US-12625133-B2 - Chemical denaturation for oligonucleotide analysis

Abstract

The present disclosure provides compositions and methods for sample processing, particularly for oligonucleotide analysis e.g. analysis of formulated nucleic acid drugs. A composition for pretreating at least one target nucleic acid in a biological mixture provided herein includes a chaotropic agent selected from a substituted guanidine, a substituted amidine, a substituted quaternary amine, or a combination thereof, an optional protease, and/or an optional disulfide-reducing agent. Methods of analyzing at least one target nucleic acid in a biological mixture is also provided herein. Furthermore, the present disclosure provides methods for quantifying at least one target cationic lipid interacting with a nucleic acid.

Inventors

• Matthew A Lauber
• Jennifer M. NGUYEN
• Xiaoxiao Liu
• Michael Donegan

Assignees

• WATERS TECHNOLOGIES CORPORATION

Dates

Publication Date

20260512

Application Date

20221028

Claims (18)

1. 1 . A composition for pretreating at least one target nucleic acid in a biological mixture prior to bioanalytical analysis, the composition comprising: a chaotropic agent, a protease, and a disulfide-reducing agent, wherein the chaotropic agent is selected from a substituted guanidine, a substituted amidine, a substituted quaternary amine, or a combination thereof, wherein the composition has a pH value of about 4 to about 10.
2. 2 . The composition of claim 1 , wherein the chaotropic agent comprises tert-butyl tetramethylguanidine, and the protease comprises Proteinase K.
3. 3 . The composition of claim 1 , wherein the substituted guanidine comprises at least one from the group of tetramethylguanidine, tertbutyl tetramethylguanidine, triazabicyclodecene, or combinations thereof.
4. 4 . The composition of claim 3 , wherein the substituted guanidine of tetramethylguanidine is 1,1,3,3-tetramethylguanidine with the chemical structure of
5. 5 . The composition of claim 3 , wherein the substituted guanidine of tertbutyl tetramethylguanidine is 2-tert-butyl-1,1,3,3-tetramethylguanidine with the chemical structure of
6. 6 . The composition of claim 3 , wherein the substituted guanidine of triazabicyclodecene is 1,5,7-triazabicyclo[4.4.0]dec-5-ene with the chemical structure of
7. 7 . The composition of claim 1 , wherein the substituted guanidine is a guanidinium cation.
8. 8 . The composition of claim 1 , wherein the substituted quaternary amine is tetramethyl ammonium or tetraethylammonium, or combination thereof.
9. 9 . The composition of claim 1 , wherein the substituted amidine comprises at least one from the group of hexanimidamide, acetamidine, propanimidamide, or combinations thereof.
10. 10 . The composition of claim 1 , wherein the protease comprises a serine protease, a threonine protease, a cysteine protease or a combination thereof.
11. 11 . A method for detecting at least one target nucleic acid in a sample comprising a biological mixture, comprising the steps of: a) incubating the sample with a composition comprising a chaotropic agent selected from a substituted guanidine, a substituted amidine, a substituted quaternary amine, or a combination thereof; and a protease, thereby disrupting one or more intermolecular interaction(s) of at least one target nucleic acid; b) optionally heating the sample for a predetermined amount of time; c) extracting the at least one target nucleic acid from the sample; and d) detecting the at least one target nucleic acid using an analytical method.
12. 12 . The method of claim 11 , wherein the analytical method comprises a mass spectroscopy.
13. 13 . The method of claim 11 , further comprising a step of quantifying the at least one target nucleic acid in the sample by using a mass spectrometry.
14. 14 . The method of claim 11 , wherein the length of at least one target nucleic acid is about 5 to about 10000 individual nucleotides.
15. 15 . The method of claim 11 , wherein the at least one target nucleic acid is selected from a DNA-based oligonucleotide or antisense oligonucleotide, a RNA-based oligonucleotide, siRNA, shRNA, RNA, mRNA, snoRNA, stRNA, smRNA, pre- and pri-microRNA, other non-coding RNAs, ribosomal RNA, derivatives thereof, amplicons, and any combination thereof.
16. 16 . The method of claim 11 , wherein the heating comprises maintaining the temperature in a range from 50° C. to 100° C.
17. 17 . The method of claim 11 , wherein the sample is or is derived from a biological fluid selected from the group consisting of blood, urine, spinal fluid, synovial fluid, sputum, semen, saliva, tears, gastric juices and extracts and/or dilutions/solutions thereof.
18. 18 . A method of quantifying at least one target cationic lipid in a sample, wherein the sample comprises at least one target cationic lipid interacting with a nucleic acid, the method comprising the steps of: a) incubating the sample with a composition comprising a chaotropic agent selected from a substituted guanidine, a substituted amidine, a substituted quaternary amine, or a combination thereof; and an optional protease, thereby displacing the at least one target cationic lipid from the nucleic acid that is interacting with the at least one target cationic lipid; b) optionally heating the sample for a predetermined amount of time; c) extracting the at least one target cationic lipid from the sample; and d) quantifying the at least one target cationic lipid using an analytical method.

Description

CROSS-REFERENCE TO RELATED APPLICATION This application claims priority and benefit to U.S. Provisional Patent Application No. 63/273,294, filed on Oct. 29, 2021, and entitled “CHEMICAL DENATURATION FOR OLIGONUCLEOTIDE ANALYSIS”, the contents of which is incorporated herein by reference in its entirety. FIELD OF THE TECHNOLOGY The present disclosure generally relates to composition and methods for sample pretreatment prior to qualitative and/or quantitative detection of oligonucleotides. Specifically, this disclosure relates to pretreating at least one target nucleic acid in a biological mixture prior to bioanalytical analysis. The compositions of the present disclosure include a chaotropic agent, an optional protease, and/or an optional disulfide-reducing agent. The present disclosure also relates to methods for detecting at least one target cationic lipid interacting with a nucleic acid. BACKGROUND Oligonucleotides are increasingly being developed as direct therapeutic agents against a wide range of disease conditions. They have attracted increasing attention from the biopharmaceutical industry due to the successes of applying this new modality for the treatment of rare diseases as well as their potential in treating common diseases and even viruses, such as SARS-CoV-2 (COVID-19). Therefore, detection and determination of oligonucleotides and their metabolites has become prominent for drug development and evaluation. However, analyzing oligonucleotides and their metabolites from complex biological matrices such as blood, plasma, tissue, urine presents significant challenges as complex matrices introduce a host of potential interferences such as salts, proteins, lipids, membrane constituents and macromolecular complexes such as ribosomes, spliceosomes, and histones. Any one of these interferences can make it a challenge to obtain accurate and reliable results. Unlike protein-based drugs that generally only bind to extracellular soluble targets or targets on the cell surface, oligonucleotide therapeutics must reach the intracellular targets in the cytoplasm and nucleus to exert pharmacological activities, and as such oligonucleotide therapeutics are by their design made to exhibit a very high degree of protein binding. With this in mind, bioanalytical analysis of oligonucleotides in a biological matrix requires extraction of these highly bound oligonucleotides from freely circulating plasma, from cell surface proteins and also from intercellular domains. The most commonly used techniques to facilitate extraction include protein precipitation (PPT), liquid-liquid extraction (LLE), and solid phase extraction (SPE). These extraction techniques can be used individually or as a combination. However, these traditional techniques suffer from low extraction recoveries, long preparation times, and significant sample manipulation, which can lead to oligo degradation, and limited ability to multiplex the method in an automated format. Critically, as the time for extraction from a biological matrix increases, considerations such as ex vivo conversion of oligonucleotides via nuclease activity could lead to obtaining inaccurate data. SUMMARY In general, it is an object of the present technology to obviate or mitigate at least one disadvantage of previous methods for detection and quantification of oligonucleotides in biological matrices. In general, sample pretreatment methods of the present technology can be utilized to control cell lysis and biological sample disruption for accurate quantification of oligonucleotides and/or lipids using bioanalytical techniques. In one aspect, provided herein is a method for disrupting the local microenvironment of at least one target nucleic acid in a biological sample in order to accurately quantify and detect the at least one target nucleic acid in the biological sample. In another aspect, the compositions and methods provided herein are useful for developing sensitive and robust analytical methods for identification, mapping, and relative quantitation of impurities in therapeutic oligonucleotides. In one aspect, methods provided herein also disrupt the interaction between at least one target cationic lipid and an oligonucleotide, e.g., lipids interacting with nucleic acids within formulated nucleic acid drugs. In some embodiments, formulated nucleic acid drugs are nucleic acid-based vaccines e.g., a coronavirus vaccine. Therefore, methods provided in the present disclosure are advantageous for bioanalytical analysis of target lipids that are interacting with nucleic acids. In one aspect, provided herein is a composition for pretreating at least one target nucleic acid in a biological mixture prior to bioanalytical analysis, the composition including: a chaotropic agent selected from a substituted guanidine, a substituted amidine, a substituted quaternary amine, or a combination thereof. In some embodiments, the composition has a pH value of about 4 to about 10. In some embodiments,