility, and maintain enzyme-substrate interactions, thereby improving digestion speed, completeness, and reproducibility in applications like sample preparation for mass spectrometry, protein characterization, peptide mapping, and bottom-up proteomics, as well as when performing proteolysis in highly viscous, particulate, or lipid-rich environments where conventional buffers underperform. In some aspects, compounds of this disclosure can be beneficial in lysing enveloped viruses while preserving viral proteins.

[0192] In some aspects, the compositions containing a compound of this disclosure may be used in sample-preparation workflows, including but not limited to lysis buffers, wash solutions, binding buffers, immunoprecipitation buffers, blocking buffers to reduce non-specific binding, cytoplasmic extraction buffers, hybridization buffers, transport media, stabilization reagents, and12 / 22 / 25 TP391474W01amplification-compatible formulations. In some aspects, the disclosed compositions can be included in electrolyte solutions contained in electrolyte reservoirs used, for example, in ion chromatography. In some aspects, the disclosed compositions may be used in various multiomic workflows. The compounds of this disclosure are highly beneficial in multiomics because they provide gentle but effective cell lysis, selectively disrupting plasma membranes while preserving proteins, metabolites, and nucleic acids for downstream analysis. Their non-denaturing nature maintains native protein structure and interactions, which is crucial for accurate proteomics and interactomics. In addition, MS-compatible nonionic surfactants improve protein solubilization and enzymatic digestion in proteomic workflows while degrading or being easily removed before mass spectrometry, preventing ion suppression. In some aspects, the compound in the disclosed composition is selected based on the compound’s propensity to dissociate or fragment under mass spectrometric conditions, thereby minimizing interference with detection, identification, or quantification of the target molecule(s). In some aspects, the compound in the disclosed composition is selected based on the compound’s propensity to be easily removed.

[0193] Native mass spectrometry to the use of native mass spectrometry to analyze membrane-associated molecules (such as membranes, proteins, lipids, or intact vesicles) without disrupting their natural structures or interactions. Nonionic surfactants play a key enabling role in native mass spectrometry of membranes and vesicles because they can extract, solubilize, or stabilize membrane proteins while still being gentle enough to preserve native structure and non-covalent interactions. In some cases, native mass spectrometry is performed in the presence of one or more compounds of this disclosure that solubilize membrane-associated components while preserving their native structural and functional states. The use of these compounds facilitates gentle extraction and stabilization of membrane proteins or vesicles and enables efficient release of intact complexes into the gas phase for accurate mass analysis.

[0194] Single-cell multiomics refers to analytical methods that concurrently measure two or more molecular modalities — such as the genome, transcriptome, proteome, or epigenome — from an individual cell, enabling high-resolution characterization of cellular heterogeneity and functional state. The compounds of this disclosure, which have been shown to be highly effective while maintaining the structural integrity of proteins, protein complexes, and epigenetic features, can facilitate controlled cellular disruption enabling concurrent measurement of cellular features with genomic and transcriptomic components in single-cell multiomics workflows. In addition to other uses disclosed herein, compounds of this disclosure can be used to coat a single cellcontainer to prevent binding, sticking or adsorption to the container to ensure high recovery. Similarly, for any of the plastics, containers, plates (e.g., those used for ELISA), or consumables used in any of the many workflows described herein or in other laboratory, research, manufacturing, and diagnostic workflows, compounds of this disclosure can be used to precoat the plastics and consumables, in some instances by forming a film to passivate surface of a container to keep proteins and other target molecules from sticking to surfaces.

[0195] T ogether, these properties make nonionic surfactants valuable for generating high-quality, integrative datasets across multiomics, proteomics, transcriptomics, and metabolomics.

[0196] In some aspects the disclosed compositions may be used in various workflows, processes, and techniques in the fields of microscopy, biochemistry, molecular biology, immunology, microbiology, genetics, cell and tissue culture, bioprocessing, pharmaceutical, and protein and nucleic acid chemistry. The inclusion of nonionic surfactants offers multiple advantages, such as facilitating efficient disruption of cellular and viral membranes while preserving the structural and functional integrity of target biomolecules. Nonionic surfactants further reduce nonspecific adsorption of nucleic acids and proteins to plastics, magnetic particles, membranes, or other surfaces encountered during purification, thereby improving analyte yield and consistency. These surfactants also mitigate aggregation and viscosity issues in complex sample matrices, promoting more uniform processing and enhanced compatibility with automated or high-throughput systems. Additionally, because nonionic surfactants exhibit minimal inhibitory effects on downstream enzymatic reactions — including amplification, sequencing, labeling, and detection chemistries — they enable seamless transition from sample preparation to analytical workflows without the need for extensive removal or dilution. Collectively, these properties contribute to improved robustness, reproducibility, and performance across diverse sample types and processing conditions.

[0197] The inclusion of nonionic surfactants offers multiple advantages, such as facilitating efficient disruption of cellular and viral membranes while preserving the structural and functional integrity of target biomolecules. Nonionic surfactants further reduce nonspecific adsorption of nucleic acids and proteins to plastics, magnetic particles, membranes, or other surfaces encountered during purification, thereby improving analyte yield and consistency. These surfactants also mitigate aggregation and viscosity issues in complex sample matrices, promoting more uniform processing and enhanced compatibility with automated or high-throughput systems. Additionally, because nonionic surfactants exhibit minimal inhibitory effects on downstreamenzymatic reactions — including amplification, sequencing, labeling, and detection chemistries — they enable seamless transition from sample preparation to analytical workflows without the need for extensive removal or dilution. Collectively, these properties contribute to improved robustness, reproducibility, and performance across diverse sample types and processing conditions. in compositions, including composition for use in various sample prep processes.

[0198] The compounds of this disclosure have demonstrated surprisingly effectiveness, achieving comparable or improved results to leading nonionic surfactants without the unfavorable toxicity and environmental profiles. Compounds of this disclosure have demonstrated comparable or improved results for, among other things, polypeptide and nucleic acid lysis and recovery, enhancement of enzyme activity, stability, protein denaturation, interactions with other commonly used agents, background signal interference in downstream processes, and toxicity and environmental profiles. In some aspects, the compounds disclosed herein provide these performance benefits at concentrations lower than those conventionally required by other nonionic surfactants, while, conversely, also exhibiting reduced interference with downstream analytical or processing steps even when employed at levels exceeding those typical of standard nonionic surfactants. Owing to their minimal interaction with proteins and other components of the composition, as well as their ability to reduce non-specific binding, the disclosed compounds can be utilized at substantially higher concentrations without adversely affecting a wide range of workflows.

[0199] In some aspects, the disclosed compositions may be widely used in proteinhandling workflows because they gently solubilize cellular membranes while preserving the native structure and function of proteins. During protein cell solubilization, the compounds of this disclosure prevent aggregation and precipitation by maintaining proteins in solution; their low critical micelle concentrations and inherently non-denaturing properties make them especially well-suited for applications in structural biology or ligand-binding assays, where maintaining correct protein conformation after extraction is essential. In protein detection assays, compounds of this disclosure help preserve functional epitopes and protein-protein complexes so that antibodies or binding partners can recognize their targets, and they are compatible with techniques such as isoelectric focusing that require proteins to retain native charge and configuration. In protein purification, compounds of this disclosure reduce nonspecific adsorption to containers or chromatography materials, minimize clumping of resins or magnetic beads, and improve overall flow and handling characteristics — leading to cleaner separations and higher-quality protein preparations. Compounds of this disclosure are valuable tools for gently disrupting and stabilizing biological structures without causing full protein denaturation. They can solubilize lipoproteins, viral envelopes, and membrane proteins in a controlled manner that preserves critical structural and functional features for downstream analysis.

[0200] In some aspects, the composition comprising compounds of this disclosure is a wash buffer, which may be used in a broad range of workflows and techniques. In samples containing polypeptides, including those used in various downstream assays, nonionic surfactants are important components of wash buffers, in part because they help reduce nonspecific binding by disrupting weak hydrophobic interactions without denaturing proteins, and because they inhibit non-specific interactions between polypeptides while maintaining specific binding between analytes and analyte binding agents. Wash buffers may also be used for sample cleanup, to remove unwanted molecules, such as lipids, nucleic acids, and membrane debris. The compounds of this disclosure improve wash efficiency, ensuring unbound or loosely associated molecules are removed while preserving desired target complexes. Additionally, they enhance surface wetting, minimize aggregation, and contribute to more consistent, lower-background assay performance. In some aspects, the disclosed wash buffer may optionally include one or more salts, chelators, stabilizers, or surfactants. In some aspects, the wash buffer may include one or more stabilizers, which optionally may be BSA. In some aspects, the disclosed composition may have a pH in the range of about 5.5 to about 10.0, or about 3.0 to about 12.0, and any pH in between. A skilled artisan will know how to ascertain the optimal concentrations through routine experimentation.

[0201] In some aspects, compounds of this disclosure or the disclosed compositions can be used in the manufacture and formulation of drug-delivery aids or adjuvant components by mildly permeabilizing cellular membranes, forming nanocarriers such as micelles, mixed micelles, and niosomes, and enhancing the uptake or stability of delivery vehicles including nanoparticles and liposomes, thereby facilitating the intracellular transport of therapeutic agents and other molecular cargo. In some aspects, compounds of this disclosure or the disclosed compositions may be used to facilitate the in vivo transport and intracellular delivery of therapeutic agents and other molecular cargo.

[0202] In some aspects, the compositions comprising compounds of this disclosure may be used to lyse cells, vesicles, or viruses. In some aspects, the compositions comprising compounds of this disclosure are lysis buffers. Nonionic surfactants are used in lysis buffers inpart to achieve improved cell disruption while maintaining compatibility with downstream analytical workflows. In some aspects, the disclosed lysis buffer may be used to lyse cells, nucleic acids, or vesicles in order to extract nucleic acids, polypeptides or other target molecules. Nonionic surfactants are key components in lysis buffers, as they enhance solubilization of lipid membranes while minimizing denaturation or degradation of target molecules, thereby improving recovery and integrity of nucleic acids, proteins, and other intracellular components. Their mild, non-denaturing properties also reduce inhibitory effects on downstream analytical or enzymatic processes, including enzymatic reactions, chromatographic separations, amplification, sequencing, labeling, and detection assays, allowing for improved workflow compatibility without the need for extensive surfactant removal. Additionally, nonionic surfactants promote uniform lysis across diverse sample types, decrease sample viscosity, and mitigate nonspecific adsorption of biomolecules to reaction vessels, purification substrates, or instrument surfaces. Collectively, these characteristics enable more consistent yields, greater reproducibility, and improved robustness in sample-preparation workflows. The compounds of this disclosure have shown to be excellent alternatives to current nonionic surfactants, demonstrating successful lysing of many types of cells and extraction of nucleic acids and polypeptides. In some aspects, the disclosed lysis buffers may include, or be used with, an enzyme, including an enzyme such as cellulase or lysozyme.

[0203] In some aspects, a method for using a disclosed lysis buffer includes contacting a sample with the disclosed lysis buffer to generate a lysed sample. In some aspects, the sample comprises cells, vesicles, or viruses. Further, nucleic acids and / or polypeptides may be isolated from the lysed sample. The use of lysis buffers on a wide number of samples and cells is well known in the art and a skilled artisan would readily understand the many uses of the disclosed method and its ability to be employed using many samples and to generate a lysed sample containing many different types of target molecules. The disclosures herein are provided merely as examples and the techniques and compositions are not limited to the examples. In some aspects, the sample may be any type of sample, including the samples disclosed herein. In some aspects, the disclosed lysis buffers may be used to lyse hard-to-lyse cells, such as organoids, gram-positive bacteria, mycobacteria, yeasts and other fungi, plant cells, bacterial and fungal spores, various archaea, and tough animal-derived tissues such as insects or parasites. In some aspects, the disclosed lysis buffers may be used to lyse cultured cells which may be processed using qPCR without performing RNA extraction. In some aspects, the disclosed lysis buffers may12 / 22 / 25 TP391474W01be used to lyse non-cultured cells, such as cells in native, fresh, or directly isolated state, which may be processed using qPCR without performing RNA extraction.

[0204] In some aspects, the disclosed lysis buffers may be used to lyse vesicles, natural and synthetic. For example, lipid nanoparticles (LNPs) are an important class of delivery systems for nucleic acids, including messenger RNA (mRNA), small interfering RNA (siRNA), and genomic editing components. Nonionic surfactants are also used, in certain concentrations, in LNP workflows in LNP encapsulation efficiency assays which test the efficiency of the encapsulation process. As demonstrated, compounds of this disclosure were shown to lyse LNPs with similar efficacy as comparable to Triton™ X-100, providing comparable alternatives with more favorable environmental and toxicity profiles than current industry standards.

[0205] In some aspects, the lysis buffer may lyse all or part of the cells in the sample. For example, the lysis buffer may be formulated for controlled disruption of cellular organelles using a defined lysis buffer to enable selective extraction of target molecules. In one aspect, the method comprises contacting a biological sample comprising intact cells with the disclosed lysis buffer formulated to modulate membrane permeability under conditions sufficient to lyse one or more intracellular organelles, such as the nucleus, mitochondria, endoplasmic reticulum, Golgi apparatus, lysosomes, peroxisomes, and related membrane-bound compartments, while substantially maintaining the integrity of other cellular compartments. In some aspects, the lysis buffer comprises one or more salts, chelating agents, or pH-modifying components that preferentially disrupt organelles such as mitochondria, nuclei, endoplasmic reticulum, lysosomes, or peroxisomes, thereby releasing organelle-associated proteins into solution. The selective lysis conditions permit enrichment of protein subsets localized to specific organelles without complete cell disruption, improving recovery, purity, and functional integrity of proteins of interest. In other aspects, the method enables sequential lysis of distinct organelles by stepwise modification of buffer composition or exposure time, allowing differential extraction of proteins based on their subcellular localization. Compounds of this disclosure support reliable subcellular fractionation by selectively lysing specific membrane components while maintaining the integrity of others. The disclosed lysis buffer compositions and methods are useful in proteomic analysis, biomarker discovery, biochemical pathway characterization, and preparation of isolated protein fractions for research, diagnostic, or therapeutic applications.

[0206] In some aspects, the disclosed compositions are employed in immunoprecipitation workflows. In some aspects, the disclosed compositions are employed in cytoplasmic proteinextraction, where they may be used to break open the plasma membrane while keeping organelles and protein complexes intact, enabling recovery of native cytosolic proteins.

[0207] In some aspects, the disclosed compositions can be employed in detergent phase separation, a biochemical technique that uses the differential interactions of membrane components with nonionic surfactants to isolate distinct membrane domains. When a biological membrane sample is exposed to a nonionic surfactant, disordered or loosely packed lipid regions readily solubilize into surfactant-lipid assemblies, while more ordered, tightly packed, or cholesterol-rich microdomains remain structurally intact and resistant to solubilization. This selective behavior causes the system to partition into at least two physically distinguishable phases: a surfactant-rich, solubilized phase and a surfactant-resistant phase enriched in membrane substructures or protein complexes that maintain their native associations under mild surfactant treatment. Compounds of this disclosure may be used to facilitate this selective phase behavior, enabling reproducible enrichment of membrane microdomains, protein complexes, or lipid assemblies of analytical or industrial interest. Compounds of this disclosure offer several advantages, including mildness toward protein structure, controlled solubilization behavior, and tunability based on environmental conditions, resulting in improved specificity and compatibility with downstream analytical workflows. The disclosed methods and compositions can be applied for biochemical fractionation, characterization of membrane-associated structures, and preparation of samples for research, diagnostic, or therapeutic development.

[0208] In some aspects, the target molecules may be isolated after lysis. In some aspects, two or more target molecules may be isolated from the same sample, either sequentially or simultaneously. For example, two or more of cfDNA, RNA, DNA, polypeptides and vesicles may be isolated from a single sample.

[0209] In some aspects, the compositions of this disclosure may be used in connection with a bead or microcarrier conjugated to an analyte binding agent for use in analytical assays requiring selective capture, isolation, or detection of one or more target analytes. The conjugated beads or microcarriers enable high-affinity binding interactions and may be incorporated into heterogeneous assay formats, including, for example, flow cytometric analysis, magnetic separation, immunoassays, high-throughput or multiplexed analytical systems, and nucleic acid detection workflows. The conjugated beads or microcarriers are compatible with automated liquidhandling platforms and diverse detection modalities, permitting integration into high-throughput or multiplexed analytical systems. In some aspects, compounds of this disclosure may be12 / 22 / 25 TP391474W01incorporated in lysis buffers or wash buffers included in a kit that also includes bead or microcarrier conjugated to an analyte binding agent. In some aspects, the bead or microcarrier may be selected from a group including, but not limited to, magnetic beads (streptavidin, Ni-NTA / His-tag, carboxylated, Protein A / G, ion-exchange, silica-coated), silica beads, agarose resins or beads including Sepharose (Protein A / G agarose, glutathione agarose, Ni-NTA agarose, activated agarose), polymer beads (ion-exchange, hydrophobic interaction, reversed-phase / C18), paramagnetic nanoparticles, specialty beads such as immunomagnetic beads, lectin beads, aptamer-coated beads, and oligo(dT) beads, hydroxyapatite beads, cellulose and modified cellulose resins, cellulose beads, dextran-based (Sephadex) beads, polyacrylamide beads, chitosan-coated beads, modified polysaccharide beads, polystyrene-divinylbenzene (XAD) beads, ceramic or zirconia beads, alumina beads, enzyme-immobilized beads, metal oxide beads (Fe3O4, TiO2, ZrO2, AI2O3), sialic-acid-binding beads, graphene oxide or carbon-based beads, MOF-based beads, polystyrene beads and resins, and many more. Examples of nonbead particles used for binding target molecules include nanoparticles (gold, silver, silica, iron oxide), quantum dots, liposomes, polymeric nanoparticles, carbon nanotubes, graphene or graphene oxide sheets, metal-organic frameworks (MOFs), micelles, dendrimers, magnetic nanorods or nanowires, hydrogels or nanogels, porous silica particles, and aptamer-coated nanoparticles. In some aspects, the solid support is selected from PEG-based beads such as TentaGel and pegylated-polystyrene beads, silica and silica-gel supports including functionalized silica beads, alumina supports, magnetic silica or polymer-coated magnetic beads, controlled-pore glass beads, functionalized magnetic beads, functionalized silica gel, chitosan beads, and pre-functionalized systems including NHS-activated beads, maleimide-activated beads, epoxyactivated supports, and streptavidin-biotin capture systems, maleimide-activated beads, and epoxy-activated supports. In some aspects, the capture antibody reagent comprises a bead or microcarrier that is internally labeled with a dye.

[0210] In some aspects, the compositions containing a compound of this disclosure is a binding buffer. Binding buffers include nonionic which provide enhanced performance during target molecule capture on to a solid support such as silica surfaces, membranes, resins, beads, particles, or chromatographic matrix or media. The inclusion of nonionic surfactants reduces nonspecific adsorption of contaminants and sample debris, thereby improving the selectivity and efficiency of nucleic acid or protein binding. These surfactants further promote more uniform wetting and dispersion of complex samples, facilitating improved contact between the analyte andthe binding substrate and reducing variability across samples of differing viscosity or composition. Nonionic surfactants additionally minimize aggregation of cellular debris, lipids, or other particulates that can impede binding efficiency or clog filtration-based systems. Because nonionic surfactants exhibit minimal inhibitory effects on downstream enzymatic reactions and do not substantially disrupt the chemical conditions required for selective binding, they support seamless integration of the binding step into broader sample-preparation workflows. Collectively, these advantages contribute to increased yield, purity, and reproducibility of analyte capture from diverse sample types. In some aspects, the disclosed binding buffer may be used to bind target molecules, including nucleic acids, polypeptides, and vesicles, to a solid support, such as silica surfaces, membranes, resins, beads, particles, or chromatographic matrix or media.

[0211] The use of silica surfaces, membranes, resins, beads, particles, or chromatographic matrix or media in methods of binding target molecules to a solid surface is well known. Common bead types used for target molecule binding include magnetic beads (streptavidin, Ni-NTA / His-tag, carboxylated, Protein A / G, ion-exchange, silica-coated), silica beads, agarose resins or beads including Sepharose (Protein A / G agarose, glutathione agarose, Ni-NTA agarose, activated agarose), polymer beads (ion-exchange, hydrophobic interaction, reversed-phase / C18), paramagnetic nanoparticles, specialty beads such as immunomagnetic beads, lectin beads, aptamer-coated beads, and oligo(dT) beads, hydroxyapatite beads, cellulose and modified cellulose resins, cellulose beads, dextran-based (Sephadex) beads, polyacrylamide beads, chitosan-coated beads, modified polysaccharide beads, polystyrene-divinylbenzene (XAD) beads, ceramic or zirconia beads, alumina beads, enzyme-immobilized beads, metal oxide beads (Fe3O4, TiO2, ZrO2, AI2O3), sialic-acid-binding beads, graphene oxide or carbon-based beads, MOF-based beads, polystyrene beads and resins, and many more. Examples of nonbead particles used for binding target molecules include nanoparticles (gold, silver, silica, iron oxide), quantum dots, liposomes, polymeric nanoparticles, carbon nanotubes, graphene or graphene oxide sheets, metal-organic frameworks (MOFs), micelles, dendrimers, magnetic nanorods or nanowires, hydrogels or nanogels, porous silica particles, and aptamer-coated nanoparticles. In some aspects, the solid support is selected from PEG-based beads such as TentaGel and pegylated-polystyrene beads, silica and silica-gel supports including functionalized silica beads, alumina supports, magnetic silica or polymer-coated magnetic beads, controlled-pore glass beads, functionalized magnetic beads, functionalized silica gel, chitosan beads, and pre-functionalized systems including NHS-activated beads, maleimide-activated beads, epoxy-12 / 22 / 25 TP391474W01activated supports, and streptavidin-biotin capture systems, maleimide-activated beads, and epoxy-activated supports.

[0212] In some aspects, the membranes may include membranes contained in spin columns, no spin columns, or the like. Spin and no-spin column technologies provide distinct mechanisms for target molecule purification in sample prep and analytical workflows. Spin columns employ centrifugation to move samples through a membrane or resin, enabling rapid binding, washing, and elution of nucleic acids or proteins; examples include silica-based DNA and RNA purification columns, plasmid miniprep spin columns, protein desalting devices such as Zeba™ spin columns, and affinity-based formats such as Ni-NTA or Protein A / G spin columns. In contrast, no-spin purification systems rely on gravity flow, vacuum-driven movement, or capillary wicking to achieve separation without centrifugation. Representative no-spin formats include gravity-flow chromatography columns used for protein purification, PD-10 desalting columns, vacuum-compatible nucleic acid purification plates for high-throughput processing, and membrane-driven or capillary-action devices that enable rapid extraction in automated or low-instrumentation settings. No spin columns are popular in workflows where gentle handling, high throughput, or automation is needed. Exemplary uses of spin columns and no spin columns include DNA extraction, for example, isolating high-quality genomic DNA from cells, tissues, and blood; RNA extraction and purification, for example, extracting and purifying total RNA or specific RNA species, such as mRNA; plasmid DNA purification, for example, purifying plasmid DNA from bacterial cultures; PCR cleanup, for example removing primers, nucleotides, and enzymes from PCR product; and other routine molecular biology, biology, biochemistry, clinical, and diagnostic applications.

[0213] In some aspects, the chromatography matrix or media may include ones commonly used in the art, including columns or matrixes used in ion exchange chromatography matrix, affinity chromatography matrix, size exclusion chromatography matrix, hydrophobic interaction chromatography matrix, immobilized metal affinity chromatography matrix, reverse phase chromatography matrix, immunoaffinity chromatography matrix, or mixed mode chromatography matrix, and chromatography matrixes where non-ionic surfactants are commonly included to maintain solubility and prevent non-specific binding. Exemplary resins include, but are not limited to, Q-type resins, Ni Sepharose™ High Performance and Ni Sepharose™ 6 Fast Flow (Cytiva), which are agarose-based nickel-charged resins with high binding capacity for histidine-tagged proteins and broad compatibility with common buffer additives, including surfactants,12 / 22 / 25 TP391474W01denaturants (e.g. urea, guanidinium-HCI), and reducing agents; Profinity™ IMAC resins (BioRad), available as Ni2+-charged iminodiacetic acid (IDA) resins that are compatible with a wide range of salts, surfactants and denaturants, including 8 M urea and 6 M guanidinium-HCI; Ni-NTA Agarose and Ni-NTA Superflow resins (QIAGEN), nickel-charged nitrilotriacetic acid (NTA) resins for purification of 6xHis-tagged proteins under native or denaturing conditions; and other commercial Ni2+resins, such as HisPur™ Ni-NTA (Thermo Fisher Scientific), Ni-NTA agarose resins from additional suppliers, Q Sepharose™ Fast Flow (Cytiva), a strong anion exchanger based on a 6% cross linked agarose matrix functionalized with quaternary ammonium (Q) groups, widely used for capture and intermediate purification of proteins at laboratory and industrial scale; related ion exchangers such as SP Sepharose™ Fast Flow (strong cation exchanger), DEAE Sepharose™ Fast Flow (weak anion exchanger), and CM Sepharose™ Fast Flow (weak cation exchanger), which share a robust agarose Fast Flow matrix and are similarly compatible with typical chromatography additives. Compounds of this disclosure may be used in binding buffers to maintain column performance and protein recovery while providing improved protein solubility and reduced non-specific adsorption, without detrimental interaction with resin ligands, resin matrices, or common buffer components (such as NaCI, phosphate, Tris, urea, guanidinium-HCI, monovalent salts, or other buffer components).

[0214] In some aspects, the binding buffer may be used to bind a single type of target molecule from a sample to a solid support. In some aspects, the method may include binding two or more target molecules from a single sample using one or more solid supports, either sequentially or simultaneously. In some aspects, multiple target molecules may be bound by a single support that provides multiple binding sites, multiple ligands, spatially separated capture zones, layered / modular ligand assembly or broad-selectivity chemistry for simultaneous extraction. In some aspects the method may include using two or more solid supports simultaneously or sequentially. For example, the method may comprise first binding exosomes to a solid support and then removing the bound exosomes, next binding polypeptides to a second support and then removing the bound polypeptides, and then binding nucleic acids to a solid support and removing the bound nucleic acids.

[0215] In some aspects, the target molecules isolated by the disclosures herein, including nucleic acids, polypeptides, vesicles and exosomes, can used in a wide range of downstream processes and assays, including detection (assays, arrays, immonoassays, diagnostic tests, IVD assays), detection and quantification (qPCR, digital PCR, ELISA), sequencing and geneticanalysis, amplification (PCR, RT-PCR), isothermal amplification, cloning, restriction digestion, molecular assembly, and nucleic acid synthesis, as well as cDNA synthesis, epigenetic analysis (methylation, ChIP), and broader molecular or functional studies. In one aspect, the disclosure provides the aforementioned methods, wherein the detection is a signal amplification assay for multiplexed gene expression quantitation. By way of example, the compounds of the present disclosure can be used in branched DNA-based assays to detect target nucleic acids present in a sample lysate, e.g., as described in US Patent No. 8426578. The skilled artisan will readily appreciate that the compounds and compositions according to the disclosure may also be useful in a variety of downstream processes and assays. They may also undergo size separation, chromatography, and mass spectrometry for characterization; be used in transfection, expression analysis, and genome editing (CRISPR, base editing); and play important roles in pharmaceutical or therapeutic formulation, including recombinant protein production, gene therapy, cell-free systems, exosome-based delivery technologies, formation and manufacturing of and nucleic acid-based therapeutics, and those that do not require downstream polymerization or amplification reactions.

[0216] In one aspect, the disclosed compounds can be incorporated in hybridization buffer compositions, methods for preparing the hybridization buffer compositions, methods of using the hybridization buffer compositions and in kits relating to the same.

[0217] In one aspect, the disclosed compounds are used in hybridization buffers that are used fully or partly in all types of hybridization techniques known in the art and used in nucleic acid amplifications and sequencing workflows. In one aspect, hybridization buffers incorporating the disclosed compounds are used in nucleic acid or polypeptide microarrays, in situ hybridization (ISH) (for example, fluorescent in situ hybridization (FISH; including multi-color FISH and Fiber-FISH), chromogenic in situ hybridization (CISH), silver in situ hybridization (SISH)), Southern blot assays, Northern blot assays, dot blot assays or other arrays.

[0218] In some aspects, the disclosed compounds are used in hybridization buffers that are used with a number of arrays, including gene or polypeptide expression microarrays, including cDNA arrays and oligonucleotide arrays used to measure transcriptional activity across the genome; single nucleotide polymorphism (SNP) genotyping arrays, in which synthetic oligonucleotides detect specific allelic variants with high stringency; comparative genomic hybridization (CGH) arrays, including oligo-CGH and bacterial artificial chromosome (BAC)-based CGH arrays for detecting chromosomal copy number variations; tiling arrays, comprising denselyspaced probes designed to interrogate entire genomic regions or whole genomes; DNA methylation and epigenetic arrays, including CpG island microarrays and bisulfite-dependent detection arrays; BAG arrays and other large-fragment DNA arrays; pathogen detection and subtyping arrays, used to differentiate viral, bacterial, fungal, or parasitic nucleic acid sequences; custom oligonucleotide arrays, including inkjet-printed, mechanically spotted, or in situ-synthesized probe arrays; NGS-target capture arrays, such as early array-based enrichment systems used to isolate genomic regions prior to sequencing; membrane-based arrays, including macroarrays, dot blots, and slot blots on nylon or nitrocellulose substrates; and bead-based arrays, including fluorescently encoded or magnetically addressable bead sets in which each bead population functions as a discrete array element. In each of these array formats, the hybridization process relies on a buffer that supports probe-target duplex formation while minimizing unwanted binding events. In some aspects, including the disclosed compounds in hybridization buffer formulations can significantly enhance the performance, specificity, and robustness of array-based nucleic acid detection technologies.

[0219] In some aspects, the disclosed compounds can be included in hybridization buffers that incorporate non-ionic surfactants for a number of reasons, including to reduce nonspecific binding, improve probe dispersion and target accessibility, stabilize hybridization components, and suppress hydrophobic interactions that cause background signal, thereby increasing hybridization specificity and overall assay performance. Incorporation of the disclosed compounds into hybridization buffers can improve targeted binding and improve stabilization of buffer components to reduce sample degradation. Hybridization buffers containing the disclosed compounds can enhance the signal-to-noise ratio in an assay and further maintain hybrid stability during post-hybridization washing steps. Hybridization buffers containing the disclosed compounds demonstrate improved shelf stability and advantageously reduce signal to noise ratio in hybridization assays. The disclosed compounds are particularly advantageous in these applications as they result in reduced background signal in downstream applications such as PGR, as compared to other publicly available non-ionic surfactants.

[0220] In some aspects, compounds of this disclosure may be present in a hybridization buffer in an amount effective to achieve hybridization between a first nucleic acid and a complementary second nucleic acid. In some aspects, compounds of the disclosure may be present in a hybridization buffer in an amount effective to reduce nonspecific probe binding while maintaining target-probe hybrid stability during hybridization and wash steps. In some aspects,12 / 22 / 25 TP391474W01the compounds of this disclosure are present in a hybridization buffer at a concentration of about 0.0001% to 10% (w / v), such as, for example, 0.0001, 0.01, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%. In some aspects, the disclosed compounds are present in hybridization buffers at concentrations from about 0.0001% to 1%(w / v), such as <0.001%, <0.01%, <0.02%, <0.03%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%, and any percentage between. The hybridization buffers described herein are provided by way of example, and the skilled person will understand that the hybridization buffer components and concentrations may be optimized as necessary for different assays, using conventional methods.

[0221] In some aspects, the disclosed hybridization buffers may also include one or more salts, hybridization enhancers, accelerating agents, chelating agents, additional surfactants, blocking agents, buffering agents, crowding agents, stabilizers, or solvents. In some aspects, the hybridization buffer may include one or more salts, which optionally may be selected from the inorganic salts disclosed herein. In some aspects, the disclosed hybridization buffer may include one or more hybridization enhancers, which optionally may be selected from the hybridization enhancers disclosed herein. In some aspects, the disclosed hybridization buffer may include accelerating agents, which optionally may be selected from the accelerating agents disclosed herein. In some aspects, the hybridization buffer may include buffering agents, which optionally may be selected from the buffering agents disclosed herein. In some aspects, the buffering agent of the hybridization buffer may be capable of maintaining the pH of the buffer composition at or near physiological pH.

[0222] In some aspects, the disclosed hybridization buffer may include agents that reduce non-specific binding, for example to a cell membrane, or blocking agents to block binding. In some aspects, the disclosed hybridization buffer may include blocking agents, which optionally may be selected from the blocking agents disclosed herein. In some aspects, the disclosed hybridization buffer may include chelating agents selected from the chelating agents disclosed herein.

[0223] In some aspects, the disclosed compounds can be used in hybridization buffers included in kits for nucleic acid hybridization. In such kits, the hybridization buffer may be formulated for use across multiple nucleic acid hybridization techniques including microarrays, ISH, including FISH, CISH, Southern blot, Northern blot, dot blots or other arrays. In some aspects, the disclosed kit may include a hybridization buffer formulated for in situ hybridization of nucleic acids in fixed cells or tissue section. In other aspects, the disclosed kits may be configured12 / 22 / 25 TP391474W01for use of two or more nucleic acid hybridization techniques and may include instructions specifying assay-specific hybridization conditions using the same hybridization buffer composition.

[0224] In some aspects, the disclosed kits may comprise a hybridization buffer comprising one or more of the disclosed compounds and one or more additional components selected from fixatives, buffers and reagents, mounting media, detection reagents, labeling reagents, sample membranes or support, positive controls, negative controls, nucleic acid probes or targets, probes comprising oligonucleotides immobilized on a solid substrate selected from glass, silica, or polymeric films, or wash buffers. In some aspects, the disclosed kits may include labeling reagents. In some aspects, the disclosed kits may include labeling reagents selected from the labeling reagents disclosed herein. In some aspects, the disclosed kits may include labeling reagents that may be incorporated directly into nucleic acids, conjugated to probes, or introduced via enzymatic or chemical labeling reactions. In some aspects, the disclosed kits may include a one or more probes, which optionally may be selected from the labeling reagents disclosed herein. In some aspects, the disclosed kits may include one or more probes that may be labeled for detecting genomic or transcriptomic targets on a membrane substrate, or reagents for target nucleic acid immobilization and post-hybridization washing.

[0225] In some aspects, the disclosed compounds are used in the manufacture of in vitro diagnostic (IVD) products, including those used for infectious disease screening, endocrinology, and oncology testing. For example, the disclosed compounds may be used for their wetting properties in the production of buffers, reagents and gel supports allowing the dissolution, the dilution and the good spreading of substrates and reagents, necessary to optimize the functioning and the sensitivity of gel electrophoresis in IVD tests. In some aspects, the disclosed compounds are incorporated in various components, including wash buffers, related to IVD kits, FISH test kits and laboratory developed test (LDT) equivalents.

[0226] In other aspects, the disclosed compounds are used as components in one or more reaction buffers, wash buffers, stain buffers, polymerase buffers, controls, extraction reagents, purification reagents, fragmentation reagents, quantification reagents, master mixes or other reagents used in nucleic acid or polypeptide amplifications and sequencing workflows, including multiplex amplification techniques and next-generation sequencing (“NGS”). In some aspects, disclosed compounds are used in loading buffers, including those for gel electrophoresis, multiplex amplification, and next-generation sequencing, and in various storage buffers andreagent mixes for particles including ion sphere particles, beads including magnetic and nonmagnetic beads, nucleic acids including variants, fragments and conjugates thereof, oligonucleotides including variants, fragments and conjugates thereof, nucleotides including variants and conjugates thereof, proteins including variants, fragments and conjugates thereof, enzymes including variants, fragments and conjugates thereof, and antibodies including variants, fragments and conjugates thereof. In some embodiments, disclosed compounds are components in various fluidic nucleic acid amplification and sequencing steps, including in loading reagents, reaction reagents, and in washes applied between workflow steps. In some embodiments, disclosed compounds are components in reagents contained in ready-to-use cartridges or kits used with nucleic acid amplification and sequencing instruments. In some embodiments, disclosed compounds are components added to a mixture of reagents, prior to loading the mixture onto a cartridge.

[0227] In other aspects, the disclosed compounds are used as performance-enhancing additives in enzymatic reaction mixtures, sample preparation workflows including those for multiplex nucleic acid amplification and next-generation sequencing, sequencing library construction, and sequencing procedures. In some aspects, the disclosed compounds enhance performance by various mechanisms including stabilizing reagents such as enzymes and other proteins, activating enzymatic reactions, reducing aggregation of reagents, improving solubility and uniformity of reagents, reducing secondary structure of nucleic acids, enhancing tolerance to reaction variables and inhibitory compounds, minimizing surface adsorption of reagents, facilitating efficient flow of reagents through amplification and sequencing systems, and enhancing cleansing of amplification and sequencing systems within, between, and following workflow procedures.

[0228] In some aspects, the compounds of this disclosure can be used to isolate nucleic acids and polypeptides for further use in downstream processes and analyses. Such downstream processes and analyses include, but are not limited to arrays, including microarrays, ISH, including FISH, CISH, Southern blot, Northern blot, dot blots or other arrays. They also may include signal amplification assays or hybridization-based assay for multiplexed gene expression quantitation. In multiplexed gene expression, in some embodiments, the analyte is RNA or RNA transcripts and the multiplexed gene expression is quantified, in optionally, up to 80 genes of interest are simultaneously detected in a sample container.12 / 22 / 25 TP391474W01

[0229] In some aspects, the compounds of this disclosure are bound to a solid support. In some aspects the compounds of this disclosure may be covalently bound to the solid support. In some aspects, the compounds are bound to the support in a manner that preserves the accessibility and functionality of their hydrophobic and hydrophilic domains, such that the compounds maintain their activity as effective surfactants. In some aspects, the solid-support bound surfactant is employed in conjunction with compositions that typically include nonionic surfactants. Following completion of the reaction or processing step, the solid-supported compound may be readily removed from the mixture prior to downstream handling. In some aspects, a solid support bearing an immobilized nonionic surfactant is included as a component of a composition such as a lysis buffer. Upon contact with a cellular, viral, or vesicular sample, the immobilized surfactant may disrupt lipid bilayers and promote release of intracellular or intra-vesicular contents, while remaining associated with the solid phase. After the lysis reaction, the solid support — and the immobilized surfactant thereon — is removed from the sample by physical separation, for example by filtration, sedimentation, magnetic separation, or centrifugation, thereby yielding a clarified lysate that is substantially free of surfactant. In this way, downstream analytical, purification, or amplification processes may be conducted without interference from residual surfactant and without additional detergent-removal or desalting steps. This approach is particularly advantageous for sensitive downstream assays and processes, including chromatographic and mass-based analytical methods.

[0230] In some aspects, the solid support (SS) may be a resin, a bead, a particle, magnetic bead, a non-magnetic bead, an ion exchange matrix, an affinity chromatography matrix, a size exclusion chromatography matrix, a hydrophobic interaction chromatography matrix, an immobilized metal affinity chromatography, a reverse phase chromatography matrix, immunoaffinity chromatography matrix, or a mixed mode chromatography matrix, or any of the solid supports disclosed herein.

[0231] In some aspects, the solid support (SS) may be selected from agarose resins such as Sepharose, polystyrene resins such as Merrifield resin, Wang resin, and Rink amide resin, polystyrene beads, PEG-based beads such as TentaGel and pegylated-polystyrene beads, silica and silica-gel supports including functionalized silica beads, alumina supports, magnetic silica or polymer-coated magnetic beads, controlled-pore glass beads, functionalized magnetic beads, functionalized silica gel, cellulose and modified cellulose resins, chitosan beads, dextran-based supports such as Sephadex, and pre-functionalized systems including NHS-activated beads,12 / 22 / 25 TP391474W01maleimide-activated beads, epoxy-activated supports, and streptavidin-biotin capture systems, maleimide-activated beads, and epoxy-activated supports.

[0232] In some aspects, artificial intelligence or a machine-learning model may be used to evaluate and screen a defined subset of candidate compounds within the disclosed group to identify those predicted to exhibit optimal nonionic surfactant functionality for different applications or workflows, wherein the model correlates molecular features with performance attributes to enable targeted selection of surfactant compositions. Data obtained from chemical, functional, and application-specific tests of surfactant candidate molecules may be collected and integrated or compiled into a training dataset used to develop, refine, or retrain artificial intelligence or machine-learning models configured to predict and optimize nonionic surfactant properties for diverse workflows and end-use conditions.Overview of Several Aspects

[0233] Disclosed herein is a compound having a structure according to Formula I,Formula Iwherein:R1is a hydrophilic group;R2is a lipophilic group;X is selected from oxygen, sulfur, or N(R5);R3is selected from heteroaliphatic, aliphatic, or aryl;each R4independently is aliphatic;Y is selected from oxygen, sulfur, or N(R5);the linker, when present, is aliphatic or has a formula -C(=Z)-W-C(=Z)-X’, wherein each Z independently is oxygen, sulfur, or NR5, W is an aliphatic or heteroaliphatic group, and X’ is oxygen, sulfur, or NR5;n is an integer selected from 0 or 1 ;m is an integer selected from 0 to 6, provided that, if n is 0, then m is 1;p is an integer selected from 0 to 2; andeach R5group independently is selected from hydrogen, aliphatic, aromatic, or heteroaliphatic.

[0234] The compound may have a structure according to any one of Formulas IA-IFFormula IE Formula IF.

[0235] The R1and R2groups may be selected to provide a hydrophilic-lipophilic balance value ranging from 10 to 18.

[0236] The R1group comprises a heteroaliphatic group.

[0237] The heteroaliphatic is a linear heteroaliphatic group.

[0238] The linear heteroaliphatic group is a polyalkylene oxide.

[0239] The linear heteroaliphatic group is a PEG group having a mass average molecular weight ranging from 500 g / mol to 800 g / mol.

[0240] R1may comprise a cyclic heteroaliphatic group.

[0241] The cyclic heteroaliphatic group is a sugar molecule.

[0242] R2may comprise an aliphatic group or an aryl group.

[0243] R2may comprise C2-2salkyl, phenyl, or naphthyl.12 / 22 / 25 TP391474W01

[0244] The C2-2salkyl group is linear, branched, cyclic, or a combination thereof.

[0245] p may be 1 or 2 and each R3independently is an alkoxy group or an aliphatic group.

[0246] Each R4independently may comprise a Ci- alkyl group.

[0247] Each R4independently may comprise methyl or ethyl.

[0248] n may be 1 and Y is oxygen or sulfur.

[0249] X may be oxygen or NH.

[0250] X may be oxygen.

[0251] The linker may not be present.

[0252] The linker may be present and is a Ci-2saliphatic group.

[0253] The linker may be present and has a structure according to the formula -C(=Z)-W-C(=Z)-X’-, wherein each Z independently is selected from O, S, or NR5; W is Ci-2salkyl, Ci- 2salkenyl, Ci-2salkynyl, ether, thioether, or amine; and X’ is O, S, or NR5.

[0254] The linker may be present and is -(CH2)q-, -C(=O)(CH2)qC(=O)-O-, or -C(=S)(CH2)qC(=S)-O-, -C(=O)N(H)(CH2)qN(H)C(=O)-O-, wherein each q independently is an integer ranging from 1 to 10.

[0255] m may be 1 and p may be 0.

[0256] The compound can have a structure according to any one of Formulas IG, IH, I J,wherein the aliphatic group is linear, branched, or cyclic, or a combination thereof; TG, if present, is a terminating group that is an aliphatic group; and r is an integer selected from 2 to 20.

[0257] The aliphatic group may comprise a linear C2-2oalkyl group; a branched C2-2oalkyl group; a cyclic C3-2oalkyl group; or a combination of any such linear, branched, and / or cyclic groups.

[0258] The aliphatic group may comprise isopentyl, 2-methylpentyl, heptyl, 2,2-dimethylbutyl, pentyl, octyl, or nonyl.

[0259] The TG group may comprise a Ci- alkyl group.

[0260] The TG group may comprise a methyl.

[0261] In any or all of the above aspects, the compound may be selected from any one of the compounds 1 to 54 disclosed herein.

[0262] In one aspect the disclosure provides a composition, comprising:(a) one or more surfactant, wherein the surfactant is a compound having a structure according to Formula I:Formula Iwherein:R1is a hydrophilic group;R2is a lipophilic group;X is selected from oxygen, sulfur, or N(R5);R3is selected from heteroaliphatic, aliphatic, or aryl;each R4independently is aliphatic;Y is selected from oxygen, sulfur, or N(R5);the linker, when present, is aliphatic or has a formula -C(=Z)-W-C(=Z)-X’, wherein each Z independently is oxygen, sulfur, or NR5, W is an aliphatic or heteroaliphatic group, and X’ is oxygen, sulfur, or NR5;n is an integer selected from 0 or 1 ;m is an integer selected from 0 to 6, provided that, if n is 0, then m is 1;p is an integer selected from 0 to 2; andeach R5group independently is selected from hydrogen, aliphatic, aromatic, or heteroaliphatic;(b) at least one polar protic solvent;(c) optionally, one or more inorganic salt;(d) optionally one or more buffering agent; and(e) optionally one or more chaotropic agents.

[0263] In one aspect the disclosure provides a composition, comprising:(a) one or more surfactant, wherein the surfactant is a compound having a structure according to Formulas IA, IB, IC, ID, IE, or IF:Formula IC Formula IDFormula IE Formula IF;(b) at least one polar protic solvent;(c) optionally, one or more inorganic salt;(d) optionally one or more buffering agent; and(e) optionally one or more chaotropic agents

[0264] In one aspect the disclosure provides a composition, comprising:(a) one or more surfactant, wherein the surfactant is a compound having a structure aFormula IKwherein the aliphatic group is linear, branched, or cyclic, or a combination thereof; TG, if present, is a terminating group that is an aliphatic group; and r is an integer selected from 2 to 20;(b) at least one polar protic solvent;(c) optionally, one or more inorganic salt;(d) optionally one or more buffering agent; and(e) optionally one or more chaotropic agents.

[0265] In one aspect the disclosure provides a composition, comprising a surfactant: (a) wherein the surfactant is a compound selected from the group consisting of compounds of compounds 1 - 54:(b) at least one polar protic solvent;(c) optionally, one or more inorganic salt;(d) optionally one or more buffering agent; and(e) optionally one or more chaotropic agents.

[0266] In one aspect the disclosure provides the aforementioned compositions, wherein the polar protic solvent is selected from a C1-C5 alcohol, C2-C6polyol, polyethylene glycol, water, and mixtures thereof.

[0267] In one aspect the disclosure provides the aforementioned compositions, wherein the polar protic solvent is selected from ethanol, isopropyl alcohol, 2-methyl-1,3-propanediol (MPD), isoamyl alcohol, water, and mixtures thereof.

[0268] In one aspect the disclosure provides the aforementioned compositions, wherein the polar protic solvent is a polyethylene glycol (PEG) selected from the group consisting of PEG1500, PEG8000 PEG6000, PEG2000, PEG1000, PEG600, and mixtures thereof.

[0269] In one aspect the disclosure provides the aforementioned compositions, wherein the polar protic solvent is present at a concentration of about 5% to about 50% v / v. In another12 / 22 / 25 TP391474W01aspect, the polar protic solvent is present at a concentration of about 10% to about 40% v / v. In another aspect, the polar protic solvent is present at a concentration of about 15% to about 35% v / v. In another aspect, the polar protic solvent is present at a concentration of about 20% to about 30% v / v. In another aspect, the polar protic solvent is present at a concentration of about 5% to about 25% v / v. In another aspect, the polar protic solvent is present at a concentration of about 25% to about 50% v / v. In another aspect, the polar protic solvent is present at a concentration of about 10% to about 30% v / v. In another aspect, the polar protic solvent is present at a concentration of about 15% to about 45% v / v. In another aspect, the polar protic solvent is present at a concentration of about 30% to about 45% v / v. In another aspect, the polar protic solvent is present at a concentration of about 5% to about 10% v / v. In another aspect, the polar protic solvent is present at a concentration of about 10% to about 15% v / v. In another aspect, the polar protic solvent is present at a concentration of about 15% to about 20% v / v. In another aspect, the polar protic solvent is present at a concentration of about 7.5% to about 17.5% v / v. In another aspect, the polar protic solvent is present at a concentration of at least about 7.5% v / v , at least about 10% v / v, such as at least about 15%, at least about 17.0%, at least about 17.5%, at least about 20%, or at least about 25% v / v. In another aspect, the polar protic solvent is MPD and is present at a concentration of at least about 11.3% v / v; at least about 13.1% v / v; at least about 13.8% v / v; or at least about 20.0%. In another aspect, the polar protic solvent is PEG1500 and is present at a concentration of at least about 7.5% v / v; at least about 10.0%; at least about 12.5% v / v; at least about 15.0% v / v; at least about 17.0% at least about 20.0%; or at least about 20.0%. In another aspect, the polar protic solvent is isopropanol and is present at a concentration of at least about 10.0% v / v; at least about 15.0%; at least about 18.0% v / v; at least about 20.0% v / v; at least about 25.0%; or from at least about 30.0% to about 45.0%.

[0270] In one aspect the disclosure provides the aforementioned compositions, wherein the inorganic salt is an alkali or alkaline earth metal salt.

[0271] In one aspect the disclosure provides the aforementioned compositions, wherein the alkali or alkaline earth metal salt is selected from the group consisting of LiCI, NaCI, KCI, MgCh, BaCh, and CaCh, and corresponding citrates, acetates, or any combination thereof.

[0272] In some aspects the disclosure provides the aforementioned compositions, wherein the inorganic salt is present at a concentration of 0.25 mM to 1000 mM. In some aspects, the composition includes one or more salts, such as alkaline metal salts (e.g, calcium and / or magnesium salts). For example, compositions provided herein can include a calcium salt inconcentrations ranging from 0 mM to 2.5 mM. In some aspects, a calcium salt is present in the composition in concentrations ranging from about 0.25 mM to about 2.5 mM. In some aspects, a calcium salt is present in the composition in concentrations ranging from about 0.25 mM to about 2.0 mM. In some aspects, a calcium salt is present in the composition in concentrations ranging from about 0.25 mM to about 1.5 mM. In some aspects, a calcium salt is present in the composition in concentrations ranging from about 0.25 mM to about 1.0 mM. The calcium salt can be any calcium salt, including but not limited to calcium chloride, calcium bromide, calcium acetate, calcium citrate, calcium formate, calcium sulfate, or calcium phosphate, for example. By way of example, compositions provided herein can include CaCI2is present at an effective concentration of about 0 mM, 0.1 mM, 0.2 mM, 0.5 mM, 1.0 mM, 1.5 mM, 2.0 mM, or 2.5 mM or any range of concentrations therebetween. In some aspects, MgCI2is present in the composition in concentrations ranging from 0 mM to 15.0 mM. In some aspects, MgCI2is present in the composition in concentrations ranging from about 0.5 mM to about 15.0 mM. In some aspects, MgCh is present in the composition in concentrations ranging from about 0.5 mM to about 12.5 mM. In some aspects, MgCI2is present in the composition in concentrations ranging from about 0.5 mM to about 10.0 mM. In some aspects, MgCI2is present in the composition in concentrations ranging from about 0.5 mM to about 7.5 mM. In some aspects, MgCh is present in the composition in concentrations ranging from about 0.5 mM to about 5.0 mM. In some aspects, MgCh is present in the composition in concentrations ranging from about 0.5 mM to about 4.0 mM. In some aspects, MgCI2is present in the composition in concentrations ranging from about 0.5 mM to about 3.0 mM. In some aspects, MgCI2is present in the composition in concentrations ranging from about 0.5 mM to about 2.0 mM. In certain aspects, the MgCI2is present at an effective concentration of about 0.1 mM, 0.2 mM, 0.5 mM, 1.0 mM, 1.5 mM, 2.0 mM, 2.5 mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, 5.0 mM, 5.5 mM, 6.0 mM, 6.5 mM, 7.0 mM, 7.5 mM, 8.0 mM, 8.5 mM, 9.0 mM, 9.5 mM, 10.0 mM, 10.5 mM, 11.0 mM, 11.5 mM, 12.0 mM, 12.5 mM, 13.0 mM, 13.5 mM, 14.0 mM, 14.5 mM, 15.0 mM or any range of concentrations there between. The sodium salt can be any sodium salt, including but not limited to sodium chloride, sodium acetate, sodium citrate, sodium formate, sodium sulfate, or sodium phosphate, for example; in certain aspects, the sodium chloride or sodium citrate is present at an effective concentration of about 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 650 mM, 700 mM, 750 mM, 800 mM, 900 mM, 950 mM, 1000 mM, or any range of concentrations there between.

[0273] In one aspect the disclosure provides the aforementioned compositions, wherein the buffering agent is selected from the group consisting of N-2-acetamido-2-aminoethanesulfonic acid (ACES), N-2-acetamido-2-iminodiacetic acid (ADA), 3-1,1-dimethyl-2-hydroxyethylamino-2-hydroxy propanesulfonic acid (AMPSO), N,N-bis2-hydroxyethyl-2-aminoethanesulfonic acid (BES), 4-cyclohexylamino-1 -butane sulfonic acid (CABS), 3-cyclohexylamino-1-propane sulfonic acid (CAPS), 3-cyclohexylamino-2-hydroxy-1-propane sulfonic acid (CAPSO), 2-N-cyclohexylaminoethanesulfonic acid (CHES), 3-N,N-bis-2-hydroxyethylamino-2-hydroxypropanesulfonic acid (DIPSO), N-2-hydroxyethylpiperazine-N-3-propanesulfonic acid (EPPS or HEPPS), N-2-hydroxyethylpiperazine-N-4-butanesulfonic acid (HEPBS), 4-N-morpholinobutanesulfonic acid (MOBS), 3-N-morpholino-2-hydroxypropanesulfonic acid (MOPSO), bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (Bis-Tris), hydroxyethylpiperazine ethane sulfonic acid (HEPES), morpholinoethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), [tris(hydroxymethyl)methylamino] propanesulfonic acid (TAPS), N-trishydroxymethyl-methyl-4-aminobutanesulfonic acid (TABS), N-trishydroxymethyl-methyl-3-aminopropanesulfonic acid (TAPS), 3-N-trishydroxymethyl-methylamino-2-hydroxypropanesulfonic acid (TAPSO), N-trishydroxymethyl-methyl-2-aminoethanesulfonic acid (TES), N-(2-acetamido)iminodiacetic acid (ADA), Piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCI), N-trishydroxymethylmethylglycine (TRICINE), or any combination thereof.

[0274] In one aspect the disclosure provides the aforementioned compositions, wherein the buffering agent is present at a concentration of about 0.5 mM to about 500 mM. In some aspects, the concentration of the buffering agent in the composition is from about 10 mM to 250 mM. In some aspects, the concentration of the buffering agent in the composition may be from about 25 mM to about 75 mM. In some aspects, the concentration of the buffering agent in the composition may be from about 5 mM to about 25 mM. In some aspects, the concentration of the buffering agent in the composition may be from about 7.5 mM to about 20 mM. In some aspects, the concentration of the buffering agent in the composition may be from about 0.5 mM to about 10 mM. In some aspects, the concentration of the buffering agent in the composition may be from about 1.0 mM to 5 mM. In certain aspects, the buffering agent is present at an effective concentration of about 1.0 mM, 1.5 mM, 2.0 mM, 2.5 mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, 5.0 mM, 5.5 mM, 6.0 mM, 6.5 mM, 7.0 mM, 7.5 mM, 8.0 mM, 8.5 mM, 9.0 mM, 9.5 mM, 10.0 mM, 10.5 mM, 11.0 mM, 11.5 mM, 12.0 mM, 12.5 mM, 13.0 mM, 13.5 mM, 14.0 mM, 14.5 mM, 15.0mM, 16.0 mM, 17.0 mM, 18.0 mM, 19.0 mM, 19.5 mM, 20.0 mM, 20.5 mM, 21.0 mM, 25.0 mM, 50.0 mM, 55.0 mM, 60.0 mM, 65.0 mM, 70.0 mM, 75.0 mM, 80.0 mM, 90.0 mM, 95.0 mM, 100.0 mM, 150.0 mM, 200.0 mM, 250.0 mM, 300.0 mM, 350.0 mM, 400.0 mM, 450.0 mM, 500 mM, or any range of concentrations there between.

[0275] In one aspect the disclosure provides the aforementioned compositions, wherein the chaotropic agent is selected from the group consisting of lithium perchlorate, lithium acetate, sodium dodecyl sulfate, thiourea, urea, guanidinium chloride, guanidinium thiocyanate, and a combination thereof. In one aspect the disclosure provides the aforementioned compositions, wherein the chaotropic agent is present at a concentration of about 0.5 M to about 8 M. In one aspect the disclosure provides the aforementioned compositions, wherein the chaotropic agent is present at a concentration of about 3.0 M to about 6.0 M. In one aspect the disclosure provides the aforementioned compositions, wherein the chaotropic agent is present at a concentration of about 1.0 M to about 6 M. In one aspect the disclosure provides the aforementioned compositions, wherein the chaotropic agent is present at a concentration of about 1.5 M to about 5.5 M. In one aspect the disclosure provides the aforementioned compositions, wherein the chaotropic agent is present at a concentration of about 3.5 M to about 4.5 M. In one aspect the disclosure provides the aforementioned compositions, wherein the chaotropic agent is present at a concentration of about 4.5 M to about 5.5 M. In one aspect the disclosure provides the aforementioned compositions, wherein the chaotropic agent is present at a concentration of about 5.5 M to about 7.5 M. In certain aspects, the chaotropic agent is present at an effective concentration of about 1.0 M, 1.5 M, 2.0 M, 2.5 M, 3.0 M, 3.5 M, 4.0 M, 4.5 M, 5.0 M, 5.5 M, 6.0 M, 6.5 M, 7.0 M, 7.5 M, 8.0 M, or any range of concentrations there between.

[0276] In one aspect the disclosure provides the aforementioned compositions, further comprising a chelating agent selected from EDTA, EGTA, or DTPA. In one aspect the disclosure provides the aforementioned compositions, wherein the chelating agent is present at a concentration of about 5 mM to about 750 mM. In one aspect the disclosure provides the aforementioned compositions, wherein the chelating agent is present at a concentration of about 50 mM to about 750 mM. In one aspect the disclosure provides the aforementioned compositions, wherein the chelating agent is present at a concentration of about 100 mM to about 750 mM. In one aspect the disclosure provides the aforementioned compositions, wherein the chelating agent is present at a concentration of about 250 mM to about 750 mM. In one aspect the disclosure provides the aforementioned compositions, wherein the chelating agent is present at a12 / 22 / 25 TP391474W01concentration of about 300 mM to about 750 mM. In one aspect the disclosure provides the aforementioned compositions, wherein the chelating agent is present at a concentration of about 400 mM to about 750 mM. In one aspect the disclosure provides the aforementioned compositions, wherein the chelating agent is present at a concentration of about 400 mM to about 600 mM. In one aspect the disclosure provides the aforementioned compositions, wherein the chelating agent is present at a concentration of about 500 mM to about 750 mM. In one aspect the disclosure provides the aforementioned compositions, wherein the chelating agent is present at a concentration of about 600 mM to about 750 mM. In certain aspects, the chelating agent is present at an effective concentration of about 5.0 mM, 25.0 mM, 50.0 mM, 75.0 mM, 85.0 mM, 90.0 mM, 100.0 mM, 125.0 mM, 150.0 mM, 175.0 mM, 200.0 mM, 225.0 mM, 250.0 mM, 300.0 mM, 350.00 mM, 400.0 mM, 450.0 mM, 500.0 mM, 550.0 mM, 600.0 mM, 650.0 mM, 700.0 mM, 725.0 mM, 750.0 mM, or any range of concentrations there between.

[0277] In one aspect the disclosure provides the aforementioned compositions, wherein the surfactant is present at a concentration of about 0.001% to about 25% v / v. In one aspect the disclosure provides the aforementioned compositions, wherein the surfactant is present at a concentration of about 0.001% to about 10% v / v. In one aspect the disclosure provides the aforementioned compositions, wherein the surfactant is present at a concentration of about 0.025% to about 0.2% v / v. In one aspect the disclosure provides the aforementioned compositions, wherein the surfactant is present at a concentration of about 0.1% to about 0.6% v / v. In one aspect the disclosure provides the aforementioned compositions, wherein the surfactant is present at a concentration of about 0.5% to about 2% v / v.

[0278] In one aspect the disclosure provides the aforementioned compositions, wherein the surfactant is present at a concentration of about 0.05% to about 25% v / v.

[0279] In one aspect the disclosure provides the aforementioned compositions, wherein the surfactant is present at a concentration of about 0.5% to about 15% v / v. In one aspect the disclosure provides the aforementioned compositions, wherein the surfactant is present at a concentration of about 0.05% to about 3% v / v. In one aspect the disclosure provides the aforementioned compositions, wherein the surfactant is present at a concentration of about 0.1% to about 3.0% v / v. In one aspect the disclosure provides the aforementioned compositions, wherein the surfactant is present at a concentration of about 0.5% to about 2% v / v. In one aspect the disclosure provides the aforementioned compositions, wherein the surfactant is present at a concentration of about 1.0% to about 10% v / v.

[0280] In one aspect the disclosure provides the aforementioned compositions, wherein the surfactant is present at a concentration of about 3.5% to about 15% v / v. In one aspect the disclosure provides the aforementioned compositions, wherein the surfactant is present at a concentration of about 5.5% to about 10% v / v. In one aspect the disclosure provides the aforementioned compositions, wherein the surfactant is present at a concentration of about 7.5% to about 10.5% v / v. In one aspect the disclosure provides the aforementioned compositions, wherein the surfactant is present at a concentration of about 10.0% to about 15% v / v. In one aspect the disclosure provides the aforementioned compositions, wherein the surfactant is present at a concentration of about 15.0% to about 20.0% v / v. In one aspect the disclosure provides the aforementioned compositions, wherein the surfactant is present at a concentration of about 17.5% to about 22.5% v / v

[0281] In certain aspects, the surfactant is present at an effective concentration of about 0.001%, 0.002%, 0.005%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10.0%, 12.0%, 14.0%, 15.0%, 16.0%, 17.0%, 18.0%, 19.0%, 20.0%, 21.0%, 22.0%, 23.0%, 24.0%, 25.0% or any range of concentrations there between.

[0282] In one aspect the disclosure provides the aforementioned compositions, wherein the inorganic salt is present at a concentration of about 0.25 mM to 1000 mM.

[0283] In one aspect the disclosure provides the aforementioned compositions, wherein the buffering agent is present at a concentration of about 0.5 mM to 500 mM.

[0284] In one aspect the disclosure provides the aforementioned compositions, wherein the chaotropic agent is present at a concentration of about 0.5 M to 8 M

[0285] In one aspect the disclosure provides the aforementioned compositions, wherein the chelating agent is present at a concentration of about 5 mM to 750 mM.

[0286] In one aspect the disclosure provides the aforementioned compositions, wherein the polar protic solvent is present at a concentration of about 5% to about 25% v / v.

[0287] In one aspect the disclosure provides the aforementioned compositions, wherein the pH is about 5.5 to about 10.0. In one aspect the disclosure provides the aforementioned compositions, wherein the pH is about 6.0 to about 9.0. In one aspect the disclosure provides the aforementioned compositions, wherein the pH is about 6.5 to about 8.5. In one aspect the disclosure provides the aforementioned compositions, wherein the pH is about 7.0 to about 8.0. In one aspect the disclosure provides the aforementioned compositions, wherein the pH is about12 / 22 / 25 TP391474W017.5 to about 8.0. In one aspect the disclosure provides the aforementioned compositions, wherein the pH is about 5.5 to about 6.5. In one aspect the disclosure provides the aforementioned compositions, wherein the pH is about 6.5 to about 7.5. In one aspect the disclosure provides the aforementioned compositions, wherein the pH is about 7.5 to about 8.5. In one aspect the disclosure provides the aforementioned compositions, wherein the pH is about 8.0 to about 8.5. In one aspect the disclosure provides the aforementioned compositions, wherein the pH is about 8.5 to about 9.0. In one aspect the disclosure provides the aforementioned compositions, wherein the pH is about 6.8 to about 7.8. In one aspect the disclosure provides the aforementioned compositions, wherein the pH is about 7.2 to about 8.2. In one aspect the disclosure provides the aforementioned compositions, wherein the pH is about 8.5 to about 9.5. In one aspect the disclosure provides the aforementioned compositions, wherein the pH is about 9.0 to about 9.5. In one aspect the disclosure provides the aforementioned compositions, wherein the pH is about 9.5 to about 10.0. In one aspect the disclosure provides the aforementioned compositions, wherein the pH is about 8.2 to about 9.2. In one aspect the disclosure provides the aforementioned compositions, wherein the pH is about 8.8 to about 9.8.

[0288] In certain aspects, the disclosure provides aforementioned compositions, wherein the pH is about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0, about 9.1, about 9.2, about 9.3, about 9.4, about 9.5, about 9.6, about 9.7, about 9.8, about 9.9, about 10.0, or any values there between.

[0289] In one aspect the disclosure provides a method of exposing a sample to a compound according to any one of the aforementioned formulas.

[0290] In one aspect the disclosure provides a method for isolating nucleic acid from a sample containing nucleic acid, the method comprising: contacting the sample with a solid support in the presence of the aforementioned compositions; thereby providing a nucleic acid-bound solid support.. In one aspect the disclosure further provides a method, wherein contacting the sample with a solid support in the presence of an aforementioned composition, includes: (a) contacting the sample with a lysis buffer to lyse cells, if present, in the sample, wherein the lysis buffer is a composition that has a concentration of surfactant according to Formula I effective to lyse the cells, and (b) contacting the sample with a binding buffer to bind thenucleic acids to the solid support, wherein the binding buffer is a composition having a concentration of the surfactant according to Formula I effective to allow binding of the nucleic acids to the solid support.

[0291] In one aspect the disclosure further provides a method, wherein the nucleic acid isolated from the sample is cell-free nucleic acids, wherein the contacting step comprises: (a) contacting the sample with a binding buffer to bind the cell-free nucleic acids to the solid support, wherein the binding buffer is a composition having a concentration of the surfactant according to Formula 1 effective to allow binding of the cell-free nucleic acids to the solid support.

[0292] In one aspect the disclosure further provides a method for isolating polypeptides and nucleic acids from a sample containing nucleic acids and polypeptides, the method comprising: contacting the sample with an aforementioned composition thereby providing a lysed sample.

[0293] In one aspect the disclosure further provides a method for isolating polypeptides from a sample comprising polypeptides, the method comprising: contacting the said sample with an aforementioned; thereby providing a lysed sample.

[0294] In one aspect the disclosure provides a method; wherein the method further comprises: (a) contacting the lysed sample with a first solid support in the presence of a binding buffer to bind the polypeptides to the solid support; (b) wherein the binding buffer is a composition having a concentration of the surfactant according to Formula I effective to allow binding of the polypeptides to the first solid support.

[0295] In one aspect the disclosure provides a method wherein the method further comprises: (a) contacting the lysed sample with a second solid support in the presence of a binding buffer to bind the nucleic acids to the second solid support, (b) wherein the binding buffer is a composition having a concentration of the surfactant according to Formula I effective to allow binding of the nucleic acids to the second solid support.

[0296] In one aspect the disclosure further provides a method for preparing nucleic acids from a sample containing nucleic acids, comprising: contacting the sample containing nucleic acids with a lysis buffer to produce a lysis mixture; and incubating the lysis mixture at an incubation temperature and for a time to produce a cell lysate, wherein the lysis buffer comprises: (a) an RNase inhibitor; and (b) an aforementioned composition; and wherein the cell lysate is compatible with in situ polymerase or reverse transcription reactions. In one aspect the disclosure further provides a method; wherein the RNAse inhibitor is an anionic oligomer. In one aspect the12 / 22 / 25 TP391474W01disclosure further provides a method wherein the anionic oligomer is selected from the group consisting of Poly(vinylphosphonic acid), Heparin, Sulfated cellulose, Sulfated nitrocarboxymethyl cellulose, Sulfated amylose, Sulfated amylopectin, Sulfated pectic acid, Sulfated nitrochitin, Sulfated nitrochitosan, Sulfated polyvinyl alcohol, Poly-p,p-dioxy-dibenzyl phosphate, Poly-p,p-dioxydiphenyldimethyl metaphosphate, Polyaspartic acid, Polyglutamic acid, Polyacrylic acid, Poly(methacrylic acid), Poly(maleic acid), Pentosan polysulfate, Chondroitin sulfate, polyglycerol sulfate, Polyethylene sulfonate, Poly(4-styrenesulfonic acid-co-maleic acid), Poly(vinyl sulfonic acid), Poly(4-styrenesulfonic acid), Dextran sulfate, or any combination thereof.

[0297] In one aspect the disclosure further provides a method; wherein the method further; optionally comprises a polypeptide having protease activity. In one aspect the disclosure further provides a method wherein the polypeptide having protease activity comprises proteinase K, a serine protease such as trypsin, chymotrypsin, elastase, subtilisin, streptogrisin, thermitase, aqualysin, plasmin, cucumisin, or carboxypeptidase A, D, C, or Y; a cysteine protease such as papain, calpain, or clostripain; an acid protease such as pepsin, chymosin, or cathepsin; or a metalloprotease such as pronase, thermolysin, collagenase, dispase, an aminopeptidase or carboxypeptidase A, B, E / H, M, T, or U. or an enzymatically active mutant or variant thereof.

[0298] In one aspect the disclosure further provides a method; wherein the method further optionally comprises a polypeptide having deoxyribonuclease activity. In one aspect the disclosure further provides a method wherein the polypeptide having deoxyribonuclease activity comprises DNase I, Nuclease BAL-31, exonuclease I, exonuclease III, Lambda exonuclease, CviKI-1 endonuclease, McrBC endonuclease, or an enzymatically active mutant or variant thereof

[0299] In one aspect the disclosure further provides a method; wherein the method further; optionally comprises admixing the lysate with a stop mixture at substantially the same temperature as the contacting step to form a stopped mixture, wherein the stop mixture comprises: a cation chelator effective to inactivate the polypeptide having deoxyribonuclease activity, and an inhibitor of the polypeptide having protease activity. In one aspect the disclosure further provides a method; wherein the inhibitor of proteinase K comprises methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone, carbobenzoxy-Ala-Ala-COCH2CI, carbobenzoxy-Ala-Ala-Phe-COCH2CI or carbobenzoxy-Phe-Pro-Arg-COCH2CI or phenylmethylsulfonyl fluoride (PMSF). In one aspect the disclosure further provides a method; wherein the inhibitor of protease comprises leupeptin as an inhibitor for serine and cysteine proteases such as plasmin, trypsin, papain, kallikrein and cathepsin B; or 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF) as an inhibitorfor serine proteases such as chymotrypsin, kallikrein, plasmin, thrombin, and trypsin; or aprotinin as an inhibitor of serine proteases such as trypsin, chymotrypsin, plasmin and kallikrein; or benzamidine as an inhibitor of trypsin; N-acetyl eglin-C as an inhibitor of chymotrypsin, subtilisin, leukocyte elastase and cathepsin G; or antipain or plasmin for inhibition of a serine or cysteine such as papain and trypsin. In one aspect the disclosure further provides a method; wherein the cation chelator effective to inactivate the polypeptide having deoxyribonuclease comprises a calcium chelator such as EGTA or EDTA, or cation exchange beads such as sulfopropyl-functionalized cross-linked agarose cation-exchange beads (SP SEPHAROSE™ beads; GE Healthcare), 1,10-phenanthroline, tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), diethylenetriaminepentaacetic acid (DTPA), or a combination thereof.

[0300] In one aspect the disclosure further provides a method of preparing endotoxin-free nucleic acid from a sample comprising a microorganism, wherein the microorganism comprises the nucleic acid, the method comprising: (a) providing a sample comprising the microorganism; (b) lysing the microorganism in the presence of a buffer comprising that has a concentration of surfactant according to Formula I effective to lyse the microorganism. In one aspect the disclosure further provides a method wherein the buffer further comprises a polymyxin. In one aspect the disclosure further provides a method, wherein the nucleic acid is a plasmid DNA (pDNA).

[0301] In one aspect the disclosure further provides the aforementioned methods, wherein the composition comprises a polar protic solvent selected from a C1-C5 alcohol, C2-C6 polyol, polyethylene glycol, water, and mixtures thereof. In one aspect the disclosure further provides the aforementioned methods, wherein the polar protic solvent is selected from ethanol, isopropyl alcohol, 2-methyl-1,3-propanediol (MPD), isoamyl alcohol, water, and mixtures thereof. In one aspect the disclosure further provides the aforementioned methods, wherein the polar protic solvent is a polyethylene glycol (PEG) selected from the group consisting of PEG 1500, PEG8000 PEG6000, PEG2000, PEG1000, PEG600, and mixtures thereof.

[0302] In one aspect the disclosure further provides the aforementioned methods, wherein the composition comprises an inorganic salt selected from an alkali or alkaline earth metal salt. In one aspect the disclosure further provides the aforementioned methods, wherein the alkali or alkaline earth metal salt is selected from the group consisting of LiCI, NaCI, KCI, MgCI2, BaCh, CaCh, and corresponding citrates, acetates, or any combination thereof.

[0303] In one aspect the disclosure further provides the aforementioned methods, wherein the composition comprises a buffering agent selected from the group consisting of N-2-acetamido-2-aminoethanesulfonic acid (ACES), N-2-acetamido-2-iminodiacetic acid (ADA), 3-1,1-dimethyl-2-hydroxyethylamino-2-hydroxy propanesulfonic acid (AMPSO), N,N-bis2-hydroxyethyl-2-aminoethanesulfonic acid (BES), 4-cyclohexylamino-1 -butane sulfonic acid (CABS), 3-cyclohexylamino-1 -propane sulfonic acid (CAPS), 3-cyclohexylamino-2-hydroxy-1-propane sulfonic acid (CAPSO), 2-N-cyclohexylaminoethanesulfonic acid (CHES), 3-N,N-bis-2-hydroxyethylamino-2-hydroxypropanesulfonic acid (DIPSO), N-2-hydroxyethylpiperazine-N-3-propanesulfonic acid (EPPS or HEPPS), N-2-hydroxyethylpiperazine-N 4-butanesulfonic acid (HEPBS), 4-N-morpholinobutanesulfonic acid (MOBS), 3-N-morpholino-2-hydroxypropanesulfonic acid (MOPSO), bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (Bis-Tris), hydroxyethylpiperazine ethane sulfonic acid (HEPES), morpholinoethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), [tris(hydroxymethyl)methylamino] propanesulfonic acid (TAPS), N-trishydroxymethyl-methyl-4-aminobutanesulfonic acid (TABS), N-trishydroxymethyl-methyl-3-aminopropanesulfonic acid (TAPS), 3-N-trishydroxymethyl-methylamino-2-hydroxypropanesulfonic acid (TAPSO), N-trishydroxymethyl-methyl-2-aminoethanesulfonic acid (TES), N-(2-acetamido)iminodiacetic acid (ADA), Piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCI), N-trishydroxymethylmethylglycine (TRICINE), or any combination thereof.

[0304] In one aspect the disclosure further provides the aforementioned methods, wherein the composition optionally comprises a chaotropic agent selected from the group consisting of lithium perchlorate, lithium acetate, sodium dodecyl sulfate, thiourea, urea, guanidinium chloride, guanidinium thiocyanate, and a combination thereof.

[0305] In one aspect the disclosure provides the aforementioned methods, wherein the composition is having a surfactant present at a concentration of 0.001 % to 25% v / v. In one aspect the disclosure provides the aforementioned methods, wherein the composition is having a surfactant present at a concentration of 0.05% to 25% v / v. In one aspect the disclosure provides the aforementioned methods, wherein the composition is having a surfactant present at a concentration of 0.5% to 15% v / v. In one aspect the disclosure provides the aforementioned methods, wherein the composition is having a surfactant present at a concentration of 3.5% to 15% v / v. In one aspect the disclosure provides the aforementioned methods, wherein the composition is having a surfactant present at a concentration of 1.0% to 10% v / v.

[0306] In one aspect the disclosure provides the aforementioned methods, wherein the inorganic salt is present at a concentration of 0.25 mM to 1000 mM.

[0307] In one aspect the disclosure provides the aforementioned methods, wherein the buffering agent is present at a concentration of 5 mM to 500 mM.

[0308] In one aspect the disclosure provides the aforementioned methods, wherein the chaotropic agent is present at a concentration of 0.5 M to 8 M.

[0309] In one aspect the disclosure provides the aforementioned methods, wherein the polar protic solvent is present at a concentration of 5% to 50% v / v.

[0310] In one aspect the disclosure provides the aforementioned methods, wherein the nucleic acid is DNA or RNA. In one aspect the disclosure provides the aforementioned methods, wherein the DNA is one or more of synthetic DNA, plasmid DNA (pDNA), genomic DNA (gDNA), viral DNA (e.g. dsDNA or ssDNA), cDNA, cfDNA, gDNA or ctDNA. In one aspect the disclosure provides the aforementioned methods, wherein the RNA is mRNA, siRNA, shRNA, self-replicating RNA (srRNA), an o-RNA, self-amplifying RNA, stRNA, trRNA, crRNA, sgRNA, RNAi molecule, an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a Dicer-substrate RNA (dsRNA), a small hairpin RNA (shRNA), or any combination thereof.

[0311] In one aspect the disclosure provides the aforementioned methods, wherein the sample comprises a biological sample, clinical sample, environmental sample, or an enzymatic reaction mixture. In one aspect the disclosure provides the aforementioned methods; wherein the biological sample is a pre-treated or untreated biological sample.

[0312] In one aspect the disclosure provides the aforementioned methods; wherein the biological sample is blood stain, cord blood, blood components (e.g., platelet concentrates), blood cultures, peripheral blood mononuclear cells, peripheral blood leukocytes, plasma lysates, leukocyte lysates, buffy coat leukocytes, serum, plasma, saliva, saliva stain, buccal cells, buccal swab, semen, semen stain, urine, fecal matter, fecal stain, cigarette butt, chewing gum, formalin-fixed paraffin-embedded (FFPE) sample, biopsy (e.g. tumor biopsy) sample, bone marrow or other tissue sample, cell lysate, bacterial culture, yeast culture, sputum, tear, throat swabs, oral rinses, nasopharyngeal swabs, nasopharyngeal aspirates, exhalates, nasal swabs, nasal washes, mucus, bronchial aspirations, bronchoalveolar lavage fluid, pleural fluid, endotracheal aspirates, cerebrospinal fluid, anal swabs, rectal swabs, vaginal swabs, endocervical swabs, vitreous fluid, amniotic fluid, breast milk, exosomes, circulating tumor cells, tissue lysates, bacterial lysates, yeast lysates, or exosomes.

[0313] In one aspect the disclosure provides the aforementioned methods, wherein the cell culture has been cultured on extracellular matrix.

[0314] In one aspect the disclosure provides the aforementioned methods, wherein the cell culture comprises primary cells.

[0315] In one aspect the disclosure provides the aforementioned methods, wherein the primary cells comprise primary hepatocytes.

[0316] In one aspect the disclosure provides the aforementioned methods, wherein the cells are selected from the group consisting of Kupffer cells, PBMCs, THP-1 cells, HL60 cells, 3D cell cultures, or any combination thereof.

[0317] In one aspect the disclosure provides the aforementioned methods; wherein the solid support may comprise one or more of particles, resin, beads, a filter, cartridge, a column, an array, a membrane, a chip, a disc, or a slide. In one aspect the disclosure provides the aforementioned methods; wherein the solid support comprises optionally monodisperse beads; wherein the beads are magnetic. In one aspect the disclosure provides the aforementioned methods; wherein the solid support is a paramagnetic bead or a superparamagnetic bead.

[0318] In one aspect the disclosure provides the aforementioned methods, wherein the isolated nucleic acids or polypeptides ares further subjected to one or more additional processes, optionally selected from detection, quantification, cloning, restriction, nucleic acid synthesis and / or assembly, analysis, epigenetic analysis, sequencing, amplification, study, transfection, hybridization, cDNA synthesis, size separation, chromatography and mass spectrometry, pharmaceutical or therapeutic formulation and genome editing. In one aspect the disclosure provides the aforementioned methods, wherein the amplification comprises PCR, qPCR, reverse transcription, in vitro transcription, or isothermal amplification. In one aspect the disclosure provides the aforementioned methods, wherein the isothermal amplification comprises loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), helicase-dependent amplification (HDA), multiple displacement amplification (MDA), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), multiple cross displacement amplification (MCDA), signal-mediated amplification of RNA technology (SMART), recombinase-polymerase amplification (RPA) or nucleic acid sequence-based amplification (NASBA). In one aspect the disclosure provides the aforementioned methods, wherein the isolated DNA is further subjected to one or more additional processes, optionally selected from a microarray, qPCR, dPCR and next generation sequencing (NGS).

[0319] In one aspect the disclosure provides the aforementioned methods, wherein the isolated RNA is further subjected to one or more additional processes, optionally selected from detection, cloning, restriction, nucleic acid synthesis and / or assembly, analysis, epigenetic analysis, sequencing, amplification, study, transfection, hybridization, cDNA synthesis, size separation, chromatography and mass spectrometry, pharmaceutical or therapeutic formulation and genome editing. In one aspect the disclosure provides the aforementioned methods, wherein the amplification comprises PCR, qPCR, reverse transcription, in vitro transcription, or isothermal amplification. In one aspect the disclosure provides the aforementioned methods, wherein the isothermal amplification comprises loop- mediated isothermal amplification (LAMP), rolling circle amplification (RCA), helicase-dependent amplification (HDA), multiple displacement amplification (MDA), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), multiple cross displacement amplification (MCDA), signal-mediated amplification of RNA technology (SMART), recombinase-polymerase amplification (RPA) or nucleic acid sequencebased amplification (NASBA). In one aspect the disclosure provides the aforementioned methods, wherein the isolated RNA is further subjected to one or more additional processes, optionally selected from a microarray, qPCR, dPCR and next generation sequencing (NGS).

[0320] In one aspect the disclosure provides the use of an aforementioned composition for lysing a cell, vesicle, or virus in a sample.

[0321] In one aspect the disclosure provides the use of an aforementioned composition for isolation of two or more polypeptides, vesicles, or nucleic acids from a sample containing polypeptides, vesicles or nucleic acids.

[0322] In one aspect the disclosure provides the use of an aforementioned composition for isolation of polypeptides and nucleic acids from a sample containing polypeptides and nucleic acids.

[0323] In one aspect the disclosure provides the use of an aforementioned composition for isolation of nucleic acids from a sample containing nucleic acids.

[0324] In one aspect the disclosure provides the use of an aforementioned composition for isolation of polypeptides from a sample containing polypeptides.

[0325] In one aspect the disclosure provides the use of an aforementioned composition comprising: (a) a surfactant, wherein the surfactant is a compound selected from the group consisting of compounds 1-54; (b) at least one polar protic solvent; (c) optionally, one or more inorganic salt; (d) optionally one or more buffering agent; and (e) optionally one or more chaotropicagents; for isolation of polypeptides and nucleic acids from a sample containing polypeptides and nucleic acids.

[0326] In one aspect the disclosure provides the use of an aforementioned composition comprising: (a) a surfactant, wherein the surfactant is a compound selected from the group consisting of compounds 1-54; (b) at least one polar protic solvent; (c) optionally, one or more inorganic salt; (d) optionally one or more buffering agent; and (e) optionally one or more chaotropic agents; for isolation of nucleic acids from a sample containing nucleic acids.

[0327] In one aspect the disclosure provides the use of an aforementioned composition comprising: (a) a surfactant, wherein the surfactant is a compound selected from the group consisting of compounds 1-54; (b) at least one polar protic solvent; (c) optionally, one or more inorganic salt; (d) optionally one or more buffering agent; and (e) optionally one or more chaotropic agents; for isolation of polypeptides from a sample containing polypeptides.

[0328] In one aspect the disclosure provides the use of an aforementioned composition, wherein the nucleic acid is DNA or RNA. In one aspect the disclosure provides the use, wherein the DNA is one or more of synthetic DNA, plasmid DNA (pDNA), genomic DNA (gDNA), viral DNA (e.g. dsDNA or ssDNA), cDNA, cfDNA, gDNA or ctDNA.

[0329] In one aspect the disclosure provides the use, wherein the RNA is mRNA, siRNA, shRNA, self-replicating RNA (srRNA), an o-RNA, self-amplifying RNA, stRNA, trRNA, crRNA, sgRNA, RNAi molecule, an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a Dicersubstrate RNA (dsRNA), a small hairpin RNA (shRNA), or any combination thereof.

[0330] In one aspect the disclosure provides a kit, comprising an aforementioned composition.

[0331] In one aspect the disclosure provides a kit, further comprising a polypeptide having protease activity.

[0332] In one aspect the disclosure provides a kit, further comprising a polypeptide having deoxyribonuclease activity.

[0333] In one aspect the disclosure provides a kit, further comprising an RNase inhibitor.

[0334] In one aspect the disclosure provides a kit, wherein the RNAse inhibitor is an anionic oligomer.Examples

[0335] A representative procedure for making compounds according to the present disclosure is described below in Examples 1-4. One or more procedures according to Examples 1-4 can be used to prepare other compounds disclosed herein by exposing a suitable starting material to different aliphatic halides and then performing similar conversion steps from the aliphatic-substituted intermediate to the final product (that is, similar conversion steps as outlined in Examples 2-4). Other examples can utilize other synthetic methods described herein.Example 1

[0336] Syringaldehyde (100.16 g, 550 mmol) was dissolved in anhydrous dimethylformamide (400 ml_) in a three neck 2000 mL RB flask. The mixture was stirred via mechanical stirrer at a rate of 100 RPM. Potassium carbonate (151.7 g, 1100 mmol) was added in one portion, and the reaction was heated to 110°C for 30 minutes. The temperature was then set to 80°C during which 1-Bromononane (209.8 mL, 1100 mmol) was added dropwise via dropper funnel over the course of 1 hr while the reaction was cooling to 80°C. Once the addition was complete, the reaction was stirred at 80°C for 3 hr at which point full consumption of the starting material was observed via TLC (50% EtOAc:Hep). The reaction was then cooled to room temperature before excess potassium carbonate was filtered off. The filtrate was collected and concentrated to dryness. The residue was then dissolved in EtOAc (400 mL), and the organic layer was washed with 1 M HCI (1 x 400 mL), followed by a wash with brine (1 x 400 mL). The organic layer was then dried with anhydrous magnesium sulfate before being concentrated to dryness to yield the crude product as a brown oil. The crude material was placed at -20°C for 12 hr at which point the material solidified. Heptane (200 mL) was added to the crude product and stirred vigorously for 30 min at RT. The material was then filtered and the solid was washed with Heptane (1 x 100 mL). The solid product was collected as a white crystallize solid (92.2 g, 54.4%).Example 2

[0337] 3,5-dimethoxy-4-(nonyloxy)benzaldehyde (139.85 g, 453 mmol) was dissolved in 200 proof ethanol (750 mL) in a three neck 2000 mL RB flask and stirred at 300 RPM. The mixture was cooled to 4°C in an ice bath. Sodium borohydride (39.77 g, 906 mmol) was added portion wise over the course of 5 min. The reaction was then stirred at 4°C for 1.5 hr until full consumption of the starting material was observed via TLC (50% EtOAc:Hep). Once complete, the reaction was concentrated to dryness, and the residue was dissolved in EtOAc (400 mL). The organic layer was washed successively with water (1 x 400 mL), and brine (1 x 400 mL), before being dried with anhydrous magnesium sulfate. The organic layer was then concentrated to dryness to yield the desired product as a yellow oil (133.48 g, 94.7%).Example 3

[0338] (3,5-dimethoxy-4-(nonyloxy)phenyl)methanol (34.01 g, 121 mmol) was dissolved in anhydrous dichloromethane (400 mL) in an oven dried single neck 1 L RB flask and stirred at 200 RPM. The mixture was cooled to 4°C in an ice bath. Triethylamine (30.3 mL, 242 mmol) was added dropwise via dropper funnel over the course of 10 minutes. The reaction was then stirred for 5 minutes before a mixture of methanesulfonyl chloride (12.7 mL, 181.5 mmol) in anhydrous dichloromethane (30 mL) was added dropwise via dropper funnel over the course of 15 minutes. The reaction was then allowed to warm to room temperature and stirred for 18 hr at which point full consumption of the starting material was observed via TLC (50% EtOAc: Hep). The reaction was then transferred to a separatory funnel and washed with brine (1 x 400 mL). The organic layer was dried with anhydrous magnesium sulfate before being concentrated to dryness to yield the crude product as a brown semisolid. The crude product was carried forward to the next reaction without purification. (44.95 g, 95.4%).Example 4

[0339] Polyethylene glycol) methyl ether average molecular weight 550 (24.4 g, 44.4 mmol) was dissolved in anhydrous tetrahydrofuran (112 ml_) in an oven dried 500 ml_ single neck RB flask and stirred at a rate of 250 RPM. Potassium tert-butoxide (7.5 g, 44.4 mmol) was added portion wise over the course of 1 minute. The mixture was then cooled to 4°C in an ice bath. 3,5-dimethoxy-4-(nonyloxy)benzyl methanesulfonate (11.5 g, 29.6 mmol) was dissolved in anhydrous tetra hydrofuran (42 ml_) and added dropwise to the reaction via dropper funnel over the course of 10 minutes. The reaction was then warmed to room temperature for 18 hr at which point full consumption of the starting material was observed via TLC (50% EtOAc:Hep). The reaction was then concentrated to dryness and the residue was dissolved in EtOAc (200 ml_). The organic layer was then washed successively with water (1 x 200 mL) and brine (1 x 200 ml_). The organic layer was dried with anhydrous magnesium sulfate before being concentrated to dryness. The crude product was then dissolved in a minimal amount of methanol before being loaded onto a Biotage Star silica column without the use of a precolumn before being separated by Biotage: MeOH:DCM 0-0% (2 CV), 0-15% (18 CV). The fractions containing the desired product, compound 2 were collected to yield pure product as a yellow oil (16.9 g, 79.0%). The structure of compound 2 was confirmed by1H NMR (400 MHz, CDCI3) 36.395 (s, 2H), 4.327 (s, 2H), 3.783-3.741 (t, J = 6.88 Hz, 2H), 3.671 (s, 6H), 3.501-3.469 (m, 22H), 3.211 (s, 3H), 1.608-1.534 (m, 2H), 1.299-1.235 (m, 2H), 1.170-1.096 (m, 12H), 0.727-0.688 (m, 3H).Example 4ASynthesis of Compound 1Step 1: Synthesis of Compound 1 (SY-2 or SY-No-dPEG9-OMe)

[0340] Starting with compounds 3,5-dimethoxy-4-(nonyloxy)benzyl methanesulfonate and 1 A, compound 1 was synthesized using the procedure described in Example 4. The structure was confirmed by1H NMR (400 MHz, CDCI3) 56.496 (s, 2H), 4.419 (s, 2H), 3.879-3.844 (t, J = 6.9 Hz, 2H), 3.765 (s, 6H), 3.618-3.561 (m, 22H), 3.307 (s, 6H), 1.700-1.629 (m, 3H), 1.535-1.465 (m, 2H), 1.379-1.330 (m, 4H), 1.264-1.186 (m, 12H), 0.822-0.788 (m, 6H).Example 5

[0341] One or more procedures according to Examples 1-4 were used to prepare other compounds disclosed herein by exposing a suitable starting material to different aliphatic halides and then performing similar conversion steps from the aliphatic-substituted intermediate to the final product (that is, similar conversion steps as outlined in Examples 2-4).Compound 3 (SY-No-PEG75Compound 3 (SY-207) (PEG750)

[0342] The structure was confirmed by1H NMR (400 MHz, CDCI3) 66.579 (s, 2H), 4.503 (s, 2H), 3.851 (m, 2H), 3.788 (s, 6H), 3.657 (m, 103H), 3.466 (s, 3H), 1.451 (m, 2H), 1.284 (m, 12H), 0.891 (m, 3H).Compound 4 (SY-Oc-dCompound 4

[0343] The structure was confirmed by1H NMR (400 MHz, CDCI3) 06.457 (s, 2H), 4.377 (s, 2H), 3.835-3.796 (t, J = 6.87 Hz, 2H), 3.721 (s, 6H), 3.580-3.504 (m, 22H), 3.259 (s, 3H), 1.657-1.585 (m, 2H), 1.355-1.279 (m, 2H), 1.235-1.147 (m, 12H), 0.779-0.735 (m, 6H).Compound 5 (SY-Pe-dPEG9-OMe):

[0345] The structure was confirmed by1H NMR (400 MHz, CDCI3) 66.403 (s, 2H), 4.324 (s, 2H), 3.811-3.778 (t, J = 6.87 Hz, 2H), 3.678 (s, 6H), 3.495-3.460 (m, 36H), 3.387-3.362 (m, 2H), 3.206 (s, 3H), 1.755-1.654 (m, J = 13.46 Hz, 1H), 1.498-1.446 (q, J = 6.88 Hz, 2H), 0.782-0.762 (d, J = 6.66 Hz, 6H).Compound 7 (SY-DMB-dPEG9-OMe):

[0346] The structure was confirmed by1H NMR (400 MHz, CDCI3) 66.441 (s, 2H), 4.358 (s, 2H), 3.886-3.842 (t, J = 8.21 Hz, 2H), 3.706 (s, 6H), 3.568-3.490 (m, 22H), 3.242 (s, 3H), 1.633-1.582 (t, J = 7.25 Hz, 2H), 0.812 (s, 9H).Compound 8 (SY-2MP-dPEG9-OMe):

[0348] The structure was confirmed by1H NMR (400 MHz, CDCI3) 06.496 (s, 2H), 4.418 (s, 2H), 3.884-3.845 (t, J = 6.87 Hz, 2H), 3.764 (s, 6H), 3.598-3.543 (m, 22H), 1.705-1.631 (m, 4H), 1.402-1.320 (m, 4H), 1.269-1.197 (m, 12H), 0.821-0.786 (m, 6H).Compound 10 (SY-No-dSPEG9-OMe):

[0349] The structure was confirmed by1H NMR (400 MHz, CDCI3) 66.534 (s, 2H), 3.936 (t, 2H), 3.902 (s, 6H), 3.597 (m, 25H), 3.549 (m, 2H), 3.368 (s, 3H), 2.649 (m, 2H), 1.730 (m, 2H), 1.403 (m, 2H), 1.277 (m, 10H), 0.889 (m, 3H).Example 6: Human gDNA Extraction from Blood

[0350] The following protocol was used to evaluate the efficacy of various compounds described in the present disclosure for extraction of human gDNA from whole blood using a commercially available genomic DNA extraction kit. The protocol evaluates the performance of the compounds of the present disclosure - as surfactants compared with conventional surfactants including EcoSurf™ EH-9 and Triton™ X-100.

[0351] Human whole blood samples were processed following the manufacturer’s kit user guide. The test surfactants were swapped into the kit Lysis / Binding Buffer at 2%, 5%, 10%, and 20% (v / v) final concentration. EcoSurf™ EH-9 and Triton™ X-100 were used as reference controls, and a no-surfactant buffer condition was included as a negative control. An automated Proteinase K digestion was performed on the blood samples to ensure complete digestion ofproteins prior to mixing the blood samples with the prepared Lysis / Binding Buffers plus magnetic DNA binding beads. The standard magnetic bead-based extraction steps and subsequent washes, elutions, and magnetic separations were performed on an automated sample preparation instrument, following the kit manufacturer’s guidelines and instrument script. Purified gDNA was eluted into elution buffer.

[0352] DNA yield and purity were determined spectrophotometrically. The absorbance ratios A260 / A280 and A260 / A230 were calculated as indicators of protein and organic compound contamination, respectively. DNA integrity was assessed using agarose gel electrophoresis and automated electropherogram analysis, with DNA Integrity Number (DIN) values reported. Extracted DNA was further evaluated by qPCR amplification of the GAPDH gene to assess downstream assay functionality.

[0353] FIG. 1 shows the comparative genomic DNA (gDNA) yield and purity obtained from 100 pL whole blood using compounds 1 (SY-2), 3 (SY-207), and 2 (SY-210) as the surfactant incorporated into a commercially available lysis / binding buffer at 10% and 20% concentrations, compared with EcoSurf™ EH-9 and Triton™ X-100 controls and a no-surfactant condition. The figure shows total DNA yield (pg) (black bars), A260 / A280 (checkered bars), and A260 / A230 (striped bars) absorbance ratios. These results demonstrate that compounds of this disclosure used as surfactants produced DNA yields and purity ratios comparable to or greater than those obtained with conventional surfactants, indicating efficient lysis and extraction of high-quality gDNA from whole blood.

[0354] FIG. 2 shows the comparative genomic DNA (gDNA) yield and purity obtained from 100 pL whole blood using compounds 1 (SY-2), 3 (SY-207), and 2 (SY-210) as the surfactants incorporated into a commercially available lysis / binding buffer at 2% and 5% concentrations, compared with EcoSurf™ EH-9 and Triton™ X-100 controls and a no-surfactant condition. The figure shows total DNA yield (pg) (black bars), A260 / A280 (checkered bars), and A260 / A230 (striped bars) absorbance ratios, respectively. The results demonstrate that gDNA yield and purity improved at 5% surfactant concentration relative to 2%, with compounds of this disclosure used as surfactants achieving comparable or greater DNA yields than conventional surfactants, indicating efficient extraction of amplifiable, high-quality DNA at optimized concentrations.

[0355] FIG. 3 shows the comparative genomic DNA (gDNA) yield and purity obtained from 200 pL whole blood using compounds of this disclosure as surfactants containing polyethylene glycol) linkers (Compound 7 (SY-DMB-dPEG9-OMe), Compound 6 (SY-iPe-dPEG9-OMe),Compound 7 (SY-No-dPEG9-OMe), Compound 4 (SY-Oc-dPEG9-OMe), and Compound 5 (SY-Pe-dPEG9-OMe)) which were incorporated into a commercially available lysis / binding buffer at 20% concentration. EcoSurf™ EH-9 and Triton™ X-100 served as positive controls, and a nosurfactant buffer condition was included as a negative control. The figure shows total DNA yield (pg) (black bars), A260 / A280 (checkered bars), and A260 / A230 (striped bars) absorbance ratios, respectively. The results demonstrated that compounds of this disclosure as surfactants produced high DNA yields with purity ratios comparable to or exceeding conventional surfactants, confirming efficient extraction of high-quality, contaminant-free genomic DNA.

[0356] FIG. 4 shows the quantitative PCR (qPCR) performance of genomic DNA (gDNA) extracted from 200 pL whole blood using compounds of this disclosure as surfactants containing polyethylene glycol) linkers (Compound 7 (SY-DMB-dPEG9-OMe), Compound 6 (SY-iPe-dPEG9-OMe), Compound 1 (SY-No-dPEG9-OMe), Compound 4 (SY-Oc-dPEG9-OMe), and Compound 5 (SY-Pe-dPEG9-OMe)) incorporated into a commercially available lysis / binding buffer at 20% concentration. EcoSurf™ EH-9 and Triton™ X-100 served as positive controls, and a no-surfactant condition was included as a negative control. The black bars represent the quantification cycle (Cq) values for amplification of the GAPDH gene. Lower Cq values correspond to higher amplifiable DNA yield. The results demonstrate that all compounds of this disclosure as surfactants produced consistent and efficient amplification, comparable to EcoSurf™ EH-9 and Triton™ X-100, whereas the no-surfactant condition exhibited a significantly delayed amplification (Cq = 25.7), confirming that the extracted DNA was of high purity and suitable for downstream molecular applicationsExample 7: Preparation of Samples for Nucleic Acid Analysis

[0357] ] Studies were conducted to assess the effectiveness of different non-ionic surfactants for the preparation of lysates for gene expression analysis by RT-qPCR. A lysis solution was prepared that consisted of 10 mM Tris pH 7.5, 5 mM MgCl2, 50 pg / mL PVSA, and 0.1% of non-ionic surfactant. The non-ionic surfactant tested included compounds 7 (SY-DMB-dPEG9-OMe), 5 (SY-Pe-dPEG9-OMe), 6 (SY-iPe-dPEG9-OMe), 4 (SY-Oc-dPEG9-OMe), and 2 (SY-No-dPEG9-OMe). The non-ionic surfactants were compared to controls: Triton X-100, Tergitol 15-S-9, no-surfactant sample, and water. 50 pL of each lysis solution and control was used to lyse 10,000 HeLa or HuH7 cells that were suspended in 5 pL of PBS. After applying thelysis buffer to the cells, the cell lysate was mixed by pipette 5 times and then incubated at room temperature for 5 minutes.

[0358] As a control for workflow inhibition, purified RNA from HeLa cells was suspended in 5 pL of PBS and the lysis buffer with 0.1% and 1% surfactant was applied to the purified RNA. The purified RNA lysate was mixed by pipette 5 times and then incubated at room temperature for 5 minutes. 5pL of cell-lysate or purified RNA lysate was used as a template in a 20 pL reaction for reverse transcription (RT) using the Invitrogen™ SuperScript™ IV VILO™ Master Mix. 2 pL cDNA from the RT step in a 10 pl reaction was used with Applied Biosystems™ TaqMan™ Fast Advanced Master Mix for qPCR and TaqMan Gene Expression Assays targeting the ACTB and GAPDH (5’ FAM or VIC-labeled TaqMan probe) genes. Reactions were run on an Applied Biosystems QuantStudio™ 5, 6 or 7 Flex Real-Time PCR Instrument.

[0359] For cell lysis, ACtwas calculated as Ct values obtained with 0.1% and 1% Tergitol 15-S-9 compared to Ctvalues obtained at 0.1% and 1% test compounds respectively. For RT-qPCR inhibition, ACtwas calculated as Ct values obtained with water compared to Ct values obtained at 0.1% and 1% test compounds, using the purified RNA.

[0360] Comparison of the ACt values obtained from RT-qPCR using each lysis buffer revealed that compounds 2 and 4 produced ACtcomparable to control at 0.1 % concentration and Compounds 6, 2, 4 and 5 compounds show ACt comparable to control at 1% concentration (FIG.5).

[0361] Compound 4 shows inhibition of the RT-qPCR workflow at 1% concentration (FIG.6). These results demonstrate that lysis buffers with compounds of the present disclosure as surfactants are effective for cell lysis and compatible with direct input into RT-qPCR at less than 1% concentration (FIGS. 5-6).Example 8: Evaluation of Surfactants for dPCR (Direct PCR) Compatibility

[0362] The following experiment was conducted to assess the compatibility of a syringic acid surfactant described in the present disclosure with a direct PCR workflow. The protocol evaluates the performance of the compounds disclosed herein as surfactant compared with conventional surfactants Triton X-100, EcoSurf™ EH-9 and a no surfactant water control.DNA direct PCR reactions were prepared by combining the following components in a total reaction volume of 10 pL:• 2.5 pL of a commercially available 1-step multiplex PCR master mix,• 0.5 pL of Xeno DNA primer / probe mix,• 0.5 pL of Xeno DNA template at 5,000 copies per reaction, and• 6.5 pL of either nuclease-free water (control) or surfactant working solutions. RNA direct PCR reactions were assembled in an identical manner, substituting the DNA reagents with Xeno RNA template (5,000 copies) and Xeno RNA primer / probe mix.

[0363] The compound 3 (SY-207) evaluated is tested at 0.1% and 0.5% surfactant concentration into the PCR reaction. Control reactions included Triton X-100 and EcoSurf EH-9, at the same concentrations, along with a no-surfactant water control. To assess surfactant interference with fluorescence background or nonspecific signal, no-template controls were prepared by combining the PCR master mix with 7.5 pL of each surfactant solution. All reactions were processed on a real-time qPCR instrument equipped with a 384-well heat block. Fluorescence data for the FAM channel (Xeno DNA target) and VIC channel (Xeno RNA target) were collected during amplification. Multicomponent analysis was used to monitor background fluorescence across PCR cycles for each surfactant condition. For template-containing reactions, amplification performance was assessed by determining ACt relative to the no-surfactant water control. ACt values were calculated separately for DNA (FAM channel) and RNA (VIC channel) reactions. Higher ACt values correspond to reduced amplification efficiency or increased reaction inhibition.

[0364] FIG. 15. Shows effect of surfactant type and concentration on direct PCR amplification efficiency for DNA and RNA targets. ACt values relative to the no-surfactant water control are shown for DNA (FAM channel) and RNA (VIC channel) direct PCR reactions containing 5,000 copies of Xeno DNA or Xeno RNA, respectively. Surfactants were tested at 0.1% and 0.5% surfactant concentration into the PCR reaction. Higher ACt values indicate greater inhibition of amplification. Compound 3 (SY-207) demonstrated minimal inhibition and generated slightly negative ACt values at both concentrations, consistent with improved amplification efficiency relative to the water control. For RNA targets, ACt values remained within approximately ±1 cycle across all surfactants and concentrations, indicating minimal impact on RNA amplification performance.

[0365] FIG. 16. shows multicomponent no-template control (NTC) fluorescence profiles for the FAM channel in the presence of surfactants. FAM fluorescence is plotted across PCR cycles for reactions containing no DNA template and 0.1% or 0.5% surfactant. Triton X-100 and EcoSurf EH-9 generated elevated background fluorescence and characteristic early-cycle peaks,indicating interaction with fluorescent probes or amplification chemistry. In contrast, Compound 3 (SY-207) produced low, stable background fluorescence profiles comparable to the water control across all PCR cycles. These results demonstrate that Compound 3 (SY-207) minimizes nonspecific FAM-channel signal and is compatible with direct PCR detection chemistry.

[0366] FIG. 17. shows multicomponent no-template control (NTC) fluorescence profiles for the VIC channel in the presence of surfactants. VIC fluorescence is plotted across PCR cycles for reactions containing no RNA template and 0.1% and 0.5% surfactant. All surfactants produced modest increases in VIC fluorescence over the course of cycling; however, Triton X-100 exhibited the largest upward drift, while EcoSurf EH-9 showed intermediate elevation relative to the water control. In contrast, Compound 3 (SY-207) produced stable, low-amplitude fluorescence signals at both concentrations, comparable to the control condition. These results indicate that Compound 3 (SY-207) does not substantially interfere with VIC-channel probe detection and is suitable for direct PCR applications.Example 9: Protein extraction from multiple samples

[0367] The following protocol was used to evaluate the efficacy of various compounds described in the present disclosure for extraction of proteins using commercially available lysis buffers. The protocol evaluates the performance of the compounds of this disclosure as surfactants compared with conventional surfactants including Tergitol™ NP-40, Triton™ X-100, and EcoSurf™ EH-9.

[0368] Formulations of commercially available buffers were prepared by substituting compounds of this disclosure for the conventional surfactant in each buffer in an equal concentration. Buffers with conventional surfactants were used as controls, with no surfactant buffer used as negative control.

[0369] Samples were lysed using the prepared buffers and a commercially available protease and phosphatase inhibitor cocktail. Sample was incubated at room temperature for ten minutes and micro-centrifuged at 15,000XG for five minutes. Remove lysate and perform BCA Assay on lysate subtracting reading from buffer only to obtain protein concentration. These concentrations were used to run SDS-PAGE gels. After running the gels, each gel was washed in ddH2O twice for five minutes. One gel was evaluated for total protein by Coomassie staining of the SDS-PAGE gel. 80 ml of stain was added to the gel and the gel was incubated at RT for two hours and washed with ddH2O overnight. The gel was imaged on the iBright™ FL1500 Imaging12 / 22 / 25 TP391474W01System using visible protein setting and bands were quantitated for each lane using iBright™ Analysis Software and expressed as % of TX100 control. For determining extraction of specific proteins, proteins were transferred onto a nitrocellulose membrane using Trans-Blot® Turbo transfer system and the protein transfer efficiency was determined using Ponceau stain for each membrane. Specific proteins were detected using target specific primary antibodies followed by corresponding HRP conjugated secondary antibodies as noted below. Bands of interest, corresponding to the target proteins, were detected using SuperSignal™ West Dura Extended Duration Substrate.Extraction of protein from mammalian cells:

[0370] Variations of Pierce™ IP Lysis Buffer were prepared with 1% (w / v) compounds of this disclosure, Tergitol™ NP-40, or no surfactant. Samples prepared from frozen HCT116 cell pellets were lysed using the prepared buffers, according to the above protocol, and the lysates analyzed according to the above protocol. Extraction of specific proteins was determined according to the above protocol, using the following antibodies: cytoplasmic protein HSP90 (Thermo PN MA5-006; 1:2,000), nuclear protein HistoneH3 (CST PN 2650; 1:2,000), and membrane proteins EGFR (CST PN 4267; 1:500), Cav-1 (CST PN 3238; 1:1,000), ATP1A1 (Thermo PN MA3-928; 1:2,000), COXI (CST PN 2650; 1:1,000), goat anti-mouse HRP (PN 32430; 1:2,000), and goat anti-rabbit HRP (PN 32460, 1:2000).

[0371] FIG. 7 shows the Western blot results for lysates prepared using Pierce™ IP Lysis Buffer, formulated using 1% surfactants or no surfactant. Lysates were probed using HSP90 and histone H3 antibodies to examine extraction of specific proteins compared to 1% Tergitol™ NP-40 control. These results demonstrate that compounds of this disclosure as surfactants support efficient extraction of high yield, high quality proteins from mammalian cells.Extraction of protein from bacterial cells and yeast cells:

[0372] Variations of B-PER™ Complete Bacterial Protein Extraction Reagents (PN 89821) (“B-PER™ buffers”) were prepared with 2% (w / v) compounds of this disclosure, Triton™ X-100, EcoSurf™ EH-9, or no surfactant. Samples prepared from either frozen E. coli DH1a bacterial cell pellets or frozen yeast pellets were lysed using the prepared buffers, according to the above protocol, and the lysates analyzed according to the above protocol. Extraction of specific proteins was determined according to the above protocol, using the following antibodies: cytoplasmic protein HSP90 (Thermo PN MA5-006; 1:2,000), HSP70 (Thermo PN MA3-006;1:1,000), GAPDH (Thermo PN MA5-15738; 1:2,000), goat anti-mouse HRP (PN 32430; 1:2,000), and goat anti-rabbit HRP (PN 32460, 1:2000). The E. coli lysates results were further analyzed for extraction of proteins using E. coli total protein antibody (DAKO PN B0357; 1:2,000).

[0373] FIG. 8 shows the Western blot results for E. coli DH1a lysates prepared using B-PER™ buffers, formulated using 2% surfactants or no surfactant. Lysates were probed using HSP90 and GAPDH antibodies to examine extraction of specific proteins compared to 2% Triton™ X-100 control, 2% EcoSurf™ EH-9 control, or no surfactant control. These results demonstrate that compounds of this disclosure as surfactants support efficient extraction of high yield, high quality proteins from bacterial cells.

[0374] FIG. 9 shows the Western blot results for yeast lysates prepared using B-PER™ buffers, formulated using 2% surfactants or no surfactant. Lysates were probed using HSP70 and GAPDH antibodies to examine extraction of specific proteins compared to 2% Triton™ X-100 control. These results demonstrate that compounds of this disclosure as surfactants support efficient extraction of high yield, high quality proteins from yeast cells.Extraction of protein from tissue:

[0375] Variations of N-PER™ Neuronal Protein Extraction Reagents (PN 87792) (“N-PER™ buffers”) were prepared with 1% (w / v) compounds of this disclosure, Triton™ X-100, EcoSurf™ EH-9, or no surfactant. Samples of frozen mouse brains were lysed using the prepared buffers, according to the above protocol, and the lysates analyzed according to the above protocol. Extraction of specific proteins was determined according to the above protocol, using the following antibodies: synaptophysin (CST PN 5461; 1:1,000), IGFR (CST PN 3027; 1:1,000), Cav-1 (CST PN 3238; 1:1,000), ATP1A1 (Thermo PN MA3-928; 1:2,000), COXIV (CST PN 2650; 1:1,000) PSD95 (CST PN 2507; 1:1,000), goat anti-mouse HRP (PN 32430; 1:2,000), and goat anti-rabbit HRP (PN 32460, 1:2000).

[0376] FIG. 10 shows the Western blot results for lysates prepared using N-PER™ buffers, formulated using 1% surfactants or no surfactant. Lysates were probed using IGFR and synaptophysin antibodies to examine extraction of specific proteins compared to 1% Triton™ X-100 control. These results demonstrate that compounds of this disclosure as surfactants support efficient extraction of high yield, high quality proteins from tissue.Extraction of protein from mammalian cells:

[0377] Variations of RIPA Lysis and Extraction Buffer (PN 89900) (“RIPA buffer”), were prepared with 1% (w / v) compounds of this disclosure, Tergitol™ NP-40, EcoSurf™ EH-9, or no surfactant. Samples prepared from frozen HCT116 cell pellets were lysed using the prepared buffers, according to the above protocol, and the lysates analyzed according to the above protocol. Extraction of specific proteins was determined according to the above protocol, using the following antibodies: cytoplasmic protein HSP90 (Thermo PN MA5-006; 1:2,000), nuclear protein HistoneH3 (CST PN 2650; 1:2,000), and membrane proteins EGFR (CST PN 4267; 1:500), Cav-1 (CST PN 3238; 1:1,000), ATP1A1 (Thermo PN MA3-928; 1:2,000), COXIV (CST PN 2650; 1:1,000), goat anti-mouse HRP (PN 32430; 1:2,000), and goat anti-rabbit HRP (PN 32460, 1:2000).

[0378] FIG. 11 shows the Western blot results for lysates prepared using RIPA buffers, formulated using 1% surfactants or no surfactant. Lysates were probed using HSP90 and histone H3 antibodies to examine extraction of specific proteins compared to 1% Tergitol™ NP-40 control. These results demonstrate that compounds of this disclosure as surfactants support efficient extraction of high yield, high quality proteins from mammalian cells.Example 10: Antibody Denaturation Assay by Immunoprecipitation

[0379] The following protocol was used to test the effect of compounds described in the present disclosure for protein denaturation of an antibody. To bind protein A / G beads an antibody must be folded properly and not denatured.

[0380] 2X lysis buffers were diluted to yield a 1X lysis buffer with a surfactant concentration of 1% v / v compounds of this disclosure or control (Tergitol™ NP-40 or sodium dodecyl sulfate (SDS)). The 1X lysis buffer (250 ul) was mixed with AKT antibody (PN CST 2920, 2 ul) then incubated with rotation at RT for at least 2 hours. Protein A / G Magnetic Beads (PN 88802, 15 ul) were pre-washed twice with 1X lysis buffer (2x250 ul). To a fresh tube, 1X lysis buffer (250 ul) was added followed by the pre-washed magnetic beads (15 ul). The sample was then vortexed for 30 seconds, followed by bead collection on the magnet and removal of supernatant. 1X lysis buffer (250 ul) was added, and this step was repeated once more. Next, the antibody solution was added to the tube containing the pre-washed magnetic bead pellet and incubated with rotation at RT for 2 hours. The magnetic beads were pelleted using a magnetic separation rack and the supernatant (flow-through, FT) removed. The flow-through (10 ul) was mixed with 2X lysis buffer (10 ul) and all loaded on an SDS-PAGE gel. The magnetic bead pellet12 / 22 / 25 TP391474W01was then washed three times with 1X lysis buffer (3x250 ul), then twice with ddH2O (2x250 ul). The magnetic bead pellet was then resuspended with 1XSDS-PAGE sample buffer (PN NP0008, 100 ul), briefly vortexed, then briefly centrifuged to collect any material on the sides. The sample was heated to 95-100C for 10 minutes. The beads were pelleted using a magnetic separation rack, and the supernatant (elution, E) was transferred to a new tube. The elution (20 ul) was loaded on an SDS-PAGE gel. The SDS-PAGE gels were then processed for Western blotting as described above. The Western blot was analyzed for unbound antibody in the flow through which represents denatured antibody, as well as bound antibody in the elution which represents normal antibody, not unfolded denatured antibody, given that protein A / G beads only bind nondenatured antibody. IP-Western blot results for the compounds of this disclosure showed that the antibody was only detected in the elution fraction and indicates that compounds of this disclosure do not denature the antibody (FIG. 14).

[0381] FIG. 12 shows IP- Western blot results for antibody denaturation assay. AKT antibody was incubated in 1X lysis buffer containing 1% surfactant or a control (NP40 or SDS), then allowed to bind to Protein A / G magnetic beads. Flow through (FT) and elution (E) fractions were analyzed for presence of unbound and bound antibody, which represent denatured and normal antibody respectively.

[0382] The protein Gaussia contains five disulfide bonds and must be fold properly to exhibit activity that can be measured as light emission. Gaussia Glow Assay Buffer was prepared by replacing the surfactant with an equal concentration v / v of compounds of this disclosure or SDS and the pH adjusted to 7.2. Gaussia Glow Assay Buffer (180 ul) was transferred to a 1.5 ml microcentrifuge tube to which coelenterazine (1.8 ul) was added and mixed well to yield the Working Solution. In triplicate, Gaussia luciferase produced by IVT (PN 88890; following IB protocol) was diluted 1:4000, then 10 ul was added to a 96-well plate followed by the Working Solution (50 ul) and allowed to incubate at room temperature for 30 minutes. The plate was then measured for luminescence with an emission maximum of 485 nm. The amount of light output generated by the Gaussia luciferase protein represents functional activity of the protein. The assay results show that Gaussia luciferase remained active in the presence of compounds of this disclosure, in contrast to the no / low activity exhibited with SDS solutions.Example 11: Microarray assay using hybridization buffer formulations12 / 22 / 25 TP391474W01

[0383] The following protocol was used to evaluate the efficacy of various compounds described in the present disclosure in hybridization buffer formulations used for Microarray assays.

[0384] Individual hybridization buffers were prepared using Tween 20 (Polysorbate 20) and compounds of this disclosure, formulated with the same non-surfactant ingredients, where each buffer comprised the same functional concentration of surfactant in the individual hybridization buffer. Functional concentration was determined using each surfactant’s corresponding critical micelle concentration (CMC). The individual hybridization buffers were stored at either -20 °C, representing standard cold storage conditions, or at 37 °C, representing accelerated aging conditions, for a period of three weeks. 100 ng of genomic DNA was amplified, fragmented, precipitated, and resuspended. The resuspended DNA was combined with either the -20 °C or 37 °C stored hybridization buffer formulation to generate a hybridization-ready target. The hybridization-ready target was then denatured and hybridized onto the microarray for a period of 24 hours. Following hybridization, the arrays were washed, stained, and scanned according to the SwiftArrayStudio Microarray Solution workflow.

[0385] The impact of the surfactant composition in the hybridization solution was assessed by (1) visual inspection of array images in the AT and GC channels to determine the presence or absence of blemishes indicative of buffer instability, and (2) analysis of microarray quality control (QC) metrics, including DishQC, QC Call Rate, Signal to Background Ratio (AT channel), Signal to Background Ratio (GC channel), varscore (AT channel), and varscore (GC channel). QC analysis was performed on the hybridization buffers with compounds of this disclosure and with Tween 20. The results of image inspection and QC analysis demonstrates that the hybridization buffers containing compounds of this disclosure demonstrated performance comparable to those containing Tween 20.

[0386] FIG. 13 shows signals in the AT and GC channels for hybridization buffer formulations prepared with either control Tween-20 or compounds of this disclosure and stored at -20 °C (storage temperature) or subjected to accelerated aging at 37 °C.

[0387] FIG. 14 shows the quality control performance of microarray hybridization buffers incorporating a compound of this disclosure or Tween 20 and stored at -20°C or accelerated aged at 37°C. Each point represents the median performance of a given surfactant formulation calculated from replicate samples. Axes correspond to the same quality control (QC) parameter measured under both treatment conditions, enabling direct visual comparison relative to the12 / 22 / 25 TP391474W01diagonal x = y reference line (dotted). For DishQC and QC Call Rate parameters, horizontal and vertical dotted lines represent defined performance cutoffs (DishQC = 0.88, QC_CR = 98.5), which delineate acceptable assay quality thresholds. For signal intensity and variability parameters (Signal to Background Ratios and Varscores in AT and GC channels), axes are constrained to start at zero to reflect absolute measurement scales.Example 12: Extraction of mRNA from LNPs

[0388] In this example, use of compounds of the present disclosure for extraction of nucleic acids from LNPs was assessed. Compounds of this disclosure were prepared at 0.5%, 2%, and 10% (v / v) concentrations in 1* TE buffer. Triton™ X-100 was prepared at identical concentrations and used as a control, representative of current industry practice. For each reagent and concentration, total mRNA release and free mRNA were quantified using a RiboGreen™ fluorescence assay, and encapsulation efficiency was calculated as a function of these measurements and compared against the Triton™ X-100 based buffers.

[0389] A multiwell plate was prepared with 50 pL of each surfactant solution in separate wells. 50 pL of 1x TE buffer without surfactant was added to a well as a control for measurement of free mRNA (mRNA not encapsulated within the LNPs). 50 pL of the 3 ug / mL mRNA-LNP sample was added to each well. The mixtures were incubated at 37 °C for 10 minutes, after which 100 pL of RiboGreen reagent (1:100 dilution in 1* TE) was added. Relative fluorescence was measured at an excitation wavelength of 485 nm using a plate reader. Free mRNA was calculated based on well with no surfactant. Total mRNA content was calculated for each of the wells containing a surfactant.

[0390] Encapsulation efficiency was determined by comparing the detectable mRNA signal in intact LNP samples with the detectable mRNA signal obtained in wells treated with a surfactant, thereby distinguishing protected, encapsulated mRNA from unencapsulated mRNA present in the formulation. Encapsulation efficiency was calculated using the following:Table 4 shows comparison of compounds of this disclosure with Triton™ X-100 in mRNA-LNP encapsulation efficiency assays.

[0391] Across all tested concentrations, compounds of this disclosure produced encapsulation-efficiency values comparable to those obtained with Triton™ X-100, demonstrating that compounds of the present invention may be used to extract nucleic acids from LNPs. Further optimization of concentrations and solutions may be done, depending on specific compounds chosen.Example 13: Isolation of RNA using different magnetic beads

[0392] To test the performance of compounds of this disclosure in the purification of RNA, a lysis binding buffer was prepared. Components of this buffer are identical to the Lysis / Binding buffer from the Dynabeads™ SILANE Viral NA Kit (Thermo Fisher Scientific catalogue No 37011D) except that the surfactant component was replaced by 12% of a compound of this disclosure or 12% of Tween-20. The original Lysis / Binding buffer was used as a control.

[0393] Human plasma (200 pL per reaction) was thawed on ice and spiked with defined amounts of MS2 RNA (final cone.: 1.54 x 106copies). Fifty microliters Proteinase K (20 mg / mL) were added to an empty tube, followed by 200 pL spiked serum, and the tube was gently tapped to mix. Then 300 pL of the respective Lysis / Binding Buffer were added, the tube was inverted 4-6 times and incubated for 5 minutes at room temperature. Next, 150 pL isopropanol and 50 pL resuspended Dynabeads™ MyOne™ SILANE (40 mg / ml, Thermo Fisher Scientific catalogue No.370-02D), or Dynagreen™ SIC; and Dynabeads™ MyOne Carboxylic Acid (10mg / mL, Fisher Scientific catalogue No. 65012) or Dynagreen™ CHI were added, and the mixture was incubated at room temperature for 20 minutes. The beads were washed several times and then 100 pL elution buffer (10 mM Tris HCI pH 8.0) was added, the beads were resuspended and incubated at 70 °C for 5 minutes, then briefly resuspended again, placed on the magnet for 2 minutes, and the supernatant containing purified MS2 RNA was transferred to a clean tube. A one-step TaqMan RT-qPCR assay was used to detect MS2 RNA on a QuantStudio 5 Real-Time PCR System generate a standard curve for quantitative analysis.

[0394] The same protocol, using Dynabeads™ MyOne Carboxylic Acid (“COOH”) or Dynagreen™ CHI was also performed for the isolation of spiked RNA from serum except that a non-alcoholic trigger, as disclosed in e.g. W02025003501A1 or W02025078670A1 was used instead of isopropanol in equal concentration. Bead concentration was 1mg MyOne COOH (100pL / 10mg / mL) or 1mg Dynagreen CHI (25pL / 40mg / ml_).

[0395] Results were compared to optimized original formula and the Tween 20-buffer. (Tables 2 and 3.) Without any optimization the compounds of this disclosure performed as well or better than an equal concentration of Tween-20. Performance was also comparable to the fully optimized original buffer (“Original Buffer”).isolation yield obtained with buffers containing on of Tween-20, a compound of this disclosure, or the optimized, original formulation.Table 6. Mean relative yield (% based on Tween 20 positive control +MyOne COOH = 100%): RNA isolation yield obtained with buffers containing one of Tween-20, a compound of this disclosure, or the optimized, original formulation.

[0396] In view of the many possible aspect to which the principles of the present disclosure may be applied, it should be recognized that the illustrated aspects are only preferred examples of the disclosure and should not be taken as limiting the scope of the present disclosure.Rather, the scope is defined by the following claims. I therefore claim as my invention all that comes within the scope and spirit of these claims.

Claims

We claim:

1. A composition comprising:(a) one or more surfactant, wherein the surfactant is a compound having a structure according to Formula I:Formula Iwherein:R1is a hydrophilic group;R2is a lipophilic group;X is selected from oxygen, sulfur, or N(R5);R3is selected from heteroaliphatic, aliphatic, or aryl;each R4independently is aliphatic;Y is selected from oxygen, sulfur, or N(R5);the linker, when present, is aliphatic or has a formula -C(=Z)-W-C(=Z)-X’ , wherein each Z independently is oxygen, sulfur, or NR5, W is an aliphatic or heteroaliphatic group, and X’ is oxygen, sulfur, or NR5;n is an integer selected from 0 or 1 ;m is an integer selected from 0 to 6, provided that, if n is 0, then m is 1;p is an integer selected from 0 to 2; andeach R5group independently is selected from hydrogen, aliphatic, aromatic, or heteroaliphatic;(b) at least one polar protic solvent;(c) optionally, one or more inorganic salt;(d) optionally one or more buffering agent; and(e) optionally one or more chaotropic agents.12 / 22 / 25 TP391474W01Formula IE Formula IF.

3. The composition of claim 1; wherein the surfactant is a compound having a structure according to Formulas IG, I H, IJ, or IK:12 / 22 / 25 TP391474W01wherein the aliphatic group is linear, branched, or cyclic, or a combination thereof; TG, if present, is a terminating group that is an aliphatic group; and r is an integer selected from 2 to 20.

4. The composition of claim 1; wherein the surfactant is a compound selected from the group consisting of compounds 1 - 54:- 118 -- 119 -- 120 -12 / 22 / 25 TP391474W015. The composition according to any one of claims 1-4, wherein the polar protic solvent is selected from a C1-C5 alcohol, C2-C6 polyol, polyethylene glycol, water, and mixtures thereof.

6. The composition according to any one of claims 1-4, wherein the polar protic solvent is selected from ethanol, isopropyl alcohol, 2-methyl-1,3-propanediol (MPD), isoamyl alcohol, water, and mixtures thereof.

7. The composition according to any one of claims 1-4, wherein the polar protic solvent is a polyethylene glycol (PEG) selected from the group consisting of PEG 1500, PEG8000 PEG6000, PEG2000, PEG1000, PEG600, and mixtures thereof.

8. The composition according to any one of claims 1-4, wherein the inorganic salt is an alkali or alkaline earth metal salt.

9. The composition according claim 8, wherein the alkali or alkaline earth metal salt is selected from the group consisting of LiCI, NaCI, KCI, MgCh, BaCh, and CaCh, and corresponding citrates, acetates, or any combination thereof.

10. The composition according to any one of claims 1-4, wherein the buffering agent is selected from the group consisting of N-2-acetamido-2-aminoethanesulfonic acid (ACES), N-2-acetamido-2-iminodiacetic acid (ADA), 3-1,1-dimethyl-2-hydroxyethylamino-2-hydroxy propanesulfonic acid (AMPSO), N,N-bis2-hydroxyethyl-2-aminoethanesulfonic acid (BES), 4-cyclohexylamino- 1 -butane sulfonic acid (CABS), 3-cyclohexylamino-1-propane sulfonic acid (CAPS), 3-cyclohexylamino-2-hydroxy-1-propane sulfonic acid (CAPSO), 2-N-cyclohexylaminoethanesulfonic acid (CHES), 3-N,N-bis-2-hydroxyethylamino-2-hydroxypropanesulfonic acid (DIPSO), N-2-hydroxyethylpiperazine-N-3-propanesulfonic acid (EPPS or HEPPS), N-2-hydroxyethylpiperazine-N-4-butanesulfonic acid (HEPBS), 4-N-morpholinobutanesulfonic acid (MOBS), 3-N-morpholino-2-hydroxypropanesulfonic acid (MOPSO), bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (Bis-Tris), hydroxyethylpiperazine ethane sulfonic acid (HEPES), morpholinoethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), [tris(hydroxymethyl)methylamino] propanesulfonic acid (TAPS), N-trishydroxymethyl-methyl-4-aminobutanesulfonic acid (TABS), N-trishydroxymethyl-methyl-3-aminopropanesulfonic acid (TAPS), 3-N-trishydroxymethyl-methylamino-2-hydroxypropanesulfonic acid (TAPSO), N-trishydroxymethyl-methyl-2-aminoethanesulfonic acid (TES), N-(2-acetamido)iminodiacetic acid (ADA), Piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCI), N-trishydroxymethylmethylglycine (TRICINE), or any combination thereof.

11. The composition according to any one of claims 1-4, wherein the chaotropic agent is selected from the group consisting of lithium perchlorate, lithium acetate, sodium dodecyl sulfate, thiourea, urea, guanidinium chloride, guanidinium thiocyanate, and a combination thereof.

12. The composition according to any one of claims 1-11, further comprising a chelating agent selected from EDTA, EGTA, or DTPA.

13. The composition according to any one of claims 1-12, wherein the surfactant is present at a concentration of 0.001% to about 25% v / v.

14. The composition according to any one of claims 1-12, wherein the surfactant is present at a concentration of 0.05% to about 25% v / v.

15. The composition according to any one of claims 1-12, wherein the surfactant is present at a concentration of 0.5% to about 15% v / v.12 / 22 / 25 TP391474W0116. The composition according to any one of claims 1-12, wherein the surfactant is present at a concentration of 3.5% to about 15% v / v.

17. The composition according to any one of claims 1-12, wherein the surfactant is present at a concentration of 1.0% to about 10% v / v.

18. The composition according to any one of claims 1-17, wherein the inorganic salt is present at a concentration of 0.25 mM to 1000 mM.

19. The composition according to any one of claims 1-18, wherein the buffering agent is present at a concentration of 5 mM to 500 mM.

20. The composition according to any one of claims 1-19, wherein the chaotropic agent is present at a concentration of 0.5 M to 8 M.

21. The composition according to any one of claims 1-20, wherein the chelating agent is present at a concentration of 5 mM to 100 mM.

22. The composition according to any one of claims 1-21, wherein the polar protic solvent is present at a concentration of 5% to 25% v / v.

23. The composition according to any one of claims 1-22, wherein the pH is in the range of about 5.5 to about 10.0.

24. A buffer comprising a composition according to any one of claims-1-23 effective to provide a lysed sample.

25. A buffer comprising a composition according to any one of claims-1-7, 10, 12-17, 19, and 21-22 effective to allow binding of nucleic acids or polypeptides to a solid support.

26. A wash buffer comprising a composition according to any one of claims 1-7, 10, 13-17, 19, and 22.

27. A method for lysing cells, vesicles, or viruses, if present, in a sample, comprising: contacting the sample with a composition according to any one of claims 1-24; thereby generating a lysed sample.

28. A method for isolating nucleic acids from a sample, comprising:generating a lysed sample by performing the method of claim 27; andisolating nucleic acids from the lysed sample.

29. A method for isolating polypeptides and nucleic acids from a sample containing nucleic acids and polypeptides, the method comprising:generating a lysed sample by performing the method of claim 27; andisolating polypeptides and nucleic acids from the lysed sample.

30. A method for isolating polypeptides from a sample comprising polypeptides, the method comprising:generating a lysed sample by performing the method of claim 27; andisolating polypeptides from the lysed sample.

31. The method of any one of claims 27-30 wherein the isolating step comprises contacting the lysed sample with a solid support in the presence of a composition according to any one of claims 1-7, 10, 12-17, 19, 21-22, and 25 having a concentration of surfactant effective to allow binding of the nucleic acids or polypeptides to the solid support, thereby generating a nucleic acid-bound or polypeptide-bound solid support.

32. The method of claim 31, wherein the contacting step comprises:(a) contacting the sample with a lysis buffer to lyse cells, if present, in the sample, wherein the lysis buffer is a composition according to any one of claims 1-24 that has a concentration of the surfactant effective to lyse the cells,(b) contacting the sample with a binding buffer to bind the nucleic acids or polypeptides to the solid support, wherein the binding buffer is a composition according to any ofclaims 1-7, 10, 12-17, 19, 21-22, and 25 having a concentration of the surfactant effective to allow binding of the nucleic acids or polypeptides to the solid support.

33. A method of isolating cell-free nucleic acids or cell-free polypeptide from a sample, comprisingcontacting the sample with a binding buffer to bind the cell-free nucleic acids to a solid support, wherein the binding buffer is a composition according to claims 1-7, 10, 12-17, 19, 21-22, and 25 having a concentration of the surfactant effective to allow binding of the cell-free nucleic acids to the solid support.

34. A method of isolating vesicles from a sample, comprisingcontacting the sample with a binding buffer to bind the vesicles to a solid support, wherein the binding buffer is a composition according to claims 1-7, 10, 12-17, 19, 21-22, and 25 having a concentration of the surfactant effective to allow binding of the exosomes to the solid support.

35. The method of any of claims 34 wherein the vesicle is an exosome.

36. The method according to claim 29 wherein the isolating step comprises:(a) contacting the lysed sample with a first solid support in the presence of a binding buffer to bind the polypeptides to the solid support;(b) and contacting the lysed sample with a second solid support in the presence of a binding buffer to bind the nucleic acids to the second solid support.

37. The method of claim 29, wherein the isolating step comprises:(a) contacting the lysed sample with a first solid support in the presence of binding buffer to bind the nucleic acids to the solid support;(b) and contacting the lysed sample with a second solid support in the presence of a binding buffer to bind the polypeptides to the second solid support.

38. The method of claim 28, wherein the isolating step comprises:(a) contacting the lysed sample with a first solid support in the presence of binding buffer to bind DNA or RNA to the solid support;12 / 22 / 25 TP391474W01(b) and contacting the lysed sample with a second solid support in the presence of a binding buffer to bind DNA or RNA to the second solid support.

39. A method for preparing nucleic acids from a sample containing nucleic acids, comprising:generating a lysed sample by performing the method of claim 27; andincubating the lysed sample at an incubation temperature and for a time to produce a treated cell lysate, wherein the composition comprises an RNase inhibitor,wherein the cell lysate is compatible with in situ polymerase or reverse transcription reactions.

40. The method of claim 39; wherein the RNAse inhibitor is an anionic oligomer.

41. The method of claim 40, wherein the anionic oligomer is selected from the group consisting of Poly(vinylphosphonic acid), Heparin, Sulfated cellulose, Sulfated nitrocarboxymethyl cellulose, Sulfated amylose, Sulfated amylopectin, Sulfated pectic acid, Sulfated nitrochitin, Sulfated nitrochitosan, Sulfated polyvinyl alcohol, Poly-p,p-dioxy-dibenzyl phosphate, Poly-p,p-dioxydiphenyldimethyl metaphosphate, Polyaspartic acid, Polyglutamic acid, Polyacrylic acid, Poly(methacrylic acid), Poly(maleic acid), Pentosan polysulfate, Chondroitin sulfate, polyglycerol sulfate, Polyethylene sulfonate, Poly(4-styrenesulfonic acid-co-maleic acid), Poly(vinyl sulfonic acid), Poly(4-styrenesulfonic acid), Dextran sulfate, or any combination thereof.

42. The method according to any one of claims 39-41, wherein the composition optionally comprises a polypeptide having protease activity.

43. The method according to any one of claims 39-41, wherein the composition optionally comprises a polypeptide having deoxyribonuclease activity.

44. The method of claim 42; wherein the polypeptide having protease activity comprises proteinase K, a serine protease such as trypsin, chymotrypsin, elastase, subtilisin, streptogrisin, thermitase, aqualysin, plasmin, cucumisin, or carboxypeptidase A, D, C, or Y; a cysteine protease such as papain, calpain, or clostripain; an acid protease such as pepsin,chymosin, or cathepsin; or a metalloprotease such as pronase, thermolysin, collagenase, dispase, an aminopeptidase or carboxypeptidase A, B, E / H, M, T, orU. or an enzymatically active mutant or variant thereof.

45. The method of claim 43 wherein the polypeptide having deoxyribonuclease activity comprises DNase I, Nuclease BAL-31, exonuclease I, exonuclease III, Lambda exonuclease, CviKI-1 endonuclease, McrBC endonuclease, or an enzymatically active mutant or variant thereof.

46. The method according to any one of claims 42-45;wherein the method optionally comprises admixing the lysed sample with a stop mixture at substantially the same temperature as the contacting step to form a stop mixture, wherein the stop mixture comprises:a cation chelator effective to inactivate the polypeptide having deoxyribonuclease activity, and an inhibitor of the polypeptide having protease activity.

47. The method of claim 46; wherein the inhibitor of the polypeptide having protease activity comprises leupeptin as an inhibitor for serine and cysteine proteases such as plasmin, trypsin, papain, kallikrein and cathepsin B; or 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF) as an inhibitor for serine proteases such as chymotrypsin, kallikrein, plasmin, thrombin, and trypsin; oraprotinin as an inhibitor of serine proteases such as trypsin, chymotrypsin, plasmin and kallikrein; or benzamidine as an inhibitor of trypsin; N-acetyl eglin-C as an inhibitor of chymotrypsin, subtilisin, leukocyte elastase and cathepsin G; or antipain or plasmin for inhibition of a serine or cysteine such as papain and trypsin.

48. The method of claim 43; wherein the inhibitor of the polypeptide having protease activity comprises methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone, carbobenzoxy-Ala-Ala-COCH2CI, carbobenzoxy-Ala-Ala-Phe-COCH2CI or carbobenzoxy-Phe-Pro-Arg-COCH2CI or phenylmethylsulfonyl fluoride (PMSF).

49. The method of claim 46; wherein the cation chelator effective to inactivate the polypeptide having deoxyribonuclease comprises a calcium chelator such as EGTA or EDTA, orcation exchange beads such as sulfopropyl-functionalized cross-linked agarose cation-exchange beads (SP SEPHAROSE™ beads; GE Healthcare), 1,10-phenanthroline, tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), diethylenetriaminepentaacetic acid (DTPA), or a combination thereof.

50. A method of preparing endotoxin-free nucleic acid from a sample comprising a microorganism, wherein the microorganism comprises the nucleic acid, the method comprising:(a) providing a sample comprising the microorganism;(b) generating a lysed sample by performing the method of claim 27, wherein the composition has a concentration of the surfactant effective to lyse the microorganism.

51. The method of claim 50, wherein the composition further comprises a polymyxin.

52. The method according to claims 50-51 , wherein the nucleic acid is a plasmid DNA (pDNA).

53. The method according to any one of claims 27-51 , wherein the nucleic acid is DNA or RNA.

54. The method of claim 53, wherein the DNA is synthetic DNA, plasmid DNA (pDNA), genomic DNA (gDNA), viral DNA (e.g. dsDNA or ssDNA), cDNA, cfDNA, gDNA or ctDNA, or any combination thereof.

55. The method of claim 54, wherein the RNA is mRNA, siRNA, shRNA, selfreplicating RNA (srRNA), an o-RNA, self-amplifying RNA, stRNA, trRNA, crRNA, sgRNA, RNAi molecule, an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a Dicer-substrate RNA (dsRNA), a small hairpin RNA (shRNA), or any combination thereof.

56. The method according to any one of claims 27-55 wherein the sample comprises a cell, cell culture, biological sample, clinical sample, environmental sample, or an enzymatic reaction mixture.12 / 22 / 25 TP391474W0157. The method according to claim 56, wherein the biological sample is a pre-treated or untreated biological sample.

58. The method according to claim 57, wherein the biological sample is blood stain, cord blood, blood components (e.g., platelet concentrates), blood cultures, peripheral blood mononuclear cells, peripheral blood leukocytes, plasma lysates, leukocyte lysates, buffy coat leukocytes, serum, plasma, saliva, saliva stain, buccal cells, buccal swab, semen, semen stain, urine, fecal matter, fecal stain, cigarette butt, chewing gum, formalin- fixed paraffin-embedded (FFPE) sample, biopsy (e.g. tumor biopsy) sample, bone marrow or other tissue sample, cell lysate, bacterial culture, yeast culture, sputum, tear, throat swabs, oral rinses, nasopharyngeal swabs, nasopharyngeal aspirates, exhalates, nasal swabs, nasal washes, mucus, bronchial aspirations, bronchoalveolar lavage fluid, pleural fluid, endotracheal aspirates, cerebrospinal fluid, anal swabs, rectal swabs, vaginal swabs, endocervical swabs, vitreous fluid, amniotic fluid, breast milk, exosomes, circulating tumor cells, tissue lysates, bacterial lysates, yeast lysates, or exosomes.

59. The method according to claim 56, wherein the cell culture has been cultured on extracellular matrix.

60. The method of claim 56, wherein the cell culture comprises primary cells.

61. The method of claim 60, wherein the primary cells comprise primary hepatocytes.

62. The method of claim 61, wherein the cells are selected from the group consisting of Kupffer cells, PBMCs, THP-1 cells, HL60 cells, 3D cell cultures, or any combination thereof.

63. The method according to any one of claims 27-49, wherein the solid support may comprise one or more of particles, resin, beads, filter, cartridge, column, chromatography column, microcolumn, array, membrane, chip, disc, or slide.

64. The method according to claim 63, wherein the solid support comprises optionally monodisperse beads and wherein the beads are magnetic.

65. The method according to claim 64; wherein the solid support is a paramagnetic bead or a superparamagnetic bead.

66. The method according to claim 63, wherein the solid support is selected from ion exchange chromatography matrix, an affinity chromatography matrix, a size exclusion chromatography matrix, a hydrophobic interaction chromatography matrix, an immobilized metal affinity chromatography, a reverse phase chromatography matrix, immunoaffinity chromatography matrix, or a mixed mode chromatography matrix.

67. The method according to claim 63; wherein microcolumn includes a silica-based membrane.

68. A hybridization buffer according to the compositions of any one of claims 1-4, wherein the surfactant is present in an amount effective to achieve hybridization between a first nucleic acid and a complementary second nucleic acid.

69. The buffer of claim 68, wherein the surfactant is present in a concentration of 0.0001% to 10% (w / v).

70. The buffer of claim 69, wherein the surfactant is present in a concentration of 0.0001% to 1% (w / v).

71. The buffer of claim 68, wherein the buffer further comprises one or more of a salt, a denaturant, a polymer, a blocking agent, a crowding agent, a hybridization enhancer, an accelerating agent, a chelating agent, a stabilizer, or buffering agent.

72. The buffer of claim 71, wherein the salt comprises monovalent and / or divalent cations.

73. The buffer of claim 71, wherein the salt is selected from sodium chloride, lithium chloride, ammonium chloride, potassium chloride, sodium acetate, potassium acetate,ammonium acetate, sodium citrate, potassium citrate, sodium phosphate, magnesium phosphate, tetramethylammonium chloride, tetraethylammonium chloride, tetrapropylammonium chloride, tetrabutylammonium chloride, guanidinium thiocyanate, sodium thiocyanate, or potassium thiocyanate.

74. The buffer of claim 71, wherein the hybridization enhancer comprises DMSO, formamide, dextran sulfate, PEG, or guanidinium thiocyanate.

75. The buffer of claim 71, wherein the buffering agent is selected from a group consisting of citrate buffers (saline-sodium citrate (SSC)), phosphate buffers (phosphate buffered saline (PBS)), Tris (tris(hydroxymethyl)aminomethane) or (2-amino-2-(hydroxymethyl)propane-1,3-diol)-based buffers, TAPS ([tris(hydroxymethyl)methylamino]propanesulfonic acid)-based buffers, HEPES (4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid)-based buffers, Bicine (2-(bis(2-hydroxyethyl)amino)acetic acid)-based buffers, tricine (N-[tris(hydroxymethyl)methyl]glycine)-based buffers, TAPSO (3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid)-based buffers, TES (2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid)-based buffers, MES (2-(N-morpholino)ethanesulfonic acid)-based buffers, MOPS (3-(N-morpholino)propanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid))-based buffers, Cacodylate (dimethylarsenic acid)-based buffers, or any combination thereof.

76. A method of increasing stability of a hybridization buffer, comprising incorporating a concentration of surfactant according to any one of claims 1-4 in a concentration of 0.0001% to 1% (w / v).

77. A method of hybridizing a first nucleic acid to a complementary second nucleic acid, comprising contacting the nucleic acids in the presence of the hybridization buffer of any of claims 68-75 under conditions permitting hybridization.

78. The method of claim 77, wherein the hybridization is performed in in situ hybridization assay.12 / 22 / 25 TP391474W0179. The method of claim 77, wherein the hybridization is performed in a fluorescence in situ hybridization (FISH) assay.

80. The method of claim 77, wherein the hybridization is performed in a Southern blot, Northern blot, or dot blot assay.

81. The method of claim 77, wherein the hybridization is performed in an array assay.

82. The method of claim 81, wherein the hybridization is performed in a nucleic acid microarray.

83. A method of enhancing the signal-to-noise ratio in an assay comprising a nucleic acid hybridization step, comprising hybridizing a first nucleic acid to a complementary second nucleic acid by contacting the nucleic acids in the presence of the hybridization buffer of any of claims 68-75 under conditions permitting hybridization.

84. The method according to any one of claims 27-67 or 77-83, wherein the isolated nucleic acids or polypeptides are further subjected to one or more additional processes, optionally selected from detection, quantification, cloning, restriction, nucleic acid synthesis and / or assembly, analysis, epigenetic analysis, sequencing, amplification, study, transfection, cDNA synthesis, size separation, chromatography and mass spectrometry, pharmaceutical or therapeutic formulation and genome editing.

85. The method of claim 84, wherein the amplification comprises PCR, qPCR, next generation sequencing (NGS), reverse transcription, in vitro transcription, or isothermal amplification.

86. The method of claim 85, wherein the isothermal amplification comprises loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), helicase-dependent amplification (HDA), multiple displacement amplification (MDA), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), multiple cross displacement amplification (MCDA), signal-mediated amplification of RNA technology (SMART), recombinase-polymerase amplification (RPA) or nucleic acid sequence-based amplification (NASBA).12 / 22 / 25 TP391474W0187. The methods of claim 84-86 wherein the nucleic acids are DNA, RNA or both.

88. A method of enhancing the activity of an enzymatic protease, comprising contacting a sample with an enzymatic protease with the composition of any of claims 1-24.

89. The method of claim 88, wherein the enzymatic protease is proteinase K.

90. Use of a composition according to any one of claims 1-24 for lysing a cell, vesicle, or virus in a sample.

91. Use of a composition according to any one of claims 1-24 for isolation of two or more polypeptides, vesicles, or nucleic acids from a sample containing polypeptides, vesicles or nucleic acids.

92. Use of a composition according to any one of claims 1-24 for isolation of nucleic acids from a sample containing nucleic acids.

93. Use of a composition according to any one of claims 1-24 for isolation of polypeptides from a sample containing polypeptides.

94. Use of a composition according to any one of claims 1-24 for isolation of vesicles from a sample containing vesicles.

95. Use of a composition comprising:(a) a surfactant, wherein the surfactant is a compound selected from the group consisting of compounds 1 to 54;(b) at least one polar protic solvent;(c) optionally, one or more inorganic salt;(d) optionally one or more buffering agent; and(e) optionally one or more chaotropic agents;for isolation of two or more polypeptides, vesicles, or nucleic acids from a sample containing two or more polypeptides, vesicles, or nucleic acids.

96. Use of a composition comprising:(a) a surfactant, wherein the surfactant is a compound selected from the group consisting of compounds 1 to 54;(b) at least one polar protic solvent;(c) optionally, one or more inorganic salt;(d) optionally one or more buffering agent; and(e) optionally one or more chaotropic agents;for isolation of nucleic acids from a sample containing nucleic acids.

97. Use of a composition comprising:(a) a surfactant, wherein the surfactant is a compound selected from the group consisting of compounds 1 to 54;(b) at least one polar protic solvent;(c) optionally, one or more inorganic salt;(d) optionally one or more buffering agent; and(e) optionally one or more chaotropic agents;for isolation of polypeptides from a sample containing polypeptides.

98. Use of a composition comprising:(a) a surfactant, wherein the surfactant is a compound selected from the group consisting of compounds 1 to 54;(b) at least one polar protic solvent;(c) optionally, one or more inorganic salt;(d) optionally one or more buffering agent; and(e) optionally one or more chaotropic agents;for isolation of vesicles from a sample containing vesicles.

99. Use according to any one of claims 91-92 or 95-96, wherein the nucleic acid is DNA or RNA.

100. Use according to claim 99, wherein the DNA is one or more of synthetic DNA, plasmid DNA (pDNA), genomic DNA (gDNA), viral DNA (e.g. dsDNA or ssDNA), cDNA, cfDNA, gDNA or ctDNA.

101. Use according to claim 99, wherein the RNA is mRNA, siRNA, shRNA, selfreplicating RNA (srRNA), an o-RNA, self-amplifying RNA, stRNA, trRNA, crRNA, sgRNA, RNAi molecule, an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a Dicer-substrate RNA (dsRNA), a small hairpin RNA (shRNA), or any combination thereof.

102. Use of a composition according to any one of claims 1-7, 10, 12-17, 19, 21-22, and 25 for binding of polypeptides, vesicles, or nucleic acids to a solid support, where in the solid support optionally is selected from one or more of particles, resin, beads, a filter, cartridge, a column, a microcolumn, an array, a membrane, a chip, a disc, or a slide.

103. Use of a composition according to any one of claims 1-24 and 65-72 for hybridizing a first nucleic acid to a complementary second nucleic acid.

104. A kit, comprising the composition of any one of the claims 1-26, and optionally a solid support or a chelating agent.

105. The kit of claim 104, further comprising one or more of a lysis buffer, binding buffer, and wash buffer comprising the composition of any one of claims 1-26.

106. The kit of claim 104, further comprising a polypeptide having protease activity.

107. The kit of claim 106, further comprising a polypeptide having deoxyribonuclease activity.

108. The kit of claim 104, further comprising an RNase inhibitor.

109. The kit of claim 108, wherein the RNAse inhibitor is an anionic oligomer.12 / 22 / 25 TP391474W01110. A kit for nucleic acid hybridization comprising: (a) the hybridization buffer of any one of claims 68-75; and (b) one or more components selected from the group consisting of: fixatives, buffers and reagents, mounting media, detection reagents, labeling reagents, sample membranes or support, positive controls, negative controls, nucleic acid probes or targets, probes comprising oligonucleotides immobilized on a solid substrate selected from glass, silica, or polymeric films, and wash buffers, or any combination thereof.

111. The kit of claim 110, wherein the probes comprise oligonucleotides immobilized on a solid substrate selected from glass, silica, or polymeric films.

112. The kit of any one of claims 110 or 111, further comprising a labeling reagent selected from fluorescent dyes, biotin, or chemiluminescent substrates.

113. The kit of claim 110, wherein the probes are provided in labeled form for detecting genomic or transcriptomic targets on a membrane substrate.

114. The kit of claim 110, further comprising reagents for target nucleic acid immobilization and post-hybridization washing.

115. The kit of claim 110, wherein the buffer is formulated for in situ hybridization of nucleic acids in fixed cells or tissue sections.

116. The kit of claim 115, further comprising fixatives, mounting media, and fluorescence detection reagents.

117. The kit of claim 115, wherein the hybridization buffer is compatible with multi-color probe hybridizations.

118. The kit of claim 110, wherein the hybridization buffer is formulated for membranebased nucleic acid screening.12 / 22 / 25 TP391474W01119. The claim of 118, wherein the hybridization buffer is formulated for high-throughput nucleic acid screening.

120. The kit of claim 118, further comprising sample membranes, positive and negative controls, and wash buffers.

121. The kit of claim 110, wherein the hybridization buffer is configured for use across one or more nucleic acid hybridization techniques including microarrays, FISH, Southern blot, Northern blot, or dot blot assays.

122. The kit of claim 121, further comprising instructions specifying assay-specific hybridization conditions using the same buffer composition.

123. A compound according to Formula I, bound to a solid support; wherein;R2is a lipophilic group;X is selected from oxygen, sulfur, or N(R5);12 / 22 / 25 TP391474W01SS is a solid-support;each R4independently is aliphatic;Y is selected from oxygen, sulfur, or N(R5);the linker, when present, is aliphatic or has a formula -C(=Z)-W-C(=Z)-X’ , wherein each Z independently is oxygen, sulfur, or NR5, W is an aliphatic or heteroaliphatic group, and X’ is oxygen, sulfur, or NR5;n is an integer selected from 0 or 1 ;m is an integer selected from 0 to 6, provided that, if n is 0, then m is 1;p is an integer selected from 0 to 2; andeach R5group independently is selected from hydrogen, aliphatic, aromatic, or heteroaliphatic.

124. The compound of claim 123; having a structure according to any one of Formulas IA, IB, IC, ID, IE, or IF:12 / 22 / 25 TP391474W01Formula IE Formula IF125. The compound of claims 123 and 124 wherein; the solid-support (SS) is selected from the group consisting of polystyrene (PS) beads, agarose beads, pegylated-polystyrene beads, controlled-pore glass beads, functionalized silica gel, sepharose, alumina supports, magnetic silica or polymer-coated magnetic beads, cellulose and modified cellulose resins, chitosan beads, dextran-based supports such as sephadex, and pre-functionalized systems including NHS-activated beads, maleimide-activated beads, and epoxy-activated supports.

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