CN118546193A

CN118546193A - Oxidizing agent for oxidizing phosphite triester and application thereof

Info

Publication number: CN118546193A
Application number: CN202410212527.4A
Authority: CN
Inventors: 黄东亮; 杨光
Original assignee: Jiangsu Genscript Biotech Co Ltd
Current assignee: Jiangsu Genscript Biotech Co Ltd
Priority date: 2023-02-27
Filing date: 2024-02-27
Publication date: 2024-08-27

Abstract

The invention relates to the technical field of chemical synthesis of nucleic acid, in particular to an oxidant for oxidizing phosphite triester and application thereof. The oxidant for oxidizing the phosphite triester is an iodine solution, and the solvent comprises Tetrahydrofuran (THF), N-Dimethylformamide (DMF) and water in a volume ratio of (7-9) (0.8-1.2); and the oxidant also contains (1-3) mL/L of organic alkali with pKa of 9-14.

Description

Oxidizing agent for oxidizing phosphite triester and application thereof

Cross Reference to Related Applications

The present application claims priority from chinese patent application number 202310171405.0 filed on 27, 02, 2023, which is incorporated herein by reference in its entirety.

Technical Field

The invention relates to the technical field of chemical synthesis of nucleic acid, in particular to an oxidant for oxidizing phosphite triester and application thereof.

Background

Primer (Primers) is a short, single-stranded DNA or RNA molecule of oligonucleotide composition that serves as a point of initiation of DNA replication in an organism. Since the last fifties of the century, the general structure of nucleic acids was proposed and the study of chemical synthesis of DNA was driven, large-scale and inexpensive oligonucleotides synthesized by chemical methods have been widely used. Including Polymerase Chain Reaction (PCR), probes, cloning, and DNA sequencing, greatly promote research and development in life science fields such as synthetic biology, protein engineering, genome engineering, metabolic engineering, etc. However, unlike primer polymerase (Primase) which is unique in organisms, which is capable of synthesizing from the 5 '. Fwdarw.3' end of nucleotides under mild conditions, the complex environment of chemical synthesis is often accompanied by a number of unwanted by-products. Thus, the development of a stable and efficient strategy for synthesizing oligonucleotide chain primers has been a hotspot in the current research field.

The solid phase phosphite triester method has become the first route in the field of commercial synthesis of oligonucleotides since the eighties of the twentieth century because of its rapid and efficient coupling and stable starting reagents. It generally involves a four-step cyclic synthetic pathway by first disengaging (deblock) the Dimethoxytrityl (DMT) of the nucleotide attached to the solid support Column (CPG) by an acid; then, mixing the target phosphoramidite protected monomer with tetrazole to obtain a 3' activated nucleoside-phosphite activated intermediate, and performing condensation reaction (coupling) with free 5' -hydroxyl (5 ' -OH); thereafter, the unreacted 5' -OH end cap (capping) is prevented from participating in the subsequent reaction by the acetylation reaction of maleic anhydride and N-methylimidazole with hydroxyl groups; finally, the unstable trivalent phosphite triester is oxidized with iodine solution to a stable pentavalent phosphate triester. Through the four steps, one target base is grafted on the solid phase carrier, and then the circulation is repeated in turn until the target base is synthesized in sequence. Wherein, the condensation efficiency of the base can be judged by the removal color of DMT cations in the deblock process. Currently, the length of oligonucleotides synthesized by phosphoramidite chemistry is generally limited to within 200 nucleotides, and theoretically cannot exceed 300 nucleotides due to the fact that chemical synthesis efficiency cannot reach 100% of its own limit. For the synthesis of long DNA, DNA assembly is required to assemble short oligonucleotides into long DNA. As can be seen, the efficient reaction of each step in the four-step cycle of the solid phase phosphite triester method is a determining factor limiting the quality of the commercial oligonucleotide chains.

Various approaches have been used to improve the synthesis efficiency of the solid-phase phosphite triester method, mainly focusing on the replacement of tetrazole activator in the condensation step to improve the condensation efficiency; however, the iodine/tetrahydrofuran/pyridine/water (I ₂/THF/pyridine/H ₂ O) reagent formulations employed in the usual oxidation steps have been found to be somewhat deficient in the actual manufacturing process.

Disclosure of Invention

In one aspect of the invention, an oxidizing agent for oxidizing phosphite triester is an iodine solution, and the solvent comprises Tetrahydrofuran (THF), N-Dimethylformamide (DMF) and water in a volume ratio of (7-9): (0.8-1.2);

And the oxidant also contains (1-3) mL/L of organic alkali with pKa of 9-14.

In some embodiments, the solvent has a volume ratio of THF, DMF and water of (7.5-8.5): 0.9-1.1; preferably, the volume ratio is 8:1:1. In some embodiments, the concentration of iodine in the iodine solution is between 0.015M and 0.03M.

In some embodiments, the organic base is selected from one or more of triethylamine, tert-butylamine, N-propylamine, N-diisopropylethylamine, N-methylimidazole, and 1, 8-diazabicyclo [5.4.0] undec-7-ene; preferably, the organic base is triethylamine.

In some embodiments, the oxidizing agent does not contain pyridine.

Another aspect of the present invention relates to a kit for synthesizing oligonucleotides by the solid phase phosphite triester method, which contains an oxidizing agent as described above.

A further aspect of the present invention relates to a process for the preparation of an oxidizing agent as described above, comprising:

a) Adding iodine simple substance into a mixed solution of THF, DMF and water and uniformly mixing;

b) Mixing an organic base with the solution obtained in a).

In some embodiments, the preparation of the oxidizing agent is performed under light-protected conditions.

Yet another aspect of the present disclosure relates to a method of synthesizing an oligonucleotide comprising the steps of: oxidizing an intermediate having phosphite triester linkages to a pentavalent triester intermediate in the presence of an oxidizing agent;

Wherein the oxidant is iodine solution, the solvent contains THF, DMF and water with the volume ratio of (7-9) (0.8-1.2), and the oxidant also contains organic base with the pKa of 9-14 with the volume ratio of (1-3) mL/L.

A further aspect of the present invention relates to the use of an oxidizing agent as described above, or a kit as described above, in the synthesis of oligonucleotides by the solid phase phosphite triester method.

In some embodiments, the oligonucleotide is selected from a primer, a probe, siRNA, miRNA, mRNA, sgRNA, a nucleic acid aptamer, or an antisense nucleic acid.

The oxidant provided by the invention effectively avoids the defects of iodine consumption, crystallization generation and the like of the traditional I ₂/THF/pyridine/H ₂ O oxidant formula, and is used for realizing efficient oxidation of the phosphite triester intermediate into the pentavalent phosphate triester intermediate.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a mass spectrum of LC-MS obtained under the synthesis conditions of example 1;

FIG. 2 is a mass spectrum of LC-MS obtained under the synthesis conditions of example 2;

FIG. 3 is a mass spectrum of LC-MS obtained under the synthesis conditions of example 3;

FIG. 4 is a mass spectrum of LC-MS obtained under the synthesis conditions of the comparative examples.

Detailed Description

Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.

Unless otherwise defined, all terms (including technical and scientific terms) used to describe the invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present invention. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The terms "comprising," "including," and "comprising," as used herein, are synonymous, inclusive or open-ended, and do not exclude additional, unrecited members, elements, or method steps.

The recitation of numerical ranges by endpoints of the present invention includes all numbers and fractions subsumed within that range, as well as the recited endpoint.

Concentration values are referred to in this invention, the meaning of which includes fluctuations within a certain range. For example, it may fluctuate within a corresponding accuracy range. For example, 2%, may allow fluctuations within + -0.1%. For values that are larger or do not require finer control, it is also permissible for the meaning to include larger fluctuations. For example, 100mM, fluctuations in the range of.+ -. 1%,.+ -. 2%,.+ -. 5%, etc. can be tolerated. Molecular weight is referred to, allowing its meaning to include fluctuations of + -10%.

In the present invention, the terms "plurality", and the like refer to, unless otherwise specified, 2 or more in number.

In the invention, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.

In the present invention, the term "oligonucleotide" refers to a compound in which 2 or more nucleotides are linked to a nucleoside. Also included in the "oligonucleotide" are: phosphorothioate oligonucleotides in which the oxygen atom of the phosphate group is replaced with a sulfur atom, oligonucleotides in which the-O-of the phosphate group is replaced with-NH-, and oligonucleotides in which the hydroxyl group (-OH) in the phosphate group is replaced with-OY (in the formula, Y represents an organic group). The number of nucleosides of the oligonucleotide of the present invention is not particularly limited, and the longer the oligonucleotide synthesized by the solid phase, the greater the probability of occurrence of a problem. Thus preferred oligonucleotides are no more than 150 bases, for example 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10 bases, preferably no more than 80 bases, more preferably 3 to 50, even more preferably 5 to 30, in length. .

In the present invention, the term "nucleic acid" or "nucleotide" refers to a straight-chain compound (oligonucleotide) in which nucleotides are linked via phosphodiester bonds, and is considered to include DNA, RNA, and the like. The nucleic acid may be single-stranded or double-stranded. Because it allows efficient synthesis using a nucleic acid synthesizer, the nucleic acid is preferably single stranded. In the present specification, "nucleic acid" includes not only oligonucleotides containing purine bases such as adenine (A), guanine (G) and the like and pyrimidine bases such as thymine (T), cytosine (C), uracil (U) and the like, but also modified oligonucleotides containing other modified heterocyclic bases.

In the present invention, the term "phosphite triester oxide" refers to the process of oxidizing an unstable trivalent phosphite triester to a stable pentavalent phosphate triester in a solid phase phosphite triester process.

The invention relates to an oxidant for oxidizing phosphite triester, which is iodine solution, wherein the solvent contains THF, DMF and water with the volume ratio of (7-9): (0.8-1.2);

And the oxidant also contains (1-3) mL/L of organic alkali with pKa of 9-14.

The product has long storage stability, selective oxidation performance and high purity of synthesized oligonucleotide.

In some embodiments, the solvent has a volume ratio of THF, DMF and water of (7.5-8.5): 0.9-1.1.

In some embodiments, the solvent has a volume ratio of THF, DMF and water of (7.7-8.3): 0.95-1.05.

In some embodiments, the volume ratio of THF, DMF and water in the solvent is 8:1:1.

In some embodiments, the concentration of iodine in the iodine solution is between 0.015M and 0.03M, such as 0.02M, 0.025M.

The organic base is usually in the form of a liquid and is contained in an amount of (1-3) mL/L, for example, 1.3mL/L, 1.5mL/L, 1.8mL/L, 2mL/L, 2.3mL/L, 2.5mL/L, 2.8mL/L. The organic base has a pKa of 9 to 14, for example 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5.

In general, organic bases contain nitrogen atoms, such as amine compounds and nitrogen-containing heterocyclic compounds. In some embodiments, the organic base is selected from one or more of triethylamine, tert-butylamine, N-propylamine, N-diisopropylethylamine, N-methylimidazole, and 1, 8-diazabicyclo [5.4.0] undec-7-ene. In a preferred embodiment, the organic base is triethylamine.

In some embodiments, the oxidizing agent is free of pyridine.

The oxidant provided by the invention can avoid the introduction of harmful reagent pyridine, has no obvious crystallization and pinhole blockage in the actual synthesis process, and has no obvious difference between the quality of the obtained product and the quality of the obtained product of the traditional oxidant.

The invention also relates to a kit for solid phase synthesis of oligonucleotides by the phosphite triester method, which contains an oxidizing agent as described above.

The kit may also contain other components common in the solid phase phosphite triester method, such as cleaning solutions, buffers, blocking agents for blocking free hydroxyl groups, vulcanizing agents, acids, etc., and optionally containers for holding the components, instructions for experiments, etc.

According to a further aspect of the present invention, there is also provided a process for the preparation of an oxidizing agent as described above, comprising:

a) Adding iodine into a mixed solution of THF, DMF and water and uniformly mixing;

b) Mixing an organic base with the solution obtained in a).

Firstly preparing stable I ₂/THF/DMF/H₂ O iodine mixed solution, and then adding organic base TEA with corresponding proportion according to the volume of the solution to prepare the oxidant which has higher I ₂ concentration than that of the traditional iodine solution formula.

In some embodiments, the preparation process is performed under light-protected conditions.

The invention also relates to a method for synthesizing the oligonucleotide, which comprises the following steps: oxidizing an intermediate having phosphite triester linkages to a pentavalent triester intermediate in the presence of an oxidizing agent;

The invention also relates to the use of an oxidizing agent as described above, or of a kit as described above, in the solid phase synthesis of oligonucleotides by the phosphite triester method.

The solid-phase synthesis of phosphite triesters is well known and the improvements of the present invention are directed to the oxidation of unstable trivalent phosphite triesters to stable pentavalent phosphotriesters, and it will be readily appreciated that modifications to the procedures otherwise or related modifications which do not materially affect the oxidation of trivalent phosphite triesters are not excluded from the context of the "solid-phase synthesis of phosphite triesters" described above.

By way of example, a typical procedure for the "phosphite triester solid phase synthesis" may include:

1) Providing a nucleoside monomer, nucleotide or oligonucleotide (a) having a hydroxyl group at the 5' position protected by a substituted or unsubstituted dimethoxytrityl group (DMT) and at least one nucleoside monomer, nucleotide or oligonucleotide bound to a solid support;

removing the substituted or unsubstituted trityl group under the action of an acid to give a free hydroxyl group, and washing the solid support;

2) Condensing a nucleoside monomer, nucleotide or oligonucleotide (b) with the nucleoside monomer, nucleotide or oligonucleotide (a) after the reaction of step 1) to obtain phosphite triester;

Wherein the 3' -position of the nucleoside monomer, nucleotide or oligonucleotide (b) has a phosphoramidite group and is previously activated by an acidic activator;

3) The phosphite triester is converted to a phosphotriester or a phosphorothioate triester using an oxidizing agent, or an oxidizing agent plus a vulcanizing agent.

Steps 1) to 3) may be subjected to a plurality of cycles to successively lengthen the oligomeric compound and further comprise step 4) after the oligomeric compound reaches a desired length: cleaving the obtained oligomeric compound from the solid phase, and removing all protecting groups thereof; and optionally purifying it. The method of separation and purification from the solid phase may be carried out using a method known in the art, and for the purification method, desalting, bioRP/OPC purification, HPLC or PAGE purification is preferable.

The solid phase used in the solid-phase phosphite triester method of the present invention is not particularly limited, and is preferably a porous particle, and more preferably a porous particle having a functional group contributing to nucleic acid synthesis. "functional group that facilitates nucleic acid synthesis" refers to a functional group that can become the origin of nucleic acid synthesis and can be added with a linker. In particular, amino groups, hydroxyl groups, and the like may be mentioned. The shape of the porous solid support is not particularly limited, and may be any shape of flat plate, particle, fiber, or the like. Since the filling efficiency of the synthesis reaction vessel can be enhanced, the reaction vessel is not easily broken, and thus a porous synthetic polymer having a particle shape is preferable. The term "particle" in the specification is not meant to be precisely spherical, but rather to have any constant shape (e.g., generally spherical, such as ellipsoidal, polygonal, cylindrical, amorphous, etc.). Although the size (volume) of one particle of the porous synthetic polymer particles is not particularly limited, when the average particle size obtained when the porous particles are measured by laser diffraction (scattering type) is less than 1 μm, inconvenience occurs when it is packed in a column, and the back pressure becomes excessively high or the solution transport speed decreases at the time of use. On the other hand, when the average particle size is more than 1000 μm, the voids between the carrier particles become large, and it becomes difficult to efficiently pack the carrier particles in a column having a predetermined volume. Accordingly, it is preferably 1 to 1000. Mu.m, more preferably 5 to 500. Mu.m, still more preferably 10 to 200. Mu.m.

Examples of the solid phase carriers include polystyrene supports, and rigid functionalized supports such as polydimethylacrylamide, silica, or microporous glass encapsulated with diatomaceous earth; the solid-phase resin matrix can be composed of amphiphilic polystyrene-PEG resin or PEG-polyamide or PEG-polyester resin; as solid phase carriers, there are also included, for example, wang-PEG resins or Rink-amide PEG resins. The most commonly used solid support is controlled microporous glass beads (CPG, controlled pore glass), the pore size of which depends on the length of the oligonucleotide synthesized, and CPG with a pore size of 500 angstroms is typically selected when the synthesized chain length is less than 60 mers; when the chain length is longer than 60mer, 1000 angstrom CPG is used. The coupling efficiency using CPG is as high as 98% -99.9%, and can meet the conditions for synthesizing oligonucleotides up to 175 mer. CPG is covalently bound to the hydroxyl group of the original nucleotide by a linker compound (linker) and the 5' -OH group of the nucleotide is protected with DMT. In some embodiments, the CPG is an unmodified naked CPG having hydroxyl groups on the surface that are covalently linked to an oligonucleotide/nucleotide or nucleotides.

Embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples, in which specific conditions are not noted, are preferably referred to in the guidelines given in the present invention, and may be according to the experimental manuals or conventional conditions in the art, and may be referred to other experimental methods known in the art, or according to the conditions suggested by the manufacturer.

In the specific examples described below, the measurement parameters relating to the raw material components, unless otherwise specified, may have fine deviations within the accuracy of weighing. Temperature and time parameters are involved, allowing acceptable deviations from instrument testing accuracy or operational accuracy.

Example 1

Preparation of novel formulation I ₂/THF/DMF/H₂ O iodine mixture and oxidizer containing organic base TEA

(1) Firstly, respectively weighing 100mL of DMF and 100mL of H ₂ O, adding into a brown glass bottle with 800mL of THF, and fully and uniformly mixing;

(2) Adding the weighed I ₂ simple substance into the solution prepared in the step (1), and uniformly mixing to obtain 0.03M iodine mixed solution;

(3) And finally, sucking 1mL of TEA solution by a pipette, adding the TEA solution into the iodine solution prepared in the step (2), and fully and uniformly mixing to obtain the novel oxidant.

Example 2

(3) And finally, sucking 2mL of TEA solution by a pipette, adding the TEA solution into the iodine solution prepared in the step (2), and fully and uniformly mixing to obtain the novel oxidant.

Example 3

(3) And finally, sucking 3mL of TEA solution by a pipette, adding the TEA solution into the iodine solution prepared in the step (2), and fully and uniformly mixing to obtain the novel oxidant.

Comparative example

Preparation of market general formula I ₂/THF/pyridine/H ₂ O iodine mixed solution oxidant

(1) Firstly, 200mL of pyridine and 100mL of H ₂ O are respectively measured and added into a brown glass bottle with 700mL of THF, and the materials are fully and uniformly mixed;

(2) Adding the weighed I ₂ simple substance into the solution prepared in the step (1), uniformly mixing to obtain 0.03M iodine mixed solution, and fully and uniformly mixing to obtain the oxidant.

Experimental example

In the specific embodiment of the invention, the identification performance evaluation is carried out according to the following method: oligonucleotide synthesis was performed using a dr.oligo 192 high throughput oligonucleotide synthesizer. A50 nmol solid phase CPG synthesis vector column was inserted into a 96 well synthesis plate, and the target oligonucleotide sequence was introduced, and the detailed synthesis procedure was as shown in Table 1. Placing the oligonucleotide carrier column after synthesis in a gas phase pot, performing cleavage and deprotection by ammonia gas phase ammonolysis (90 ℃ for 60 min), washing with acetonitrile after nucleotide cleavage, and eluting the oligonucleotide from the synthesis carrier by 80 mu L of TE buffer; the eluent obtained in the last step is respectively subjected to LC-MS detection, detection of OD ₂₆₀ value by using an enzyme-labeled instrument, high Performance Liquid Chromatography (HPLC) purification analysis detection, and the influence of different proportions on the synthesis quality of the oligonucleotide according to the purity and MS spectrum analysis.

TABLE 1 circulation table for DNA oligonucleotide synthesis on solid phase synthesizer

TABLE 2 product detection method and qualification criteria decision table

Synthetic oligonucleotide sequences: GAAGGTGATGTTGAACACGA A

The detection results of the synthesized oligonucleotides under the condition of the oxidizing agents prepared in experimental examples 1/2/3 and comparative examples are shown in the following Table 3, and the obtained LC-MS mass spectra are shown in FIGS. 1 to 4, respectively;

TABLE 3 oligonucleotide synthesis sequence detection results Table

HPLC analysis the purity and cost of oligonucleotides synthesized using different ratios of oxidants are compared to Table 4 below.

Table 4 comparative synthetic purity and cost for each experimental example

Conclusion:

The synthesis result of the iodine solution of the best example 2 (namely 1L of 0.03M I ₂/THF/DMF/H₂ O plus 2ml of TEA) as an oxidant shows that no obvious impurities are generated under the condition of gas-phase ammonolysis except for the occurrence of a depurination front impurity peak of a small quantity of bases, and the purity of the synthesized oligonucleotide reaches 87.05% at most; in terms of cost, the comparative example traditional formulation costs 129.93 yuan/L, while the new formulation was only about 88.17 yuan/L. The conventional technology represented by the comparative example has the problems of reduced actual concentration of iodine, introduction of pyridine as a harmful reagent, easy pinhole crystallization of a synthesizer in the actual solid phase synthesis process, incomplete blockage of CPG pore canal due to residual reagent cleaning, and the like. Experimental results show that the novel oxidant provided by the application has obvious advantages compared with the traditional iodine liquid oxidant formula, not only has long-term storage stability and selective oxidation performance, but also has higher purity of the synthesized oligonucleotide, and in addition, the introduction of harmful reagent pyridine is effectively avoided, and the production cost is greatly reduced.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. The scope of the invention is therefore intended to be covered by the appended claims, and the description and drawings may be interpreted in accordance with the contents of the claims.

Claims

1. An oxidant for oxidizing the phosphite triester, which is an iodine solution, wherein the solvent comprises THF, DMF and water with the volume ratio of (7-9): (0.8-1.2);

And the oxidant also contains (1-3) mL/L of organic alkali with pKa of 9-14.

2. The oxidant of claim 1, wherein the solvent has a volume ratio of THF, DMF and water of (7.5-8.5): 0.9-1.1; preferably, the volume ratio is 8:1:1.

3. The oxidizing agent according to claim 1, wherein the concentration of iodine in the iodine solution is 0.015M to 0.03M.

4. The oxidant of claim 1, the organic base being selected from one or more of triethylamine, tert-butylamine, N-propylamine, N-diisopropylethylamine, N-methylimidazole, and 1, 8-diazabicyclo [5.4.0] undec-7-ene; preferably, the organic base is triethylamine.

5. The oxidizing agent according to any of claims 1 to 4, which does not contain pyridine.

6. A kit for solid phase synthesis of oligonucleotides by the phosphite triester method, comprising the oxidizing agent of any one of claims 1 to 5.

7. The method for producing an oxidizing agent according to any one of claims 1 to 5, comprising:

b) Mixing an organic base with the solution obtained in a).

8. The process according to claim 7, which is carried out under light-protected conditions.

9. A method of oligonucleotide synthesis comprising the steps of: oxidizing an intermediate having phosphite triester linkages to a pentavalent triester intermediate in the presence of an oxidizing agent;

10. Use of the oxidizing agent of any one of claims 1 to 5, or the kit of claim 6, in solid phase synthesis of oligonucleotides by the phosphite triester method.

11. The use according to claim 10, wherein the oligonucleotide is selected from the group consisting of a primer, a probe, siRNA, miRNA, mRNA, sgRNA, a nucleic acid aptamer or an antisense nucleic acid.