WO2025259669A1 - Methods and systems for characterizing modified oligonucleotides - Google Patents

Abstract

Provided herein are methods for characterizing a sample containing a modified oligonucleotide. The methods may include: subjecting a sample including the modified oligonucleotide and an isomer of the modified oligonucleotide to a digestion condition to form a digested sample comprising an oligonucleotide fragment of the isomer; subjecting the digested sample to liquid chromatography to form an eluate; subjecting the eluate to cyclic ion-mobility mass spectrometry to obtain a separation profile of the oligonucleotide fragment; comparing the separation profile of the oligonucleotide fragment to a separation profile of an oligonucleotide standard, wherein the oligonucleotide standard includes: a nucleic acid sequence identical to a nucleic acid sequence within the oligonucleotide fragment; and a nucleic acid modification identical to a nucleic acid modification of the oligonucleotide fragment; and characterizing the isomer based on a comparison of the separation profile of the oligonucleotide fragment to the separation profile of the oligonucleotide standard.

Description

METHODS AND SYSTEMS FOR CHARACTERIZING MODIFIED OLIGONUCLEOTIDES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63/658,243, filed on June 10, 2024.

FIELD OF THE DISCLOSURE

[0002] The present disclosure relates to methods for characterizing isomers and backbone modifications of an oligonucleotide.

INTRODUCTION

[0003] Oligonucleotide therapeutics are a class of biopharmaceutical products that are designed to modulate gene expression. In general, oligonucleotides are synthesized as unmodified phosphodiester (PO) oligos, but these unmodified oligonucleotides are unstable and easily degraded by nucleases. Chemical modifications to the phosphate backbone are required to improve the oligonucleotide’s stability against nuclease digestion. One such chemical modification is phosphorothioate (PS), which is synthesized by replacing one of the nonbridging oxygen atoms in the PO linkage with a sulfur atom. Remarkably, this substitution has been found to help extend the effective molecular lifetime of the oligonucleotide by minimizing extracellular and intracellular nuclease degradation.

[0004] PS oligonucleotides are susceptible to oxidation, which results in the desulfurization of the PS linkage, that is, the conversion of PS to PO. The stability and gene editing efficiency of the oligonucleotide may be adversely affected by the conversion of the nuclease resistant PS linkage to the more susceptible PO linkage. Additionally, the stability and efficacy of the nucleotide may be further diminished by the formation of new constitutional isomers.

[0005] Since the purity and chemical identity of PS oligonucleotides is crucial to confirming product quality and thereby ensuring patient safety, the ability to characterize these oligonucleotides on a routine basis is important. However, developing such analytical methods is a challenging task since it is difficult to distinguish all the isomers of a typical PS oligonucleotide. Therefore, it will be appreciated that a need exists for improved methods for characterizing such isomers and the backbone modifications of an oligonucleotide.

SUMMARY

[0006] Provided herein are methods for characterizing a sample containing a modified oligonucleotide. The method may include: subjecting a sample including the modified oligonucleotide and an isomer of the modified oligonucleotide to a digestion condition to form a digested sample comprising an oligonucleotide fragment of the isomer; subjecting the digested sample to liquid chromatography to form an eluate; subjecting the eluate to cyclic ion-mobility mass spectrometry to obtain a separation profile of the oligonucleotide fragment; comparing the separation profile of the oligonucleotide fragment to a separation profile of an oligonucleotide standard, wherein the oligonucleotide standard includes: a nucleic acid sequence identical to a nucleic acid sequence within the oligonucleotide fragment; and a nucleic acid modification identical to a nucleic acid modification of the oligonucleotide fragment; and characterizing the isomer based on a comparison of the separation profile of the oligonucleotide fragment to the separation profile of the oligonucleotide standard.

[0007] In some aspects, the nucleic acid modification may be a backbone modification. The nucleic acid modification may be a phosphorothioate modification. Subjecting the sample to a digestion condition may include contacting the sample to a digestive enzyme. Subjecting the sample to a digestion condition may include contacting the sample to RNAaseTl. Subjecting the sample to a digestion condition may include contacting the sample to RNaseA. Subjecting the sample to a digestion condition may include contacting the sample to RNAaseTl and RNaseA. The separation profile may include a mobiligram.

[0008] In some aspects, the method may further include identifying the nucleic acid sequence within the oligonucleotide fragment. The method may further include quantifying an amount of the oligonucleotide fragment in the eluate. The method may further include quantifying an amount of the isomer in the sample based on a quantification of an amount of the oligonucleotide fragment in the eluate. The method may further include subjecting the sample to a denaturing condition, prior to subjecting the sample to a digestion condition.

[0009] In some aspects, subjecting the digested sample to liquid chromatography may include subjecting the sample to ion-pairing reversed phase liquid chromatography. Subjecting the eluate to cyclic ion-mobility mass spectrometry may include subjecting the eluate to a cyclic ion-mobility mass spectrometer comprising a time-of-flight mass analyzer. The modified oligonucleotide may be a single guide RNA (sgRNA).

BRIEF DESCRIPTION OF THE DRAWINGS

[0001] FIG. 1 is a schematic illustration of chemical structures of phosphodiester (PO) linkages in oligonucleotides and the corresponding Sp or Rp diastereomers upon conversion to phosphorothioate (PS) linkages, according to aspects of the present disclosure.

[0002] FIG. 2 A is a panel of extracted ion chromatograms of 5 ’-end fragments of an sgRNA with various PS linkages obtained using IPRP-LC/MS analysis, according to aspects of the present disclosure. FIG. 2B is a panel of extracted ion chromatograms of 3 ’-end fragments of an sgRNA with various PS linkages obtained using IPRP-LC/MS analysis, according to aspects of the present disclosure. FIG. 2C is a panel of extracted ion chromatograms of 5 ’-end fragments of an sgRNA with various PS linkages obtained using IPRP-LC/MS analysis, according to aspects of the present disclosure. [0003] FIG. 3A is a mobiligram of 5’-end fragments of an sgRNA with two PS linkages obtained using four passes of cyclic ion-mobility mass spectrometry (cIMS), according to aspects of the present disclosure. FIG. 3B is a panel of three mobiligrams of oligonucleotide standards.

[0004] FIG. 4 A is a mobiligram of 5 ’-end fragments of an sgRNA with two PS linkages obtained using twenty passes of cIMS, according to aspects of the present disclosure. FIG. 4B is a panel of three mobiligrams of oligonucleotide standards.

[0005] FIG. 5 A is a mobiligram of 5 ’-end fragments of an sgRNA with one PS linkage obtained using cIMS, according to aspects of the present disclosure. FIG. 5B is a panel of three mobiligrams of oligonucleotide standards.

[0006] FIG. 6 A is a mobiligram of 3 ’-end fragments of an sgRNA with two PS linkages obtained using cIMS, according to aspects of the present disclosure. FIG. 6B is a panel of three mobiligrams of oligonucleotide standards.

[0007] FIG. 7A is a mobiligram of 3 ’-end fragments of an sgRNA with one PS linkage obtained using cIMS, according to aspects of the present disclosure. FIG. 7B is a panel of three mobiligrams of oligonucleotide standards.

[0008] FIG. 8 is a panel of nine total ion current (TIC) profiles of stereoisomers of 5 ’-end fragments of an sgRNA with two PS linkages, according to aspects of the present disclosure.

[0009] FIG. 9 is a panel of two TIC profiles of stereoisomers of 5 ’-end fragments of an sgRNA with two PS linkages acquired using an extended separation time, according to aspects of the present disclosure.

[0010] FIG. 10 is a panel of six TIC profiles of stereoisomers of 5 ’-end fragments of an sgRNA with one PS linkage, according to aspects of the present disclosure.

[0011] FIG. 11 is a panel of nine TIC profiles of stereoisomers of 3’-end fragments of an sgRNA with two PS linkages, according to aspects of the present disclosure.

[0012] FIG. 12 is a panel of six TIC profiles of stereoisomers of 3’-end fragments of an sgRNA with one PS linkage, according to aspects of the present disclosure.

[0013] FIG. 13A is a panel of three extracted ion chromatograms of 5’-end fragments of an sgRNA with two PS linkages, according to aspects of the present disclosure. FIG. 13B is a panel of three extracted ion chromatograms of two overlapping stereoisomers of 5 ’-end fragments of an sgRNA with two PS linkages (isomers 3 and 4 in top panel; isomer 3 in middle panel; isomer 4 in bottom panel), according to aspects of the present disclosure. FIG. 13C is a panel of three extracted ion chromatograms of 5 ’-end fragments of an sgRNA with one PS linkage, according to aspects of the present disclosure.

[0014] FIG. 14A is a graph of relative abundance of stereoisomers of various PS linkages as a function of acquisition scan time during cIMS, according to aspects of the present disclosure. FIG. 14B is a panel of six graphs of normalized ion abundance of a 5 ’-end fragment of an sgRNA having one PS linkage across various acquisition scan times, according to aspects of the present disclosure. FIG. 14C is a graph of integrated peak areas from FIG. 14B as a function of acquisition scan time, according to aspects of the present disclosure.

[0015] FIG. 15 is a graph of ion abundance of a 5 ’-end fragment of an sgRNA with one PS linkage as a function of injection amount, according to aspects of the present disclosure.

[0016] FIG. 16 is a graph of observed relative abundance of 5 ’-end fragments of an sgRNA having various PS linkages as a function of expected relative abundance, according to aspects of the present disclosure.

[0017] FIGS. 17A-17C are graphs demonstrating oxidation kinetics of PS modifications in 5’-end fragments of an sgRNA under stress conditions over 72 hours, according to aspects of the present disclosure. FIG. 17A is a graph of relative abundance as a function of incubation time for precursor and oxidative impurities based on the number of PS linkages. FIG. 17B is a graph of relative abundance as a function of incubation time for individual constitutional isomers with one or two PS to PO conversions. FIG. 17C is a graph of relative abundance as a function of incubation time for PS to PO conversion at individual linkage positions.

DETAILED DESCRIPTION

[0018] Oligonucleotide therapeutics are synthetically modified nucleic acids that have the ability to modulate gene expression via a number of mechanisms, including RNA interference or degradation, splicing modulation, gene editing, and gene activation. Oligonucleotide therapeutics can execute these functions by selectively targeting RNA or DNA sequences through Watson-Crick base pairing. Consequently, oligonucleotides have the potential to target any gene of interest, including those that have been traditionally considered “undruggable” by small molecule or protein therapeutics. However, the use of oligonucleotides in vivo presents a number of challenges, including intracellular delivery and nuclease degradation.

[0019] In an effort to modify the chemical properties of native-state oligonucleotides and overcome the challenges posed by the use of oligonucleotides in vivo, several phosphate backbone variants have been developed. One of the earliest and still widely used variant is phosphorothioate (PS), which is synthesized by replacing one of the nonbridging oxygen atoms in the phosphodiester (PO) linkage with a sulfur atom. Remarkably, this substitution has been found to help extend the effective molecular lifetime of the oligonucleotide by minimizing extracellular and intracellular nuclease degradation. Thus, to increase resistance to nucleases, such as exonucleases, oligonucleotides are often chemically synthesized to include PS linkages at the 3’ and 5’ ends. [0020] Although PS oligonucleotides provide increased nuclease resistance, the introduction of PS linkages can reduce the affinity of the oligonucleotide to the target RNA or DNA sequences. One reason for this reduction is the prochiral nature of the phosphorous atom. Introduction of a sulfur atom to the phosphate backbone of PS oligonucleotides creates a chiral center at the phosphorous atom, resulting in two chemical configurations known as the Rp and Sp diastereomers, as shown in FIG. 1. As a result, synthesis of PS oligonucleotides using traditional approaches can produce a racemic mixture having up to 2ⁿ possible diastereomers, where n is the total number of PS linkages. For example, the synthesis of a typical 100 nucleotide single guide RNA (sgRNA), which has three consecutive PS linkages on both the 5’ and 3’ end of the sequence, can generate a total number of 2^? or 8 stereoisomers on each end of the sequence, as shown in Table 1, below. In view of this, in a racemic mixture, only a small percentage of the PS oligonucleotides are likely to bind to the target RNA or DNA sequences with sufficient affinity. Indeed, fully Sp-configured oligonucleotides are known to be resistant to nuclease degradation but have poor binding affinity, while fully Rp-configured oligonucleotides are known to be less resistant to nuclease degradation but to bind more strongly to the RNA target. See, for example, Hannauer et al., “Review of fragmentation of synthetic single-stranded oligonucleotides by tandem mass spectrometry from 2014 to 2022”, Rapid Communications in Mass Spectrometry, 2023, volume 37, issue 17, article no. e9596, pages 1-20, which is incorporated by reference herein in its entirety.

[0021] Further, it is known that PS oligonucleotides are susceptible to oxidation during manufacturing and storage, which results in desulfurization of the PS linkage, that is, the conversion of the PS linkage to the PO linkage. The stability and gene editing efficiency of the oligonucleotide can be adversely impacted by the conversion of the nuclease resistant PS linkage to the more susceptible PO linkage. Further, since desulfurization could be a random process and not all PS linkages will desulfurize, this could lead to the formation of new constitutional isomers, which would further reduce the stability and efficacy of the oligonucleotide.

[0022] Considering this, desulfurization of a PS oligonucleotide can produce up to C(n, r) * 2ⁿ stereoisomers, where n is the total number of PS linkages, and r is the number of remaining PS linkages that have not been desulfurized. See, for example, the estimated total stereoisomers of an oligonucleotide based on the number of PS linkages as shown in Table 1, below. If one of the three PS linkages in a sgRNA was desulfurized, the total number of stereoisomers would be C(3, 2) * 2² or 12. Similarly, if two of the three PS linkages in a sgRNA were desulfurized, the total number of stereoisomers would be C(3, 1) * 2¹ or 6. Further, if all three PS linkages in a sgRNA were desulfurized, the total number of products would be C(3, 0) * 2° or 1. Thus, for a sgRNA with 3 PS linkages, desulfurization of PS could result in the formation of up to 19 total final products with a different number of PS-related stereoisomers. Table 1

[0023] Since the purity and chemical identity of PS oligonucleotides is crucial to confirming product quality and thereby ensuring patient safety, the ability to characterize these oligonucleotides on a routine basis is important. Oligonucleotides are typically characterized by polymerase chain reaction (PCR)- based assays to verify their composition. However, such assays cannot be used to analyze or quantify oligonucleotides that carry backbone modifications, such as PS. Liquid chromatography/mass spectrometry (LC/MS) remains the only possible method to characterize oligonucleotides with PS modifications. However, many LC/MS methods cannot distinguish all the isomers of a typical PS oligonucleotide. Thus, the wide use of LC/MS for characterizing PS oligonucleotides has been limited due to the heterogeneity of PS stoichiometry and isomerization. Therefore, it will be appreciated that a need exists for improved methods for characterizing such isomers and the backbone modifications of an oligonucleotide.

[0024] To address this challenge, ion-mobility mass spectrometry can be added as an additional dimension to the LC-MS analysis of oligonucleotides. Ion-mobility mass spectrometry separates ions based on their mobility in a chamber filled with an inert buffer gas (e.g., N2), where ions are moved forward by an electrical field along the chamber. Thus, the 10ns of each stereoisomer could be further separated by their drift time in the chamber. However, established ion-mobility spectrometry techniques possess limited resolving powers.

[0025] Cyclic ion-mobility spectrometry (cIMS) offers increased resolving power compared to linear ion-mobility spectrometry. Higher resolving powers can be achieved because cIMS can significantly increase the path length while preserving ion transmission and sensitivity. Cyclic IMS consists of three main regions: trap region, cyclic ion-mobility device, and transfer region. Ions are first accumulated in the trap region, which allows for fragmentation of the ions prior to cyclic ion-mobility. Following accumulation of the ions in the trap region, the ions are injected into the cyclic ion-mobility device which consists of a 98 cm path length, closed-loop traveling wave (TW)-enabled ion-mobility separator. The cyclic geometry of the ion-mobility device enables ions to travel multiple passes, increasing their drift times to the detector and relative separation. After separation in the cyclic ion-mobility device, the ions are further fragmented in the transfer region.

[0026] The disclosure herein provides methods for characterizing isomers and backbone modifications of an oligonucleotide. In the examples set forth below, a sgRNA oligonucleotide was analyzed by ionpairing reverse phased liquid chromatography, followed by cIMS. It was discovered that the constitutional isomers and/or stereoisomers of the sgRNA can be separated by cIMS. The isomers were then identified by comparing the retention and drift times of the separated isomer to an oligonucleotide standard. Further analysis of the data obtained by cIMS was used to quantify the relative abundance of the isomers, as well as the relative abundance of PS at each linkage position. These results establish cIMS as an analytical approach for characterizing oligonucleotides.

[0027] Unless described otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in practice or testing, particular methods and materials are now described.

[0028] The term “a” should be understood to mean “at least one” and the terms “about” and “approximately” should be understood to permit standard variation as would be understood by those of ordinary skill in the art, and where ranges are provided, endpoints are included. As used herein, the terms “include,” “includes,” and “including” are meant to be non-limiting and are understood to mean “comprise,” “comprises,” and “comprising” respectively.

[0029] As used herein, a “sample” refers to a mixture of molecules that comprises at least an oligonucleotide, such as an sgRNA oligonucleotide, that is subjected to manipulation in accordance with the methods of the disclosure, including, for example, separating, analyzing, extracting, concentrating, profiling and the like.

[0030] As used herein, the term “isomer” refers to compounds with the same chemical formula but which are structurally distinguishable. Isomers include stereoisomers and constitutional isomers. The term “stereoisomer” refers to compounds with the same chemical formula and connectivity, but have different arrangements of atoms or groups in space. Stereoisomers include enantiomers, which are stereoisomers that are non-superimposable mirror images of one another, and diastereomers, which are stereoisomers that are superimposable mirror images of one another. A mixture of enantiomers is called a “racemic mixture” or a “racemate.” The term “constitutional isomer” refers to compounds with the same chemical formula, but different connectivity.

[0031] As used herein, the terms “nucleic acid,” “polynucleotide,” or “oligonucleotide” refer a polymer composed of nucleotides or nucleosides (including ribonucleotides, deoxyribonucleotides, or analogs or modified versions thereof), which have nitrogenous heterocyclic bases or base analogs linked together along a backbone. Oligonucleotides may be isolated from genes, or chemically synthesized by methods known in the art. A nucleic acid “backbone” may be made up of a variety of linkages, including one or more of sugar-phosphodi ester linkages, peptide-nucleic acid bonds (“peptide nucleic acids” or PNA; PCT No. WO 95/32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. Sugar moieties of a nucleic acid may be ribose, deoxyribose, or similar compounds with optional substitutions, e.g., methoxy or 2’ halide substitutions. Oligonucleotides may be single-stranded, double-stranded, or multi-stranded. Oligonucleotides may be of a variety of different lengths, depending on the form. Oligonucleotides often range in size from a few monomeric units, e.g. 3-4, to hundreds of monomeric units. Whenever an oligonucleotide is represented by a sequence of letters, such as “ATGCCTG,” it will be understood that the nucleotides are in 5'— >3' order from left to right and that “A” denotes adenosine, “C” denotes cytosine, “G” denotes guanosine, and “T” denotes thymidine, unless otherwise noted. The letters A, C, G, and T may be used to refer to the bases themselves, to nucleosides, or to nucleotides comprising the bases, as is standard in the art.

[0032] The term “DNA (deoxyribonucleic acid)” or “DNA molecule” refers to a chain of nucleotides comprising deoxyribonucleotides that each comprise one of four nucleobases, namely, adenine (A), thymine (T), cytosine (C), and guanine (G). The term “RNA (ribonucleic acid)” or “RNA molecule” refers to a chain of nucleotides comprising four types of ribonucleotides that each comprise one of four nucleobases, namely; A, uracil (U), G, and C. Certain pairs of nucleotides specifically bind to one another in a complementary fashion (called complementary base pairing). In DNA, adenine (A) pairs with thymine (T) and cytosine (C) pairs with guanine (G). In RNA, adenine (A) pairs with uracil (U) and cytosine (C) pairs with guanine (G).

[0033] Oligonucleotides may be modified, e.g., comprise a modified nucleotide, a modified internucleoside linkage, and/or a modified sugar moiety, or combinations thereof. In some embodiments, particular nucleotide modification(s) may be incorporated that render an oligonucleotide more resistant to nuclease digestion than the native oligoribonucleotide or oligodeoxynucleotide molecules; such modified polynucleotides survive intact for a longer time than unmodified polynucleotides. Exemplary modified polynucleotides include those comprising modified backbones, for example, modified internucleoside linkages such as, methyl phosphonates, phosphotriesters, phosphorothioates short chain alkyl or cycloalkyl inter-sugar linkages heterocyclic inter-sugar linkages or short chain heteroatomic or. As such, the oligonucleotide may be stabilized against nucleolytic degradation, e.g., via incorporation of a modification, e.g., a nucleotide modification.

[0034] A “phosphorothioate linkage” or “phosphorothioate bond” refers to a bond where a sulfur is substituted for one nonbridging oxygen in a phosphodiester linkage, for example in the bonds between nucleotides bases. When phosphorothioates are used to generate oligonucleotides, the modified oligonucleotides may also be referred to as S- oligos. The terms A*, C*, U*, or G* denote a nucleotide that is linked to the next (e.g., 3’) nucleotide with a phosphorothioate bond.

[0035] In some exemplary embodiments, the oligonucleotide is a guide RNA (e.g., that is a single guide RNA molecule). The terms “guide RNA” or “gRNA” is an RNA molecule that binds to a Cas protein (e.g., Cas9 protein) and targets the Cas protein to a specific location within a target DNA. Guide RNAs can comprise two segments: a “DNA-targeting segment” (also called “guide sequence”) and a “proteinbinding segment.” “Segment” includes a section or region of a molecule, such as a contiguous stretch of nucleotides in an RNA. Some gRNAs, such as those for Cas9, can comprise two separate RNA molecules: an “activator-RNA” (e.g., tracrRNA) and a “targeter-RNA” (e.g., CRISPR RNA or crRNA). Other gRNAs are a single RNA molecule (single RNA polynucleotide), which can also be called a “single-molecule gRNA,” a “single-guide RNA,” or an “sgRNA.” See, for example, International Patent Application Publication Nos. WO 2013/176772, WO 2014/065596, WO 2014/089290, WO 2014/093622, WO 2014/099750, WO 2013/142578, and WO 2014/131833, each of which is herein incorporated by reference in its entirety for all purposes. A guide RNA can refer to either a CRISPR RNA (crRNA) or the combination of a crRNA and a trans-activating CRISPR RNA (tracrRNA). The crRNA and tracrRNA can be associated as a single RNA molecule, herein referred to as “single guide RNA” or “sgRNA” or in two separate RNA molecules, herein referred to as “dual guide RNA” or “dgRNA”. For Cas9, for example, a sgRNA can comprise a crRNA fused to a tracrRNA (e.g., via a linker).

[0036] In some exemplary embodiments, the sample can be prepared prior to LC/MS analysis. Preparation steps can include denaturation, dilution, digestion, and separation (for example, centrifugation).

[0037] As used herein, the terms “denaturing” or “denaturation” refers to a process in which the secondary structure of an oligonucleotide is disrupted, such that the double-stranded oligonucleotide is transformed into two complimentary single-stranded oligonucleotides. Denaturation may be full or partial. Denaturation may be performed by a variety of methods including heating double-stranded nucleic acid molecules (e.g., by heating the sample to 85° C. or higher), treating double-stranded nucleic acid molecules with one or more organic solvents (e.g., 0.5 M NaOH, DMSO, formamide, urea, etc.), changing the salt concentration of double-stranded nucleic nucleus molecules, and/or changing the pH of double-stranded nucleic acid molecules. Denaturation may also be performed through enzymatic denaturation, such as through the use of helicases, or other enzymes with helicase activity. Oligonucleotides may also be denatured through interaction with a surface or by a physical process such as stretching beyond a critical length.

[0038] As used herein, the terms “digesting” or “digestion” refers to hydrolysis of one or more phosphodiester bonds in the backbone of an oligonucleotide. Digestion may be performed by physical methods, such as sonication or physical shear, by chemical methods, such as an alkaline compound, piperidine formate, piperidine, dimethyl sulfate, hydrazine, sodium chloride, or combinations thereof, or by enzymatic digestion with a nuclease. The nuclease may be an endonuclease that cleaves the phosphodiester bond within the polynucleotide chain or an exonuclease that cleaves the phosphodiester bond at the end of the polynucleotide chain. Nucleases may cleave non-specifically or at specific sites of the oligonucleotides. Nonlimiting examples of nucleases include non-specific deoxyribonuclease I (DNasel), ribonuclease A or T1 (RNAse A or Tl), and restriction endonucleases.

[0039] In some exemplary embodiments, enzymatic digestion is performed in a buffer solution, most preferably in Tris-EDTA. In some exemplary embodiments, the concentration of Tris-EDTA is about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, or about 100 mM. In some exemplary embodiments, the pH of the buffer solution is about pH 7.0, about pH 7.5, about pH 8.0, or about pH 8.5. Digestion may also be carried out in ambient (room) temperature or above ambient temperature. In some exemplary embodiments, digestion is carried out at about 25 °C, about 37 °C, about 45 °C, or about 55 °C. The digestion mixture may be incubated for a period of time in order to ensure complete digestion. In some exemplary embodiments, the digestion mixture may be incubated for about 1 hour, about 2 hours, about 5 hours, about 8 hours, or about 10 hours.

[0040] As used herein, the term “liquid chromatography” refers to a process in which a biological and/or chemical mixture carried by a liquid can be separated into components as a result of differential distribution of the components as they flow through (or into) a stationary liquid or solid phase. Nonlimiting examples of liquid chromatography include reverse phase liquid chromatography, ion-exchange chromatography, ion-pairing chromatography, ion-pairing reversed-phase liquid chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, or mixed-mode chromatography. In some aspects, the sample or eluate can be subjected to any one of the aforementioned chromatographic methods or a combination thereof.

[0041] In some exemplary embodiments, the liquid chromatography can be ion-pairing reversed phase liquid chromatography. The term “ion-pairing reversed-phase liquid chromatography” refers to a specific form of reversed-phase liquid chromatography in which an ion with a lipophilic residue and positive charge such as an alkylammonium salt, e.g. triethylammonium acetate, is added to the mobile phase as counter ion for the negatively charged oligonucleotide. When used with common hydrophobic mobile phases in the reversed-phase mode, ion pair reagents can be used to selectively increase the retention of the oligonucleotide.

[0042] In an exemplary embodiment, the separation was carried out using a mobile phase comprised of acetonitrile (ACN), methiopropamine (MPA), triethanolamine (TEA), and hexafluoro-2-propanol (HFIP). In one embodiment, the concentration of ACN is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% including any and all values in between. In one embodiment, the concentration of MPA is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% including any and all values in between. In a one embodiment, the concentration of TEA 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM or about 20 mM, including any and all values in between. In a one embodiment, the concentration of HFIP is 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 20 mM, including any and all values in between. In one embodiment, the column temperature can be about 55 °C, about 56°C, about 57 °C, about 58 °C, about 59 °C, about 60 °C, about 61 °C, about 62 °C, about 63 °C, about 64 °C, or about 65 °C.

[0043] As used herein, the term “mass spectrometry” includes the use of a device capable of identifying specific molecular species and measuring their accurate masses. The term is meant to include any molecular detector into which an oligonucleotide may be characterized. The mass spectrometer can be coupled to a liquid chromatography system, wherein the mass spectrometer is capable of performing LC-MS (liquid chromatography-mass spectrometry). A mass spectrometer can include three major parts: the ion source, the mass analyzer, and the detector. The role of the ion source is to create gas phase ions. Analyte atoms, molecules, or clusters can be transferred into gas phase and ionized either concurrently (as in electrospray ionization) or through separate processes. The choice of ion source depends on the application. Non-limiting examples of ion sources include electrospray ionization (ESI), atmospheric pressure ionization (API), matrix assisted laser desorption ionization (MALDI), laser desorption ionization (LDI), and desorption electrospray ionization (DESI). The term “mass analyzer” refers to a device that can separate species, that is, atoms, molecules, or clusters, according to their mass. Nonlimiting examples of mass analyzers include time-of-flight (TOF), magnetic/electric sector, quadrupole mass filter (Q), quadrupole ion trap (QIT), orbitrap, Fourier transform ion cyclotron resonance (FTICR), and also the technique of accelerator mass spectrometry (AMS). [0044] As used herein, term “ion-mobility mass spectrometry” or “IMS” includes the use of a device that combines ion-mobility separation with mass spectrometry, the ion-mobility separation principle being based on the difference in collision cross section of ions in a drift tube when they collide with a buffer gas, the ions being separable according to size and shape. The term “ion-mobility”, also known as ion-mobility, refers to the velocity of movement of positive or negative ions in nW at an electric field strength of IV/m or an electric field force of IN. Ion-mobility separation is based primarily on the shape and size of the ions, and is of particular advantage for analysis of isomers or complexes, etc., which cannot be distinguished by conventional mass spectrometry. After the ions are pre-separated according to mobility, the mass number is obtained through the mass-to-charge ratio of each component, and then the two-dimensional spectrum or the three-dimensional spectrum of the ion-mobility mass spectrum can be obtained.

[0045] As used herein, the terms “cyclic ion-mobility mass spectrometry” or “cIMS” refer to a specific ion-mobility mass spectrometry technique, which separates ions according to their ion-mobility using a closed loop (cyclic) ion-mobility separation device. The cyclic geometry of the ion-mobility device enables ions to travel multiple passes or cycles, increasing their drift times to the detector and relative separation.

[0046] As used herein, the term “mobiligram” refers to a representation of mass spectrometry data as retention and drift times. The term “retention time” refers to the time an analyte spends in a column or the time spent in the stationary and mobile phases. The term “drift time” refers to the time an ion takes from the start of the ion-mobility separation device to the detector.

[0047] It is understood that the present disclosure is not limited to any of the aforesaid sample(s), isomer(s), ohgonucleotide(s), phosphorothioate linkage(s), single guide RNA(s), denaturing, digesting, liquid chromatography, ion-pairing reversed-phase liquid chromatography, mass spectrometry, ionmobility mass spectrometry, cyclic ion-mobility mass spectrometry, or mobiligram(s), and any sample(s), isomer(s), oligonucleotide(s), phosphorothioate linkage(s), single guide RNA(s), denaturing, digesting, liquid chromatography, ion-pairing reversed-phase liquid chromatography, mass spectrometry, ion-mobility mass spectrometry, cyclic ion-mobility mass spectrometry, or mobiligram(s) can be selected by any suitable means.

EXAMPLES

[0048] Sample preparation. To analyze 5’- and 3 ’-end PS modifications, single-guide RNA (sgRNA) sequences were subjected to endoribonuclease digestion by RNaseTl or RNaseAto produce oligonucleotide fragments. RNaseTl specifically cleaves single-stranded RNA at guanine (G) residues whereas RNaseA cleaves at cytosine (C) and uracil (U) residues. Prior to digestion, the sgRNA samples were diluted to 1 mg/rnL in a 10 mM Tris and 0.1 mM Tris-ethylenediamine tetraacetic acid (EDTA) buffer, pH 8.0, and denatured at 95 °C for 3 minutes before rapid cooling on ice. To digest the sgRNA sample, 25 units of RNaseTl or RNaseA were added per 1 pg of substrate, and the mixture was incubated at about 37° C for about 1 hour. A combination of RNaseTl and RNase could also be used to generate fragments with the shortest length.

[0049] Ion-pairing reversed-phase liquid chromatography-mass spectrometry (IPRP-LC/MS) analysis. The sgRNA fragments were analyzed by IPRP-LC-MS. LC separation of the sgRNA fragments was performed on a UPLC system equipped with an oligonucleotide column for separating oligonucleotides via ion-pairing reversed-phase chromatography (2.1 x 150 mm, 200 A, 1.7 pm, WATERS™ ACQUITY® Premier Oligonucleotide BEH Cl 8 column). The column temperature was set at 60 °C. The injection loads consisted of 1 pg of the digested sgRNA. Triethanolamine (TEA), hexafluoro-2-propanol (HFIP), and acetonitrile (ACN) were used for mobile phase compositions as follows. Mobile phase A (15 mM TEA (0.2%), 50 mM (0.5%) HFIP in H₂O) and B (15 mM TEA (0.2%), 50 (0.5%) HFIP in MPA/ACN 80/20 (v/v) at a flow rate of 0.2 mL/min was applied to separate the stereoisomers of the sgRNA fragments. The separated stereoisomers were monitored by UV (260 nm) using a photodiode array (PDA) detector or by MS using a cyclic ion-mobility mass spectrometer. [0050] Cyclic ion-mobility mass spectrometry (cIMS) analysis. The sgRNA fragments separated by IPRP-LC were monitored by cIM device connected to a time-of-flight (TOF) cyclic ion-mobility mass spectrometer. The sgRNA fragments were subjected to negative electrospray ionization at a flow rate of 0.2 mL/min, and the following cIMS source parameters were used: capillary 2 kV, cone 40 V, source offset 10 V, and source temperature 100 °C, desolvation temperature 100 °C, desolvation gas 800 L/hour, nebulizer gas 6 Bar, and reference capillary 2.5 kV. Mass spectra was collected over a mass range between 400 m'z to 2000 m/z.

[0051] Example 1: Characterization of sgRNA Stereoisomers by IPRP-LC/MS Without cIMS [0052] To evaluate the impact of cyclic ion-mobility separation on the characterization of sgRNA stereoisomers, sgRNA sequences containing one, two, and three PS linkages were first analyzed by LC/MS without cIMS. The sgRNA sequences were digested by RNaseTl and/or RNaseA to generate short oligonucleotide fragments, and the fragments were then subjected to IPRP-LC/MS.

[0053] Full nucleic acid sequences for sgRNA molecules labelled as sgRNA- 1 and sgRNA-2 are provided in Table 2, below, along with the endonuclease used for digestion of the full sequences into 5’- and 3 ’-end fragments.

Table 2

[0054] Model molecule sgRNA-1 was digested into 5 ’-end and 3 ’-end fragments containing three PS linkages (N*N*N*), two PS linkages ((NNN)**), one PS linkage (NNN), or zero PS linkages (NNN). Model molecule sgRNA-2 was digested into 5 ’-end fragments which similarly varied in their PS linkages. 5’-end fragments of sgRNA-1 and sgRNA-2 are described in Table 3A and 3’-end fragments of sgRNA-1 are described in Table 3B, below. Fragments containing two PS linkages and one PS linkage each had 3 constitutional isomers, whereas sequences with three PS linkages or zero PS linkages did not have any constitutional isomers. Each constitutional isomer had 2^r diastereomers due to the S_P/R_P configuration, where ‘r’ denotes the number of PS present in the sequences. Therefore, each sequence contained 8 isomers with three PS linkages, 12 isomers with two PS linkages, 6 isomers with one PS linkage, and a single species with zero PS linkages. In other words, up to 27 species could potentially coexist in each complex mixture. All constitutional isomers and stereoisomers with the same number of PS had identical molecular masses, resulting in the same m'z values observed during MS analysis.

Table 3A

Table 3B

[0055] The mixtures were then subjected to IPRP-LC/UV/MS under optimized separation conditions. The artificial PS degradation during digestion and LC separation as well as the potential non-specific digestion were confirmed to be negligible. Extracted ion chromatograms (XICs) obtained from IPRP- LC/MS analysis of sgRNA fragments with zero PS linkages (NNN), one PS linkage ((NNN)*), two PS linkages ((NNN)**), and three PS linkages ((NNN)***) at 5’ or 3’ ends. FIG. 2A shows XICs for the 5’- end fragment of sgRNA- 1 and its PS — > PO derivatives (bottom three panels) upon IPRP-LC/MS separation and detection. FIG. 2B shows XICs for the 3 ’-end fragment of sgRNA- 1 and its PS — > PO derivatives (bottom three panels) upon IPRP-LC/MS separation and detection. As expected, separation of sgRNA fragments containing zero PS linkages confirmed the presence of one stereoisomer, as evidenced by the appearance of a single peak in the bottom panel of each of FIG. 2A and FIG. 2B. Further, separation of sgRNA containing one or two PS linkages confirmed the presence of several stereoisomers, as evidenced by the appearance of multiple peaks in the middle panels of each of FIG. 2A and FIG. 2B. However, several isomers appeared to coelute as one peak, as the 6 isomers for 5’-(NNN)* and 3’-(NNN)* appeared as 3-4 peaks and the 12 isomers for 5’-(NNN)** and 3’-(NNN)*** appeared as 5-6 and 9 peaks, respectively. As shown in FIG. 2C, similar results were observed for the 5’-end fragment of sgRNA-2, 5’-mG*mU*mA*C. Thus, these results suggested that separation by IPRP- LC/MS could not resolve all the stereoisomers of the sgRNA fragments. Without complete separation of individual PS-induced isomers, the co-eluting peaks could lead to misidentification and inaccurate quantification, eventually compromising the reliability of the analytical results.

[0056] To determine relative abundance of fragments, the total area under the curve (AUC) for each 5’- end and 3 ’-end fragment of sgRNA- 1 was quantified based on the XICs shown in FIG. 2 A and FIG. 2B. Relative abundance values are provided in Table 4, below. Relative abundance of each individual stereoisomer of (NNN)* and (NNN)** could not be quantified since the stereoisomers could not be resolved.

Table 4

[0057] Example 2: Characterization of sgRNA Stereoisomers by IPRP-LC/MS with cIMS

[0058] To resolve separation of the sgRNA stereoisomers, the sgRNA fragments were analyzed by IPRP-LC/MS using a cyclic ion-mobility mass spectrometer (cIMS). The sgRNA fragments were first separated by IPRP-LC, then further separated by cIMS followed by TOF analysis. Because cIMS further separates fragment ions by drift time, a mobiligram (drift time versus retention time) was used to visualize the mass spectra data in addition to XIC.

[0059] A 5 ’-end fragment of sgRNA- 1 was modified to have two PS linkages and analyzed as described above, using a total of 4 cIMS passes. FIG. 3A shows both the XIC and mobiligram of a mixture of a 5’- end sgRNA fragment modified to have two PS linkages. The mobiligram revealed that twelve dots could be distinguished in the sgRNA fragment, each of which represented a unique stereoisomer. To identify each dot, the mobiligram was compared to mobiligrams of synthetic oligonucleotide standards, shown in FIG. 3B. Analysis of the stereoisomers as compared to the oligonucleotide standards confirmed the presence of four stereoisomers for 5-NN*N*, four stereoisomers for 5-N*N*N, and four stereoisomers for 5-N*NN*. [0060] The total number of stereoisomers identified from the oligonucleotide standards was greater than that of the sgRNA fragment, as two of the isomers (5-N*N*N and 5-N*NN*) overlapped at a retention time of 24.6 minutes and a drift time of 18 ms. To improve the resolution between the two isomers, separation time was extended by performing 20 cIMS passes with the same 5 ’-end sgRNA fragment. FIG. 4A shows the mobiligram of the sgRNA isomers with an extended separation time, as compared to mobiligrams of synthetic oligonucleotide standards shown in FIG. 4B. Analysis of the stereoisomers as compared to the oligonucleotide standards indicated that the 5’-N*N*N and 5’-N*NN* isomers were well-resolved.

[0061] In addition to the sgRNA fragment described above, other sgRNA fragments were analyzed by cIMS.

[0062] FIG. 5A shows the mobiligram of the 5’-end fragment of sgRNA-1 modified to have one PS linkage. The mobiligram obtained by 13 cIMS passes revealed the presence of six stereoisomers. Comparison of the mobiligram of the sgRNA fragments to the mobiligrams of synthetic oligonucleotide standards (FIG. 5B) confirmed the presence of two stereoisomers for 5’-NNN*, two stereoisomers for 5- NN*N, and two stereoisomers for 5’-N*NN.

[0063] FIG. 6A shows the mobiligram of the 3 ’-end fragment of sgRNA-1 modified to have two PS linkages. The mobiligram obtained by cIMS revealed the presence of eleven stereoisomers. Comparison of the mobiligram of the sgRNA isomers to the mobiligrams of synthetic oligonucleotide standards (FIG. 6B) confirmed the presence of three stereoisomers for N*N*N-3’, four stereoisomers for NN*N*- 3’, and four stereoisomers for N*NN*-3’.

[0064] FIG. 7A shows the mobiligram of the 3 ’-end fragment of sgRNA-1 modified to have one PS linkage. The mobiligram obtained by cIMS revealed the presence of six stereoisomers. Comparison of the mobiligram of the sgRNA isomers to the mobiligrams of synthetic oligonucleotide standards (FIG. 7B) confirmed the presence of two stereoisomers for N*NN-3’, two stereoisomers for NN*N-3’, and two stereoisomers for NNN*-3’.

[0065] Together, these results demonstrated that cIMS improved the identification fidelity of stereoisomers of sgRNA fragments.

[0066] Example 3: Quantification of sgRNA Stereoisomers by cIMS

[0067] To assess the relative abundance of each stereoisomer present within the sgRNA fragments after ion-mobility separation, the mobiligram data from Example 2 was extracted to produce total ion current (TIC) profiles of the stereoisomers. FIG. 8 shows the TIC profiles of stereoisomers 1-12 of the 5 ’-end fragment of sgRNA-1 modified to have two PS linkages. For the majority of the stereoisomers, the resolution was sufficient to produce a TIC profile for each stereoisomer. However, for stereoisomers 3 and 4, 8 and 9, and 11 and 12, the resolution was not sufficient to obtain individual TIC profiles, and the relative abundance of these stereoisomers could not be determined. As demonstrated in Example 2, the resolution between stereoisomers 3 and 4 was improved by extending the separation time. The mobiligram data obtained from this analysis was then extracted in order to obtain TIC profiles of stereoisomers 3 and 4. FIG. 9 shows the individual TIC profiles of stereoisomers 3 and 4 that was acquired due to the improved resolution.

[0068] The peak areas of the TIC profiles were integrated and used for quantifying the amount of each stereoisomer in the sgRNA fragment. Comparison of the observed relative abundance of the isomers from the mobiligram data and the expected relative abundance from OD260 measurements of the 5 ’-end fragment of sgRNA-1 with two PS linkages is shown in Table 5, below. Table 5 shows the abundance of each stereoisomer, as well as the abundance of the constitutional isomers, as determined by integration of the TIC profile. The observed relative abundance of the constitutional isomers was then determined by calculating the ratio of the peak area sum of the constitutional isomers to the total peak area sum.

Table 5 further shows the observed relative abundance of the constitutional isomers from the mobiligram data compared to the expected relative abundance from OD measurements at 260 nm. The observed relative abundance obtained from the mobiligram data was consistent with the expected relative abundance obtained from the OD260 measurements, suggesting the high accuracy of this quantification method.

Table 5

[0069] The same quantification method was used to evaluate the relative abundance of isomers in other sgRNA fragments. The observed relative abundance of the isomers of the other sgRNA fragments correlated well with the expected relative abundance. FIG. 10 shows the TIC profiles of stereoisomers 1- 6 of the 5’-end fragment of sgRNA-1 with one PS linkage. As shown in FIG. 10, an individual TIC profile was obtained for each stereoisomer. Comparison of the observed relative abundance of the isomers from the mobiligram data and the expected relative abundance from OD260 measurements of a 5 ’-end sgRNA fragment with one PS linkage is shown in Table 6, below. Table 6 shows the observed relative abundance of the constitutional isomers of the sgRNA fragment, as determined by adding the peak area of the stereoisomers, compared to the expected relative abundance, as determined by OD260 measurements.

Table 6

[0070] FIG. 11 shows the TIC profiles of stereoisomers 1-11 of the 3 ’-end fragment of sgRNA-1 with two PS linkages. For most of the stereoisomers, an individual TIC profile was obtained. However, resolution of stereoisomers 5, 6 and 7 was not sufficient to produce individual TIC profiles. Comparison of the observed relative abundance of the isomers from the mobiligram data and the expected relative abundance from OD260 measurements of a 3 ’-end sgRNA fragment with two PS linkages is shown in Table 7, below. Table 7 shows the observed relative abundance of the constitutional isomers of the sgRNA fragment compared to the expected relative abundance.

Table 7

[0071] FIG. 12 shows the individual TIC profiles of stereoisomers 1-6 of the 3’-end fragment of sgRNA- 1 with one PS linkage. Comparison of the observed relative abundance of the isomers from the mobihgram data and the expected relative abundance from OD260 measurements of 3 ’-end sgRNA fragment with two PS linkages is shown in Table 8, below. Table 8 shows the observed relative abundance of the constitutional isomers of the sgRNA fragment compared to the expected relative abundance.

Table 8

[0072] The peak area data from the TIC profiles was further used to calculate the abundance of each constitutional isomer relative to the total abundance of isomers present in the sgRNA sequence. From these calculations, the relative abundance of PS at each linkage position was also determined.

Comparison of the observed relative abundance of the isomers of an sgRNA sequence and the expected relative abundance from OD260 and LC/UV measurements is shown in Table 9, below. Table 9 shows the observed relative abundance of isomer and PS at each linkage position compared to the expected relative abundance, as determined by OD260 and LC/UV measurements. The observed experimental measurements aligned well with the expected values, demonstrating the feasibility of this method for quantifying isomers of sgRNA sequences and PS linkages.

Table 9

[0073] Example 4: Relative Quantification of PS Modification

[0074] With the complete separation of all isomers from mixtures achieved, the relative quantification of PS was performed using a two-step method. First, the total peak areas of sequences with different numbers of PS were obtained from the extracted ion chromatograms of Example 1 in an MS-only channel. The relative abundances were then calculated by dividing the peak area of the species of interest by the sum of the peak areas of all the species. Next, each isomer of interest was further extracted from the ion mobiligram of Example 2 to obtain the individual or total peak areas, as shown in FIGS. 13A-13C. In cases where isomers 3 and 4 overlapped (FIG. 13B, top panel) with different PS positions, the relative abundances of both isomers was calculated from the XIC peak areas (FIG. 13B, middle and bottom panels) generated in the separate high-pass cIMS channel. The combined peak area (FIG. 13B, top panel) was then fractioned to individual diastereomers accordingly. Thus, the relative abundance of each constitutional isomer with the same number of PS was obtained through cIMS. Finally, the relative abundance of each species was determined by multiplying the total relative abundance calculated using MS-only XIC with the relative percentage obtained from cIMS. As indicated in Table 10, below, each species measured nearly 12.5%, consistent with the feed ratio in the mixtures.

Table 10: Relative Abundance Calculation of Isomers

[0075] In order to achieve reliable quantification, the data acquisition rates in both the LC retention time and cIMS drift time dimensions were evaluated. As shown in FIG. 14A, the relative abundance of each standard remained consistent when extending the acquisition time to 1000 milliseconds. Above this value (slower acquisition rate), fewer data points failed to capture the actual peak profile in EIC, which eventually resulted in higher variations in both quantification precision and accuracy (FIG. 14B). Meanwhile, higher sensitivity was observed with longer acquisition scan time with the same injection amount of samples, possibly due to the benefits of reporting “packaged” ions (FIG. 14C). Based on these evaluations, we decided to use 1000 ms as the acquisition scan time for subsequent experiments. In the other dimension, the interval of measuring drift time is mainly determined by the scan rate in time of flight (TOF) mass analyzer, which is kept constant at 0.17 ms and remains independent of other variables.

[0076] After determining the acquisition scan time, its sensitivity, precision, dynamic range, repeatability, and accuracy was evaluated. We prepared mixtures of eight synthetic standards of sgRNA- 1 at a series of dilutions. Following digestion and IPRP-LC/cIMS analysis in triplicate, the mean and standard deviation of ion abundances for each species were plotted against the injection amounts, exemplified by the representative data from 5’-mAmCmA*AAG in FIG. 14A. The limit of detection (LOD) was observed to be close to 9.7 finol, as the signal-to-noise ratios (S/N) were approximately 3 across different species. The results also exhibited excellent linearity for injection amounts ranging from 9.7 to 7730 femtomoles, with an R² value consistently exceeding 0.99. The only exception was the 5’- mAmCmAAAG, where the ion abundance began to plateau above 1900 finol in the MS-only channel, likely due to signal saturation at high injection amounts. However, since this is the end-product with complete PS PO conversions, which typically appeared in low percentages in real scenarios, this slightly narrower dynamic range was unlikely to compromise the quantification performance. Overall, this high degree of linearity over an 800-fold range indicates that this analytical method was capable of reliably quantifying PS modifications with high sensitivity and consistency, covering a wide range of approximately 0.2% to 100%. In addition to low signal-to-noise ratios resulting from a low injection amount, peak distortion was observed in cIMS with high injection amounts. Without being limited by theory, this distortion could be attributed to the space charge effect within the cyclic separation cell, which could potentially degrade the desired separation resolution in certain cases.

[0077] The relative standard deviations (RSD) of the ion abundances for all eight synthetic standards were categorized by different injection amounts, as shown in FIG. 15. The mean and standard deviation of the RSDs for each group are presented. With the exception of the 9.7 fmol injection amount, which exhibits slightly higher variation, all other injection amounts demonstrate excellent repeatability, with an overall RSD of approximately 2.5%. It is important to note that the slightly higher variations observed at the 9.7 fmol injection amount (up to 16.5%) are primarily due to the low S/N, which is near the LOD. Meanwhile, higher injection amounts achieve more precise quantifications with low RSD.

[0078] The accuracy of this relative quantification method was assessed by preparing six different mixtures with varying ratios of synthetic standards. The experimental values of each standard in the mixtures were plotted against the expected values in FIG. 16. All data points were centered near the diagonal dashed line, indicating that the experimental values closely matched the expected values, with the average error smaller than 0.5%. This alignment demonstrated the favorable accuracy of the method, as well as the robustness supported by the consistent results across different mixtures.

[0079] Example 5: PS to PO Conversion Under Oxidative Stress

[0080] PS linkages are known to be susceptible to oxidation during various stages, including synthesis, storage, processing, and in vivo conditions, where PS can convert back to PO. To better understand the stability of multiple PS in sgRNA, the methods described in Examples 1 and 2 were utilized to monitor the kinetics of PS — > PO under oxidative forced degradation conditions. The model sgRNA- 1 with full PS at the first three linkages was stressed using 0.1% (v/v) H2O2 at room temperature over 72 hours. As shown in FIG. 17A, the relative abundance of the precursor with three PS linkages (3 x PS) gradually decreased, while the overall relative abundances of the oxidized products increase, with the order of 2 x PS > 1 x PS > 0 x PS. This result aligned was obtained using a standard bottom-up LC/MS oligonucleotide mapping method. However, due to the limited characterization resolution, detailed information regarding the position and extent of individual PS — > PO conversion was not available for the oxidized products with 2 x PS and 1 x PS. [0081] With the newly developed method, all the PS-induced isomers were fully separated and accurately quantified. This allowed for distinguishing kinetics of forming individual constitutional isomers for the oxidized products with 1 x and 2 x PS — > PO conversions, as shown in FIG. 17B. Additionally, the relative abundance of each oxidized product was calculated to determine the rates of PS PO conversion at different linkage positions, as shown in FIG. 17C. Interestingly, the PS — > PO conversion rates followed the order: 1st linkage > 2nd linkage > 3rd linkage, indicating that terminal PS groups were oxidized more rapidly. This suggested that PS oxidation was not entirely random in solution but could be correlated with the structural context or accessibility of the PS position. These fundamental understandings, which highlight the non-random nature of PS oxidation process, were uniquely enabled by the novel analytical methods described herein.

[0082] These experiments demonstrate the successful use of the method described herein to obtain information about the isomers and backbone modifications of oligonucleotide sequences. These methods provided high-resolution separation of the stereoisomers of the sgRNA sequence. Further, information obtained from the analysis was used to identify the stereoisomers and quantify the relative abundance of isomer and PS backbone modification at each linkage position. This information could be used to, for example, monitor and/or modify the production process of an oligonucleotide therapeutic in order to achieve acceptable product consistency.

[0083] The subject matter described herein can be understood further with reference to the following enumerated items.

[0084] Item 1. A method for identifying and/or quantifying an isomer of an oligonucleotide, comprising:

(a) subjecting a sample including the isomer of the oligonucleotide to digestion conditions to form a digested sample, wherein the digested sample comprises at least one oligonucleotide fragment;

(b) subjecting the digested sample to liquid chromatography;

(c) subjecting the eluate of (b) to cyclic ion-mobility mass spectrometry analysis to obtain a separation profile of the at least one oligonucleotide fragment;

(d) comparing the separation profile of the at least one oligonucleotide fragment to the separation profile of at least one oligonucleotide standard to identify and/or quantify the isomer of the oligonucleotide; wherein the at least one oligonucleotide standard comprises the same nucleotide sequence and modification(s) as the at least one oligonucleotide fragment.

[0085] Item 2. The method of item 1, further comprising identifying a modification of the isomer of the oligonucleotide based on the identity of the isomer of the oligonucleotide.

[0086] Item 3. The method of item 1, wherein the sample is subjected to denaturing conditions prior to digestion. [0087] Item 4. The method of item 1, wherein subjecting the sample to denaturing conditions includes contacting the sample to a temperature of about 95 °C.

[0088] Item 5. The method of item 1, wherein the oligonucleotide is a sgRNA.

[0089] Item 6. The method of item 1, wherein the oligonucleotide comprises at least one backbone modification.

[0090] Item 7. The method of item 6, wherein the backbone modification is phosphorothioate (PS). [0091] Item 8. The method of item 1, wherein subjecting the sample including the isomer of the oligonucleotide to digestion conditions includes contacting the sample to one or more digestive enzyme. [0092] Item 9. The method of item 8, wherein the one or more digestive enzyme comprises RNaseTl and/or RNaseA.

[0093] Item 10. The method of item 1, wherein the isomer of the oligonucleotide is a stereoisomer or a constitutional isomer.

[0094] Item 11. The method of item 1 , wherein the liquid chromatography step comprises ion-pairing reversed phase liquid chromatography.

[0095] Item 12. The method of item 1 , wherein the liquid chromatography comprises reversed phase liquid chromatography, ion-pairing reversed phase liquid chromatography, ion exchange chromatography, anion exchange chromatography, weak cation exchange chromatography, strong cation exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction liquid chromatography (HILIC), mixed-mode chromatography, or a combination thereof.

[0096] Item 13. The method of item 1 , wherein the cyclic ion-mobility mass spectrometer comprises a time-of-flight mass analyzer.

[0097] Item 14. The method of item 1, wherein the cyclic ion-mobility mass spectrometer comprises an electrospray ionization ion source, a nano-electrospray ionization ion source, or a desorption electrospray ionization ion source.

[0098] Item 15. The method of item 1, wherein the cyclic ion-mobility mass spectrometer is coupled to the liquid chromatography system.

[0099] Item 16. The method of item 1, wherein an amount of the sample is about 1 ug.

[0100] Item 17. The method of item 1, wherein the separation profile is a mobiligram.

[0101] Item 18. A method for identifying and/or quantifying a modification of an oligonucleotide, comprising:

(a) subjecting a sample including the oligonucleotide to digestion conditions to form a digested sample, wherein the digested sample comprises oligonucleotide fragments;

(b) subjecting the digested sample to liquid chromatography; (c) subjecting the eluate of (b) to cyclic ion-mobility mass spectrometry analysis to obtain separation profiles of the oligonucleotide fragments;

(d) comparing the separation profiles of the oligonucleotide fragments to the separation profiles of oligonucleotide standards to identify and quantify each oligonucleotide fragment; and

(e) identifying and/or quantifying the modification of the oligonucleotide based on the identity and quantity of each oligonucleotide fragment, wherein the oligonucleotide standards comprise the same nucleotide sequence and modification(s) as the oligonucleotide fragments.

[0102] Item 19. The method of item 19, wherein the oligonucleotide comprises isomers thereof.

[0103] Item 20. The method of item 20, wherein the isomers of the oligonucleotide are stereoisomers and/or constitutional isomers.

[0104] Item 21. The method of item 19, wherein the sample is subjected to denaturing conditions prior to digestion.

[0105] Item 22. The method of item 19, wherein subjecting the sample to denaturing conditions includes contacting the sample to a temperature of about 95 °C.

[0106] Item 23. The method of item 19, wherein the oligonucleotide is a sgRNA.

[0107] Item 24. The method of item 19, wherein the oligonucleotide comprises at least one backbone modification.

[0108] Item 25. The method of item 25, wherein the backbone modification is phosphorothioate (PS). [0109] Item 26. The method of item 19, wherein subjecting the sample including the isomer of the oligonucleotide to digestion conditions includes contacting the sample to one or more digestive enzyme. [0110] Item 27. The method of item 27, wherein the one or more digestive enzyme comprises RNaseTl and/or RNaseA.

[0111] Item 28. The method of item 19, wherein the liquid chromatography step comprises ion-pairing reversed phase liquid chromatography.

[0112] Item 29. The method of item 19, wherein the liquid chromatography comprises reversed phase liquid chromatography, ion-pairing reversed phase liquid chromatography, ion exchange chromatography, anion exchange chromatography, weak cation exchange chromatography, strong cation exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction liquid chromatography (HILIC), mixed-mode chromatography, or a combination thereof.

[0113] Item 30. The method of item 19, wherein the cyclic ion-mobility mass spectrometer comprises a time-of-flight mass analyzer. [0114] Item 31. The method of item 19, wherein the cyclic ion-mobility mass spectrometer comprises an electrospray ionization ion source, a nano-electrospray ionization ion source, or a desorption electrospray ionization ion source.

[0115] Item 32. The method of item 19, wherein the cyclic ion-mobility mass spectrometer is coupled to the liquid chromatography system.

[0116] Item 33. The method of item 19, wherein an amount of the sample is about 1 ug.

[0117] Item 34. The method of item 19, wherein the separation profile is a mobiligram.

[0118] Item 35. A method for quantifying an isomer of an oligonucleotide, comprising:

(a) subjecting a sample including the isomer of the oligonucleotide to digestion conditions to form a digested sample, wherein the digested sample comprises at least one oligonucleotide fragment;

(b) subjecting the digested sample to liquid chromatography;

(c) subjecting the eluate of (b) to cyclic ion-mobility mass spectrometry analysis to obtain a separation profile of the at least one oligonucleotide fragment; and

(d) quantifying the isomer of the oligonucleotide based on the separation profile of the at least one oligonucleotide fragment.

[0119] The present disclosure is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the disclosed methods in addition to those described herein will be apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

Claims

1. A method for characterizing a sample containing a modified oligonucleotide, the method compnsing: subjecting a sample including the modified oligonucleotide and an isomer of the modified oligonucleotide to a digestion condition to form a digested sample comprising an oligonucleotide fragment of the isomer; subjecting the digested sample to liquid chromatography to form an eluate; subjecting the eluate to cyclic ion-mobility mass spectrometry to obtain a separation profile of the oligonucleotide fragment; comparing the separation profile of the oligonucleotide fragment to a separation profile of an oligonucleotide standard, wherein the oligonucleotide standard comprises: a nucleic acid sequence identical to a nucleic acid sequence within the oligonucleotide fragment; and a nucleic acid modification identical to a nucleic acid modification of the oligonucleotide fragment; and characterizing the isomer based on a comparison of the separation profile of the oligonucleotide fragment to the separation profile of the oligonucleotide standard.

2. The method of claim 1, wherein the nucleic acid modification is a backbone modification.

3. The method of claim 1, wherein the nucleic acid modification is a phosphorothioate modification.

4. The method of claim 1, wherein subjecting the sample to a digestion condition includes contacting the sample to a digestive enzyme.

5. The method of claim 1, wherein subjecting the sample to a digestion condition includes contacting the sample to RNAaseTl.

6. The method of claim 1, wherein subjecting the sample to a digestion condition includes contacting the sample to RNaseA.

7. The method of claim 1, wherein subjecting the sample to a digestion condition includes contacting the sample to RNAaseTl and RNaseA.

8. The method of claim 1, wherein the separation profile includes a mobiligram.

9. The method of claim 1, further comprising identifying the nucleic acid sequence within the oligonucleotide fragment.

10. The method of claim 1, further comprising quantifying an amount of the oligonucleotide fragment in the eluate.

11. The method of claim 1, further comprising quantifying an amount of the isomer in the sample based on a quantification of an amount of the oligonucleotide fragment in the eluate.

12. The method of claim 1, further comprising subjecting the sample to a denaturing condition, prior to subjecting the sample to a digestion condition.

13. The method of claim 1, wherein subjecting the digested sample to liquid chromatography comprises subjecting the sample to ion-pairing reversed phase liquid chromatography.

14. The method of claim 1, wherein subjecting the eluate to cyclic ion-mobility mass spectrometry comprises subjecting the eluate to a cyclic ion-mobility mass spectrometer comprising a time-of-flight mass analyzer.

15. The method of claim 1, wherein the modified oligonucleotide is a single guide RNA.