Patent Application
20250230179
The content of the electronic sequence listing (File name: 178571_01609.xml; Size 88,650 bytes; and Date of Creation: Jan. 16, 2025) is herein incorporated by reference in its entirety.
The present disclosure relates to phosphoramidite compounds, their production, and their use in molecular biology especially in the field of nucleic acid detection and sequencing. The disclosure also relates to methods of synthesizing nucleic acids, including without limitation oligonucleotides that are oligoribonucleic acids (oligo-RNAs) or oligodeoxyribonucleic acids (oligo-DNAs).
Oligonucleotides containing various reporter groups have widespread application in molecular biology and medicine as tools for the specific detection of sequences, in DNA sequencing, and in other molecular biology methods. These labeled oligonucleotides are also useful in the research and production of pharmaceutical materials such as without limitation antisense nucleic acids, ribozymes, and RNA for RNAi-mediated control of gene expression. Non-nucleoside-based phosphoramidites that bear the 2,4-dinitrophenyl group (“DNP”) are known. These labelled phosphoramidites have been used in solid-phase oligonucleotide synthesis to attach single and multiple dinitrophenyl groups to the 5′ end of oligonucleotides, and have been shown to be compatible with the normal oligonucleotide synthesis cycle. Will et al., “The synthesis of oligonucleotides that contain 2,4-dinitrophenyl reporter groups,” Carbohydrate Res. Vol 216 (1992), 315-322. The DNP-labelled oligonucleotides have been detected using monoclonal and polyclonal anti-dinitrophenyl antibodies. Tatulchenkov et al. disclose synthesis of phosphoramidite reagents for introduction into oligonucleotides. Russian J. Bioinorg. Chem. 2017, 43, 4, pp 386-396.
Analytical tests based on detection of DNP-labelled oligonucleotides with anti-DNP antibodies have been proposed. One example of a DNP labeling reagent is 1-dimethoxytrityloxy-3-O—[N-(2,4-dinitrophenyl)-3-N-aminopropyl-(triethyleneglycol)]-glyceryl-2-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite, which is a DNP phosphoramidite with a spacer that is a branched triethylene glycol (TEG):
(glenresearch.com/10-1985.html accessed Dec. 17, 2023; chemgenes.com/products.php?product=CLP-9907, accessed Dec. 17, 2023). “DMT” is dimethoxytrityl, a protecting group used often for the 5′-hydroxy group in nucleosides and nucleoside analogs particularly in oligonucleotide synthesis, having the following structure:
One significant problem with DNP-TEG phosphoramidite is that it may allow for an intra-molecular cyclization reaction which, to be avoided, requires a step for the removal of the cyanoethyl protection group prior to the DMT group deprotection to prevent the loss of the DNP label during synthesis via intra-molecular cyclization. It is an object of the invention to provide a DNP-labelled nucleic acid that can be prepared more efficiently and less expensively than DNP-TEG phosphoramidite nucleic acids.
US Pat. App. Pub. 20200199174A1 published Jun. 25, 2020 discloses methods for fluorescently labeling an intracellular protein that includes obtaining a fusion protein of a protein to be labeled and an anti-DNP antibody, and then bringing a compound represented by this formula
in which S is a fluorescent group, or the salt of the compound into contact with the cell, and fluorescently labeling the protein to be labeled by reacting the fusion protein and the compound or its salt. Anti-DNP antibodies and antigen-binding fragments thereof are disclosed in US Pat. App. Pub. 20200199174A1, e.g. at paras. [0056]-[0061] and are commercially available as would be known to one skilled in the art.
One application for labeled oligonucleotides is in situ hybridization, which is a method employed for the precise spatial localization and identification of distinct DNA and RNA sequences within cells, conserved tissue sections, or complete tissue samples. This technique involves the binding of a nucleic acid probe's complementary strand to a specific target sequence, thus facilitating the identification of the sought-after genetic material. These resulting hybrids are capable of providing detectable signals through various methods, dependent on the labeling strategy employed. Probes labeled with radioactive elements can be visualized utilizing autoradiography, while probes labeled with haptens can be detected through affinity-based mechanisms, wherein enzyme-mediated colorimetric signals are generated. Furthermore, for direct visualization, fluorescent markers may be attached to the probes, enabling direct detection using fluorescence, a technique commonly referred to as “fluorescent in situ hybridization” (FISH).
A significant advantage of in situ hybridization is that it facilitates the elucidation of interrelationships between the spatial distribution of precise nucleic acid sequences and the corresponding protein products of the designated gene. Moreover, this technique extends to exploring the associations between these molecular components and the underlying cellular structures. Presently, this method serves as a pivotal tool within both scientific research and clinical contexts, effectively contributing to the comprehensive understanding of diverse domains. The spatial data garnered through this approach has substantially enriched researchers' comprehension in fields such as viral infection, genetic mapping, cytogenetics, gene expression dynamics, and prenatal diagnosis and developmental processes. Methods to improve the detection of nucleic acids using in situ hybridization, such as by allowing for faster detection, detection of smaller nucleic acid sequences, detection at lower concentrations of the nucleic acid target sequence, or improved reproducibility of the detection are desired.
The present disclosure relates to novel phosphoramidite compounds, their synthesis, their use, and synthetic nucleic acids, for example synthetic oligonucleotides, containing these novel phosphoramidites. The present disclosure also provides methods for the preparation and use of synthetic nucleic acids relating to or involving the phosphoramidite compounds disclosed herein. An object of the present disclosure is to provide useful and novel phosphoramidite compounds for efficient, high-yield synthesis of labeled synthetic nucleic acids, such as without limitation oligoRNAs, for use as probes in in situ hybridization. A further object is to provide a phosphoramidite compound that can be attached to an oligoRNA probe used in nucleic acid detection and screening methods, such as in situ hybridization, wherein the phosphoramidite may be easily detected at high efficiency. The compounds of the present disclosure solve a problem of providing improved labeled oligo probes for accurate in situ hybridization and other nucleic acid sequencing and screening assays.
The present disclosure concerns a compound represented by the structure of formula (I):
In another embodiment, the present disclosure concerns a method of preparing a polynucleotide, the method comprising:
In another embodiment, the present disclosure is directed to method of making a compound represented by the formula:
with 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamidite, wherein R1 is selected from the group consisting of C1-C20-alkyl preferably C1-C6-alkyl, C1-C20-alkoxy preferably C1-C6-alkoxy, C2-C20-alkenyl preferably C2-C6-alkenyl, C2-C20-alkynyl preferably C2-C6-alkynyl, C6-C14 aryl preferably C6 aryl, —C(═O)—NRaRb wherein Ra and Rb are identical or different and are C1-C4-alkyl optionally substituted by halogen, and —((CH2)nO)m wherein n is 1, 2, 3, or 4 and m is 1, 2, 3, 4, 5, 6, 7, 8, or 9,
In a further embodiment, the present disclosure is directed to a method of making a compound represented by the formula:
In a further embodiment, the disclosure concerns a polynucleotide, optionally, a nucleic acid hybridization probe, comprising the compound represented by the formula:
In another embodiment, the present disclosure is directed to polynucleotide comprising a compound represented by the formula:
As used herein, alkyl represents a straight-chain or branched alkyl. By way of example (C1-C20) alkyl can have 1 to 20 carbon atoms. In an aspect, an alkyl can include, but is not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, 1-ethylpropyl, n-pentyl, iso-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, or n-dodecyl.
As used herein, alkoxy refers to an alkyl group which is singularly bonded to oxygen. As used herein, cycloalkyl refers to a saturated monocyclic carbocyclic group having 3 to 6 carbon atoms and includes for example cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
As used herein, polyether represents a chain having a repeating unit of 1 to 4 carbon atoms and an oxygen. By way of example, the polyether units can include two carbons —(CH2CH2O)—, three carbons (CH2CH2CH2O), or four carbons (CH2CH2CH2CH2O) and the number of repeating polyether units in the chain is 1, 2, 3, 4, 5, 6, 7, 8, or 9.
As used herein, aryl or aromatic group refers to an optionally substituted monocyclic or fused bicyclic or polycyclic ring system having the well-known characteristics of aromaticity, wherein at least one ring contains a completely conjugated pi-electron system. Typically, aryl groups contain 6 to 20 carbon atoms (“C6-C20 aryl”) as ring members, preferably 6 to 14 carbon atoms (“C6-C14 aryl”) or more preferably, 6 to 12 carbon atoms (“C6-C12 aryl”). By way of example, aromatic groups can include phenyl, pyrrole, furan, anthracene, and pyrene. As used herein, heterocycle refers to a non-aromatic, saturated or partially unsaturated ring system containing the specified number of ring atoms, including at least one heteroatom selected from N, O and S as a ring member, wherein the heterocyclic ring is connected to the base molecule via a ring atom, which may be C or N. Examples of saturated heterocycles are azetidinyl, pyrrolidinyl and piperidinyl.
As used herein, the terms “oligo,” “oligonucleotide,” “polynucleotide,” and “nucleic acid,” include polymer sequences of two or more monomers, wherein the monomer is selected from the group consisting of nucleotides (whether DNA or RNA) and nucleotide analogs, wherein the nucleotide analog may be any nucleotide analog known in the art or may be a phosphoramidite compound of the present disclosure, preferably the compound of formula (I) or formula (II). The nucleotides of the present disclosure may be isolated from natural sources, recombinantly produced, or artificially synthesized.
As used herein, a probe refers to a nucleic acid capable of binding to a target nucleic acid of substantially complementary sequence through complementary base pairing, usually through hydrogen bond formation. As used herein, a probe may include natural (i.e. A, G, U, C, or T) or modified bases (7-deazaguanosine, inosine, etc.).
The term “target nucleic acid” or “target sequence” refers to a nucleic acid or nucleic acid sequence which is to be analyzed or detected in a sample. A target can be a nucleic acid to which a probe will hybridize. The probe may or may not be specifically designed to hybridize to the target. It is either the presence or absence of the target nucleic acid that is to be detected, or the amount of the target nucleic acid that is to be quantified. The term target nucleic acid may refer to the specific subsequence of a larger nucleic acid to which the probe is directed or to the overall sequence (e.g., gene or mRNA) it is desired to detect. The difference in usage will be apparent from context.
The term “hybridization” refers to the process in which two single-stranded polynucleotides bind non-covalently to form a stable double-stranded polynucleotide. The resulting double-stranded polynucleotide is a “hybrid.” The proportion of the population of polynucleotides that forms stable hybrids is referred to herein as the “degree of hybridization.” Hybrids can contain two DNA strands, two RNA strands, or one DNA and one RNA strand.
One aspect of the current disclosure is a compound of the formula (I):
In some embodiments, R1 is a C1-C20-alkyl. In another embodiment, R1 is a substituted or unsubstituted C1-C6-alkyl. In a further embodiment, R1 is an unsubstituted C5-alkyl. In one embodiment, the compound is (3R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-1-(6-((2,4-dinitrophenyl)amino)hexanoyl)pyrrolidin-3-yl (2-cyanoethyl) diisopropylphosphoramidite of the formula (II):
The phosphoramidite compounds of the present disclosure may be used in the synthesis of oligo- and poly-nucleotides, such as oligonucleotide probes. These nucleic acids may be prepared according to standard phosphoramidite synthesis procedure, e.g., automated synthesis. In one embodiment, the present disclosure is directed to an oligo- or poly-nucleotide prepared by reacting the compound of formula (I) with one or more nucleic acids, nucleic acid analogs, or other compounds of formula (I). In one embodiment, the present disclosure concerns a compound of formula (III):
For example, in one embodiment the dinitrophenyl moiety of the present disclosure can be positioned between two nucleotides within the polynucleotide chain such that the polynucleotide has the structure shown here:
or other substituents as known in the art in which * is the linkage point to the ribose backbone. It should be understood that any combination of natural or unnatural nucleic acids or nucleic acid analogs can be used in the synthetic nucleic acids of the present disclosure. Additionally, and alternatively, the dinitrophenyl moiety can be included at a 3′ terminus or a 5′ terminus of the synthetic nucleic acids of the present disclosure. Suitable non-naturally occurring nucleic acids and nucleic acid analogs for the compounds and methods of the present disclosure include those having one or more of the following non-natural bases, where —X indicates the linkage to the ribose or deoxyribose backbone:
The polynucleotide can include radiolabelled atoms at any position. For example, at least one of 14C, 15N, 3H, 18F, 19F, 32p, 35S isotopes can be substituted for non-radiolabeled atoms in the polynucleotide at any position.
Polynucleotide Synthesis with the Disclosed Phosphoramidite Compounds
In an aspect of the current disclosure, methods of synthesizing a polynucleotide, e.g., nucleic acid hybridization probes, comprising a 2,4-dinitrophenyl moiety of the present disclosure are provided. In some embodiments, the methods comprise performing a phosphoramidite nucleic acid sequencing using the disclosed compounds to synthesize a polynucleotide comprising a 2,4-dinitrophenyl moiety.
Automated and manual methods of synthesizing polynucleotides using phosphoramidites are known in the art. The compounds of the present disclosure, which are phosphoramidites comprising nucleoside analogues, may be substituted for standard phosphoramidites in such reactions and incorporated into polynucleotides. The methods may further comprise isolating, enriching, or purifying the polynucleotide to generate an isolated, enriched, or purified polynucleotide, respectively.
In one embodiment, polynucleotide synthesis may comprise (1) linking a first nucleotide to a solid support at the 3′-OH of the nucleotide, and (2) deprotection by detritylation of the 5′-4,4′-dimethoxytrityl (DMT) moiety which is used to link the first nucleotide to, for example, the next nucleotide or nucleoside analog in a polynucleotide. This is followed by (3) coupling: once the DMT has been removed, the free 5′-OH of the solid-support-linked nucleotide or nucleoside is able to react with the next nucleotide or nucleoside, which is added as a phosphoramidite monomer. The diisopropylamino group of the incoming phosphoramidite monomer in the solvent acetonitrile is ‘activated’ (protonated) by an acidic additive such as tetrazole or ETT [5-(ethylthio)-1H-tetrazole], allowing the 5′-OH to form a bond with the phosphorous of the incoming nucleotide or nucleoside. The mixing is carried out in the fluid lines of the synthesis instrument as the reagents are delivered to the solid support. The activated phosphoramidite is delivered in a many-fold excess over the solid-support-linked nucleoside to drive the reaction to as close to completion as possible. The products include a dinucleoside or dinucleotide with a phosphite triester linkage and a free diisopropylamino group. This is followed by step (4) oxidation: The phosphite triester formed during the coupling reaction is unnatural and unstable; therefore, it must be converted to a more stable phosphorus species prior to the start of the next cycle. Oxidation converts the phosphite triester to the stable phosphate triester. Oxidation of the phosphite triester is achieved with iodine in the presence of water and pyridine. The product is the phosphate triester, which is essentially a standard DNA/RNA backbone with a β-cyanoethyl protecting group on one oxygen of the phosphate group. This is followed by step (5) capping: since 100% coupling efficiency is impossible, there are always some solid-support-linked nucleosides with unreacted 5′-OH. If not blocked, these hydroxyl groups will react during the next cycle, and hence, lead to a missing base. The accumulation of these deletion mutations through successive cycles would create a complex mixture of ‘shortmers’ that are difficult to purify and therefore could render the oligonucleotide useless in the subsequent application. Capping is required to prevent shortmer accumulation. Acetic anhydride and N-methylimidazole react to form an intermediate in the solvent tetrahydrofuran, which contains a small quantity of pyridine. The mixing is carried out in the fluid lines of the synthesis instrument as the reagents are delivered to the solid support. The product is the solid-support-linked nucleoside with an acetylated 5′-OH (pyridine maintains a basic pH thereby preventing detritylation of the phosphoramidite monomer by the free acetate/acetic acid). This is followed by step (6) deprotection: after cleavage, the solution of oligonucleotide in concentrated aqueous ammonia is heated to remove protecting groups from the bases and phosphates. The oligonucleotide in concentrated aqueous ammonia is heated. The protecting groups can include any appropriate protecting groups for bases, and can for example include: N(6)-benzoyl A, N(4)-benzoyl C, and N(2)-isobutyryl G. The product of the reaction is fully-deprotected A, C, and G bases. The β-cyanoethyl group on the free oxygen of the phosphate must be removed to convert it from a phosphate triester to a phosphate diester (phosphodiester). The cyanoethyl groups are removed in concentrated aqueous ammonia via β-elimination. The reaction is quick because the hydrogen atoms on the carbon adjacent to the electron-withdrawing cyano group are highly acidic. The products are the oligonucleotide with a native phosphodiester backbone and acrylonitrile.
In addition to the standard protecting groups, the labile dimethylformamidyl G and the ‘ultramild’ protecting groups can be used for modified oligonucleotides that are sensitive to ammonia. The dimethylformamidyl protecting group is typically removed in concentrated aqueous ammonia via heating but in significantly less time than is the isobutyryl group. The ultramild protecting groups include: N(6)-phenoxyacetyl A, N(2)-acetyl C, and N(2)-isopropylphenoxyacetyl G. They are typically removed at room temperature in a concentrated aqueous ammonia/methylamine solution.
Modifications to these methods are known to those of skill in the art and may be used to prepare polynucleic acids from the compounds of the disclosure. The solid support be any one suitable, such as without limitation a polymer support such as polystyrene, a silica support, and a controlled pore glass (CPG).
In an aspect of the current disclosure, the present disclosure is directed to a probe of the formula (III):
In some embodiments, the probe comprises a polynucleotide of formula (IV):
wherein R3 and R4 are as defined above in formula (III).
As will be understood by one skilled in the art, the disclosed phosphoramidite compounds may be incorporated into polynucleotides using standard phosphoramidite synthesis methods, resulting in polynucleotides comprising 2,4-dinitrophenyl moieties.
As the 2,4-dinitrophenyl moiety-containing polynucleotides are detectable using anti-2,4-dinitrophenyl detection reagents, e.g., antibodies, the polynucleotides may be used in ISH methods. The detection methods used may be any of those known to one of skill in the art that can be used in conjunction with DNP.
The polynucleotides synthesized with the 2,4-dinitrophenyl moiety according to an aspect as disclosed herein were obtained in higher amount at lower cost that when a commercially available DNP phosphoramidite was used. For example, use of the commercially available P TEG-phosphoramidite from Lumiprobe requires removal of cyanoethyl protection group prior to DMT group deprotection. In contrast, use of the DNP-Phosphoramidite of formula (I) does not require this extra precautionary step as this intra-molecular cyclization reaction in not possible due to proper separation between DMT protection alcohol and cyanoethyl group.
The target nucleic acid sequence may be any natural or non-natural nucleotide sequence. In one embodiment, the probes of the present disclosure are designed to detect a naturally occurring nucleic acid sequence. For the design of such oligo probes of the present disclosure, the nucleic acid sequence of the desired target may be drawn from public sources such as the NCBI database and UCSC genome browser covering most of the transcript variation for the gene of interest. Probe sequences may then be designed with optimized properties, such as a GC content in the range of 30-70% and Tm in the range of 35-65° C. with the selected target sequences. Presented as an example, SEQ ID NO:1 is the antisense sequence that selectively binds to HPV6 mRNA copies within the desired targets. This sequence served as the basis for preparing oligonucleotide probes for detecting HPV6 that were used in the examples.
Similarly, SEQ ID NO:2 is the antisense sequence that selectively binds to EGFR gene consensus that served as the basis for preparing oligonucleotide probes shown in the examples for detecting mRNA of EGFR. Similar approach was used to prepare probes for hybridization used in the other examples herein.
The nucleic acids such as the probes of the present disclosure may be tagged with a detectable compound directly or indirectly via the DNP moiety. In one embodiment, the nucleic acid of the present disclosure is incubated with an anti-dinitrophenyl antibody-detectable label conjugate (“antibody conjugate”) under conditions such that the antibody conjugate selectively binds to the nucleic acid and thereby a detectable label is indirectly incorporated into the nucleic acid or probe of the present disclosure. The detectable label may be fluorescein, horseradish peroxidase, alkaline phosphatase, or any other label or tag known in the field that provides a component for a detection system to identify which nucleic acids in a mixture are hybridized to the probe. Other labeling systems are known that may be used by conjugating to the anti-DNP antibody to detect the hybridized nucleic acids of the present disclosure, such as labeling systems and reagents from Enzo Life Sciences, Inc., Farmingdale NY. Anti-Dinitrophenyl antibodies are commercially available, such as the mouse monoclonal anti-dinitrophenyl antibody IgE isotype sold by Sigma-Aldrich (accessed 12 Jan. 2024); DNP monoclonal antibody (LO-DNP-2 and LO-DNP-30) sold by Invitrogen; and a goat DNP polyclonal antibody from Bethyl Laboratories.
In one embodiment, the present disclosure concerns a non-naturally occurring composition of matter comprising a nucleic acid of the formula (III), preferably of the formula (IV), hybridized to a preselected target sequence. In one embodiment, the composition of matter further comprises an anti-DNP antibody-detectable label conjugate that is selectively bound to the nucleic acid of formula (III) or (IV) hybridized to the target sequence.
In one embodiment, the nucleic acids of the present disclosure are used for detection of nucleic acids with in situ hybridization. A major advantage of in situ hybridization is that it enables maximum use of tissue that is difficult to obtain (e.g., embryos and clinical biopsies). Hundreds of different hybridizations can be performed on the same tissue. Libraries of tissues can be formed and stored in the freezer for future use.
A further embodiment of the invention provides an in vitro hybrid composition of matter that includes:
A still further embodiment of the invention provides a method for detecting a target nucleic acid sequence in a sample that includes the steps of:
The method may further include a washing step after the contacting step and before the detecting step to remove non-naturally occurring linear nucleic acid molecule/probe that is not specifically hybridized to a target nucleic acid molecule in the sample/specimen. The detecting step may include a 2D or 3D visualization/image capture using microscopy as known in the art.
Methods for conducting polynucleotide hybridization assays have been well developed in the art. Hybridization assay procedures and conditions will vary depending on the application and are selected in accordance with the general binding methods known including those referred to in: Molecular Cloning, A Laboratory Manual, Second Ed., J. Sambrook, et al., Eds., Cold Spring Harbor Laboratory Press, 2012 (“Sambrook et al.”); Berger and Kimmel, “Methods in Enzymology,” Vol. 152, “Guide to Molecular Cloning Techniques”, Academic Press, Inc., San Diego, Calif., 2010; Young and Davis, Proc. Natl. Acad. Sci., U.S.A., 80:1194 (1983), each of which are incorporated herein by reference.
It is appreciated that the ability of two single stranded polynucleotides to hybridize will depend upon factors such as their degree of complementarity as well as the stringency of the hybridization reaction conditions.
As used herein, “stringency” refers to the conditions of a hybridization reaction that influence the degree to which polynucleotides hybridize. Stringent conditions can be selected that allow polynucleotide duplexes to be distinguished based on their degree of mismatch. High stringency is correlated with a lower probability for the formation of a duplex containing mismatched bases. Thus, the higher the stringency, the greater the probability that two single-stranded polynucleotides, capable of forming a mismatched duplex, will remain single-stranded. Conversely, at lower stringency, the probability of formation of a mismatched duplex is increased.
Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.”
As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus ≤10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.
As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of” should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term “consisting essentially of” should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.
All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
Synthesis of DNP-phosphoramidite derivative for oligonucleotide preparation is described below. Other reagents for oligonucleotide synthesis were purchased from commercial sources.
An aqueous solution of potassium carbonate (110.6 g K2CO3 in 400 mL water) was added to a mixture of caproic acid (36 g, 200 mmol) and 1-fluoro-2,4-dinitrobenzene (52 g, 400 mmol) in acetone (300 mL). After stirred at room temperature of 1 hour, the reaction mixture was acidified to pH 2 by slow addition of concentrated hydrochloric acid. The precipitate was collected by filtration and then washed with 0.1 N hydrochloric acid (1 L) and cold acetone (1 L). The yellow solid was dried under vacuum to provide 56.4 g (95%) product. The chemical structure of Compound 1 is shown below:
Compound 1 was characterized by LC-MS analysis showing mass observed as 296.03 with calculated value of 296.08.
Sodium borohydride (25 g, 0.66 mol) was added cautiously into a mixture of (2S, 4R)-4-hydroxyproline methyl ester hydrochloride (24 g, 0.13 mol) in dioxane (260 mL) under vigorous stirring. The mixture was heated to reflux, followed by dropwise addition of methanol (90 mL). After the addition, the mixture was refluxed for 4 hours, then stirred at room temperature overnight. The reaction was quenched by adding concentrated hydrochloric acid (100 mL). The solid was removed by filtration, and the filtrate was evaporated to dryness under vacuum. Isopropanol (50 mL) was added to the residue and stirred for 10 min. The insoluble solid was filtered off. After methanol (75 mL) and concentrated HCl (10 mL) were added to the filtrate, the whole mixture was refluxed for 30 min and then evaporated to dryness under vacuum. The residue was recrystallized with isopropanol (300 mL) to provide product (8.5 g) as a white powder. The chemical structure of Compound 2 is shown below:
Compound 2 was characterized by LC-MS analysis showing mass observed as 116.07 with calculated value of 116.07.
TSTU (11.6 g, 38.5 mmol) and DIPEA (13.5 g, 105 mmol) were added sequentially into a mixture of 6-[(2,4-Dinitrophenyl)amino]hexanoic acid (10.4 g, 35 mmol) in acetonitrile (180 mL). The reaction was stirred at room temperature and monitored by TLC (dichloromethane/methanol, 95/5, v/v). The reaction was done in about 30 min. (2S,4R)-4-Hydroxyprolinol Hydrochloride (5.4 g, 35 mmol) was added into the above reaction mixture. The mixture was stirred at room temperature and monitored by TLC (dichloromethane/methanol, 95/5, v/v). After stirred at room temperature for 3 hours, the mixture was evaporated under vacuum. The residue was dissolved in dichloromethane (100 mL), and then added slowly with stirring into ethyl ether (800 mL). The ethyl ether layer was discarded. The precipitate sticky oil was again dissolved in dichloromethane (100 mL) and then added to ethyl ether (800 mL). The precipitate oil was collected and purified by flash chromatography (300 g silica gel column; 0% to 10% methanol in dichloromethane in 20 column volumes). The fractions were checked by TLC (10% methanol in dichloromethane), combined and evaporated to give product (18.4 g). The chemical structure of Compound 3 is shown below:
Compound 3 was characterized by LC-MS analysis showing mass observed as 395.16 with calculated value of 395.15.
DMAP (catalytic amount) was added to a solution of starting diol (18.4 g, 46.5 mmol) in pyridine (150 mL). A solution of DMTrCl (17.3 g, 51.0 mmol) in pyridine (100 mL) was added dropwise while the reaction mixture was cooled by water bath. The mixture was stirred at room temperature overnight. The reaction was monitored by TLC (5% methanol in dichloromethane). The solvent was removed under vacuum. The residue was dissolved in dichloromethane (300 mL) and washed with brine (2×200 mL), dried with anhydrous sodium sulfate. The solvent was removed under vacuum. The residue was purified by flash chromatography (440 g silica gel, 0% to 10% of methanol in dichloromethane containing 5% of triethylamine). The fractions were checked by TLC (5% methanol in dichloromethane), combined and evaporated under vacuum to give product as yellow solid (15 g). The chemical structure of Compound 4 is shown below:
Compound 4 was characterized by LC-MS analysis showing mass observed as 697.35 with calculated value of 697.29.
Tetrazole (0.45 M in acetonitrile, 3.6 mL, 1.6 mmol) was added to a solution of starting alcohol (4.0 g, 5.7 mmol) and 2-cyanoethyl N,N,N′,N′-tetraisopropyl-phosphorodiamidite (2.1 g, 6.9 mmol) in dichloromethane (60 mL) under argon. The mixture was stirred at room temperature overnight. TLC (5% methanol in dichloromethane) showed the reaction was complete. The mixture was washed with saturated sodium bicarbonate solution (2×100 mL) and brine (100 mL), then dried with anhydrous sodium sulfate. The solvent was removed under vacuum. The residue was purified by flash chromatography (120 g silica gel column, 2% triethylamine in toluene as mobile phase). The fractions were checked by TLC (5% methanol in dichloromethane), combined and then evaporated under vacuum. The yellow solid (6.7 g) obtained was dried under high vacuum for 1 day and then stored under argon at −20° C. HPLC showed purity over 99% of the combined isomers. The chemical structure of the compound of formula (II) is shown below:
The compound of formula (II) produced was characterized by LC-MS analysis showing mass observed as 897.36 with calculated value of 897.39. The purity of the compound of formula (II) was determined by HPLC (reversed-phase) and was found to be 99.62%.
A plurality of oligonucleotides was prepared using the Dr. Oligo 48 synthesizer, employing universal CPG support at a synthesis scale of 200 nmol. The synthesis process adhered to a protocol supplied by the manufacturer of the aforementioned apparatus. To incorporate DNP moieties into the oligonucleotides for the purpose of establishing an antibody recognition locus, Compound of formula (II) was introduced into the synthesis, alongside other adjusted phosphoramidites of conventional bases. Subsequent to the synthetic procedure, detachment of the oligonucleotides from the solid surface and deprotection of the nucleotide bases were accomplished utilizing an aqueous ammonia medium. The resulting crude oligonucleotides were subsequently purified on reverse phase silica columns using standard methods. Evaluation and characterization of these oligonucleotides were conducted through Electrospray Ionization Mass Spectrometry (ESI-MS). A composite assortment of 12 to 24 (or higher) highly specific, labeled oligonucleotides, in a buffer solution of TE-8, formed the Hybridization Probe Stock Solution. These oligonucleotides were designed to target distinct nucleic acid sequences such as DNA and various forms of RNA. An example of such oligonucleotide probes directing towards EGFR mRNA target is shown below in the list of oligo probe sequences (m is 2′-methoxy modification, r is ribo bases).
Procedure shown in Example 6 was followed to synthesize oligonucleotides containing Biotin modification using commercially available phosphoramidite derivatives.
Due to incompatibility of Digoxigenin during solid phase oligonucleotide synthesis an alternate method involving copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC) was performed. Desired oligonucleotides sequences were synthesized using commercially available alkyne phosphoramidite that were then conjugated to Digoxigenin-azide (Enzo Life Sciences) using CuAAC. The resulting crude oligonucleotides were subsequently purified on reverse phase silica columns using standard methods. Evaluation and characterization of these oligonucleotides were conducted through Electrospray Ionization Mass Spectrometry (ESI-MS).
For the purpose of assessing binding between oligonucleotide probes and plasmid targets, a succession of plasmid target dilutions was applied onto a MaxSense® hybridization membrane (Enzo Catalog Number 45701) subsequent to the implementation of standard membrane blocking procedures. Following this, the membrane was subjected to treatment with HPV6 oligonucleotide RNA probes for a period of 2 hours at a temperature of 50° C., allowing for hybridization to occur. The HPV6 oligonucleotide RNA probe was labeled with biotin phosphoramidite (hydroxyprolinol) that was purchased from Lumiprobe Corporation, Hunt Valley, Maryland, which had the following structure:
Subsequent to the hybridization step, the membrane was subjected to a series of washes using a TBS-T buffer, followed by incubation with a linker antibody directed against Biotin, maintained for a duration of 1 hour at room temperature. Further washing with TBS-T buffer succeeded by an incubation with PolyView Plus-HRP (anti-rabbit, Enzo). Following additional washing with TBS-T buffer, signal detection was executed utilizing a DAB chromogen (Enzo). Notably, the detected signal exhibited a clear dependence on the concentration of the plasmid target. No binding interaction was observed with the single-stranded DNA (ssDNA) utilized as a negative control.
The methodology employed for the detection of signals through In Situ Hybridization (ISH) encompasses a series of procedural stages, including slide preparation and pre-treatment, probe hybridization and its subsequent wash, along with primary and secondary antibody incubation, culminating in either Horseradish Peroxidase (HRP) or Alkaline Phosphatase (AP) detection. The preparation and pre-treatment of tissue slides adhered to conventional procedures well-documented in relevant literature. The hybridization of the probes was executed at a temperature of 45° C. for a duration of 2 hours. Subsequent to the hybridization procedure, a sequence of washing steps was performed, followed by the application of linker and detection antibodies. Ultimately, signal generation was obtained utilizing DAB chromogen system. The outcomes of this process are shown and elaborated upon in
The probe sequences used in the data reported in
This application claims the benefit of and priority to U.S. Application Ser. No. 63/621,604, filed Jan. 17, 2024. The entirety of which is incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 63621604 | Jan 2024 | US |
