Enzymatic and Chemical Labeling Strategies for Oligonucleotides

Abstract

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Labeling strategies for oligonucleotides (oligos) differ somewhat from those for other commonly encountered biopolymers, such as proteins and carbohydrates, since oligos are exceptionally inert to common bioconjugation reagents under standard reaction conditions. While reagents are available that can modify oligo base units, such base modification usually adversely impact the binding capacity of the oligo for its target sequence. Further complicating matters, many haptens and fluorophores cannot withstand the final base cleavage conditions employed in solid-phase oligo synthesis, thus requiring that the bioconjugation step be performed after the oligo has been released from the resin. However, certain small molecules can often be incorporated directly during the oligo synthesis process. Oligos are usually first modified at either their 5' or 3' end during synthesis, using suitable functional groups for bioconjugation such as phosphate, thiol or amine moieties. Then, functional group-reactive probes are introduced to label the targeted site. Oligos may also incorporate modified bases, such as modified CPGs or phosphoramidites, directly during solid-phase oligo synthesis.

Introduction

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DNA is composed of a 2'-deoxyribose 5'-phosphate backbone and four heterocyclic bases: adenine (A), guanine (G), thymine (T), and cytosine (C) linked through an N-glycosidic bond, while RNA contains a ribose 5'-phosphate backbone and the same bases, except thymine is replaced with uracil (U). Oligonucleotides (oligos) are short-chain nucleic acid polymers containing ~10 to 100 monomer building blocks. While proteins typically contain inherently fluorescent amino acids like tryptophan and tyrosine, the intrinsic fluorescence emission of the five most common nucleotides (A, G, T, C, U) is too weak to be of analytical utility in detecting oligos for most biological applications. Advances in organic chemistry and in polymerase engineering have broadened the applications of nucleic acids well beyond their native roles in living organisms. To that end, a wide range of chemical and enzymatic methods have been devised to facilitate the detection of oligos. Radioactive phosphate (32P, 33P) is commonly inserted at the 5' or 3' end of an oligo post-synthesis using enzyme-catalyzed reactions. In recent decades, labeling with haptens and fluorophores has gained in popularity relative to incorporating such radioisotopes, due to the inherent hazards and high disposal costs associated with handling radioactivity. However, alternative probes, such as fluorescent dyes and small molecule haptens, may adversely influence the conformation and binding properties of an oligo, reducing the specificity of hybridization with its target. Thus, the challenge is to develop nucleic acid analogs and labeling procedures for different applications that do not substantially perturb hybridization. In this context, pre- and post-synthetic routes, as well as automated solid phase-based syntheses and enzymatic labeling methods, have been devised, as summarized in this article. Oligos conjugated with fluorophores can readily be detected in assays involving either gel- or microplate readers, while oligos conjugated with biotin or resin are especially useful for affinity purification procedures. Oligo conjugates are increasingly finding application in molecular diagnostics, and nanotechnology and nucleic acid-based therapeutic applications.

Enzymatic Conjugation Methods

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Molecular probes and chemically reactive functional groups are often incorporated into oligos during their synthesis (Wojczewski et al. 1999). While convenient, this strategy can incur additional costs, mainly if an experimental procedure requires both labeled and unlabeled versions of the same oligo sequence (Zearfoss and Ryder, 2012). End-labeling oligos after synthesis provide a simple and easily adapted alternative to probe incorporation during oligo synthesis. By this route, oligo conjugate preparation is achieved by including synthetic steps after the primary oligo synthesis process has been completed.

5' end labeling

An example of an enzymatic approach to labeling the 5' end of an oligo employs a two-step reaction scheme (Czworkowski et al. 1991; Zearfoss and Ryder, 2012). The strategy takes advantage of the ability of bacteriophage T4 polynucleotide kinase (T4 PNK) to transfer a phosphate group to the 5' end of RNA or DNA oligos. For the reaction, ATP is substituted with adenosine 5'-[?-thio] triphosphate (ATP?S), an ATP analog in which the gamma phosphate has been replaced with phosphorothioate. The reaction product is a phosphorylated oligo containing a single reactive sulfur group at the 5' end of the oligo. The oligo may subsequently be incubated with a haloacetamide or maleimide derivative of a molecular probe (Table 1), which then reacts with the phosphorothioate to generate the labeled oligo product. For example, the fluorophore, 5-(iodoacetamido ) fluorescein (5-IAF) (Cat No. 222), can be conjugated to the 5' end of a DNA or RNA oligo by this basic procedure.

1. The Tide Fluor™ WS [TFWS] dyes have enhanced water solubility.

Another enzymatic approach to labeling the 5'end of an oligo is post-synthetic phosphorylation and substitution of the synthesized and purified oligo (Mergny et al. 1994). Much like the previous example, the DNA oligo is enzymatically phosphorylated at the 5'-hydroxyl group using T4 PNK and native ATP. Subsequently, the phosphate residue is activated with N-methyl-imidazole, which is then readily substituted by ethylenediamine to generate a free primary amino group for fluorophore labeling (Table 2).

1. The Tide Fluor™ WS [TFWS] dyes have enhanced water solubility.

3' end labeling

The template-independent DNA polymerase, terminal deoxynucleotidyl transferase (TdT), catalyzes repetitive addition of mononucleotides from dNTPs or NTPs to the terminal 3'-OH of a DNA initiator, accompanied by the release of inorganic phosphate (Guerra, 2006; Sarac and Hollenstein, 2019). 4-thiouridine may be introduced at the 3'-terminus of DNA using the cited enzyme, followed by treatment with ribonuclease and reaction with thiol-reactive probes (Guerra, 2006; Sarac and Hollenstein, 2019, Table 1).
TdT can also directly incorporate fluorophore-labeled nucleotides to the 3'-hydroxyl terminus of DNA oligos. In the presence of a labeled nucleotide-triphosphate (dNTP or NTP, Table 3), such as Cy3-UTP, the enzyme incorporates ?20 to 100 labeled bases per 3' end (Guerra, 2006). This cited approach can easily be customized to the production process and quality control of oligo microarrays since oligos are typically directionally coupled to the solid-phase substrate using a 5' end linkage (Guerra, 2006).
Recently, a one-step enzymatic method to modify RNA 3' termini using recombinant human polymerase theta (Pol?) has been reported (Thomas et al. 2019). Pol? efficiently appends 30–50 2' -deoxyribonucleotides to the 3' terminus of RNA oligos, extending the termini with any of a variety of modified 2'-deoxy and 2',3' -dideoxy ribonucleotide analogs containing fluorophores or affinity tags (Table 3). Pol? displays a strong preference for adding deoxyribonucleotides to RNA, but additionally can add ribonucleotides with relatively high efficiency for certain sequence contexts. As TdT is not able to act on RNA, Pol? provides a unique capability as an RNA 3'-terminal extension enzyme, capable of efficiently adding canonical nucleotides and a variety of nucleotide analogs to RNA (Thomas et al., 2019)

Chemical Conjugation Methods

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Indirect Modification of Oligos with Probes Bearing Reactive Groups

Chemical conjugation methods typically require the synthesis of modified oligos containing susceptible pendant groups that can subsequently be chemically modified with a molecular probe. Oligos are typically synthesized by the phosphoramidite approach on a solid phase support with controlled pore width (Beaucage et al. 1993). The synthesis is initiated with the 5'-end deprotection of the CPG-bound nucleoside. The free hydroxyl group generated then reacts with the 3'-phosphoramidite of the subsequently introduced nucleoside, activated with 1H-tetrazole to promote the reaction. Any remaining hydroxyl groups are capped to prevent elongation of missense strands. The dinucleoside phosphite generated is then oxidized to the corresponding phosphate using a mixture of iodine in water, and the cycle can then be repeated to add more bases. Once the synthesis is complete, ammonia is employed to release the product from the solid-phase surface and to deprotect the exocyclic amino groups of the nucleobases. The modular nature of the synthetic procedure allows the covalent introduction of probes for detection at specific positions along the length of a DNA or RNA oligo. A wide range of substituted fluorophores suitable for labeling oligos is available, including acridine, coumarin, cyanine, dansyl, fluorescein, oxazine, phenazine, and rhodamine dye derivatives.

Amino-Modified Oligos

Modified nucleotides, such as 5-aminohexylacrylamido-dUTP and 5-aminohexylacrylamido-dCTP, can be utilized to generate amine-modified DNA using a variety of enzymatic incorporation methods, including nick translation, random primed labeling, reverse transcription, and PCR. The amine-modified DNA can then be labeled with any of a wide variety of amine-reactive dyes or haptens. Certain 5'-amino-modifiers have been specifically designed for use in automated synthesizers to functionalize the 5'-terminus of a target oligo with a primary amine moiety. The resulting amino-modified oligos can be conjugated to various tag molecules such as fluorophores, biotins, alkaline phosphatase, and HRP. Due to the increased possibility of side reactions during the deprotection of modified oligos, it is recommended that the ammonium hydroxide treatment be carried out at a lower temperature than that used for unmodified oligos. Amine-reactive fluorescent probes are widely used to modify amino-modified oligos at the introduced amino residue. Many fluorescent amino-reactive dyes have been developed to label various oligos, and the resultant conjugates are widely used in biological applications (Table 2). Two major classes of amine-reactive fluorophores are extensively used to label oligos: succinimidyl esters (SE) and sulfonyl chlorides (SC). Succinimidyl esters (SE) are considered more suitable for modifying amine groups since the amide bonds formed are generally quite stable. By contrast, sulfonyl chlorides (SC) are highly reactive but unstable in water, especially at the higher pH values required for reactions with aliphatic amines. Molecular modifications by sulfonyl chlorides need to be carefully performed, preferably at low temperatures. Sulfonyl chlorides may also react with phenols (such as tyrosine), aliphatic alcohols (such as polysaccharides), thiols (such as cysteine), and imidazoles (such as histidine, a consideration when labeling hybrid peptide- or oligosaccharide-oligos.

Thiol-Modified Oligos

Thiol-modified oligos can be synthesized by incorporating the thiol modification during solid-phase phosphoramidite oligo synthesis at either the 5'- end or the 3'-end of the oligo (Beaucage and Iyer, 1993). Thiol residues must be protected during oligo synthesis since they are strong nucleophiles that can interfere with phosphoramidite chemistry, and unprotected thiol groups spontaneously form disulfide crosslinks under neutral aqueous solution conditions. Two methods are routinely employed to protect thiol residues during oligo synthesis, disulfide protection, and trityl protection. After oligo synthesis and deprotection, reaction byproducts are removed by gel filtration chromatography. Since free thiol (SH) groups are not present as abundantly as other groups in biopolymers, thiol-reactive reagents often provide a means of selectively modifying a hybrid oligo at a defined site. Therefore thiol-reactive dyes are often used to prepare fluorescent oligos for probing biological structure, function, and interactions (Table 1). Many types of thiol-reactive dyes are available, including iodoacetamides, disulfides, maleimides, and vinyl sulfones, as well as various electron-deficient aryl halides and sulfonates. Maleimides are by far the most popular thiol-reactive moiety, since they react readily with thiol moieties of biopolymers to form very stable thioether conjugates, even under neutral conditions. Maleimides require conjugation conditions less stringent than those of iodoacetamides, and do not react with histidine and methionine residues under physiological conditions. Most conjugation reactions can be performed at room temperature and neutral pH conditions.

Click Chemistry Oligos

Click chemistry is a two-step labeling process involving the quantitative chemical reaction of alkyne and azide moieties to create covalent carbon-heteroatom bonds (Rostovtsev et al. 2002; Moses et al. 2007). The reaction employs the catalyst, copper(I) to form a 1,2,3-triazole bond between an azide and a terminal alkyne moiety. The process has proven itself to be reliably performed, and the resulting bond is relatively stable, making it especially suitable in demanding biological applications. The advantages of the method are that the reaction is a robust catalytic process that can be performed in aqueous solution and at room temperature, generating no side reactions, exhibiting no functional group interference, and producing a thermally and hydrolytically stable triazole linkage. Click chemistry reactions are stereospecific, simple to perform, and can be conducted in easily removable or benign solvents. Click chemistry has proven to be an efficient process for the conjugation of peptides, small molecules, fluorophores, and carbohydrates to oligos.

Direct Incorporation of Modified Bases into Oligonucleotides

5'-Amino-modifying bases may be composed of a ?-cyanoethyl phosphoramidite for automated DNA synthesis and a primary amine serving as a reactive group for post-labeling strategies. Alternatively, 3'-phosphoramidites of 5'-amino-5'-deoxynucleosides can be applied for DNA sequencing with labeled primers (Wojczewski et al. 1999). Since the necessary hydroxyl group for further chain elongation is lacking with 5'-modifying bases, they can only be employed as the final step of oligonucleotide synthesis.

When modification at the 3'-terminus of the oligonucleotide is required, leaving the 5'-end unmodified, a controlled pore glass (CPG) as a solid-phase support is employed, functionalized with a protected amine for post-synthetic probe coupling. Additionally, a dimethoxytrityl-protected hydroxyl group for standard nucleoside coupling can be employed (Wojczewski et al. 1999).

Internal sequence modification permits incorporating a molecular probe at a defined position within the oligo (Wojczewski et al. 1999). This can be necessary when the position of the probe is critical to the application, such as in energy transfer experiments involving donor and acceptor dyes. Internal modifying nucleoside 3'-phosphoramidites typically possess a dimethoxytrityl group that can be removed after the coupling step, unmasking a free hydroxyl group to continue oligo synthesis in the 5'-direction. The modified base employed is typically thymine (T), functionalized with a C-2 or C-6 linker terminating in a primary amino group.

Finally, approaches involving fluorescent probe-modified phosphoramidites may be employed as "pre-synthetic labeling building blocks" during oligo synthesis (Wojczewski et al. 1999). Since conventional automated oligo synthesis proceeds from 3' to 5', the 5'-terminus is usually selected for modification. A general approach to modifying the 5'-terminus is to use reagents that couple to the 5'-hydroxyl of an oligo. Fluorophore phosphoramidite reagents are readily adapted for use in automated synthesizers, with little or no modification to existing protocols. Typically, these reagents are highly compatible with automated DNA synthesizers. In general, fluorophore-labeled oligos can be deprotected at room temperature in concentrated ammonium hydroxide. FAM, Dabcyl, and Tide Quencher™ (TQ)-labeled oligos can be heated to 55 °C in ammonium hydroxide for extended periods of time. However, TET, TF-3, and Cy-3 labeled oligos are less stable and survive only for a few hours at 55 °C. HEX, TF-5, and Cy-5 labeled oligonucleotides must be deprotected at room temperature, and the residual ammonia should be removed immediately after deprotection. Numerous fluorophore-containing phosphoramidites are commercially available (Table 5). In most instances, these fluorophores are incorporated into oligos without substantially impacting hybridization efficiency. Simple fluorophore phosphoramidites can be used as 5'-labeling reagents, while fluorophore phosphoramidites additionally containing a dimethoxytrityl protected hydroxyl group provide an option when the label is to be located at an internal position within the oligo. For 3'-labeling, fluorophores can also be coupled to the solid-phase support.

In this context, Tide Fluor™ dyes (such as TF1, TF2, TF3, TF4, TF5, TF6, TF7, and TF8) are particularly well suited to labeling oligos, offering stronger fluorescence and higher photostability than the classical fluorophores such as fluorescein, rhodamine, and cyanine dyes. For instance, TF2 features similar excitation and emission wavelength maxima as carboxyfluoresceins (FAM), making it readily used for the biological applications that are conducted with fluoresceins. Compared to FAM probes, TF2 has much stronger fluorescence under physiological conditions, and it is much more photostable. Compared to other fluorescent dye alternatives to fluoresceins and Cy^® dyes (such as Alexa Fluor™ and DyLight^® dyes), Tide Fluor™ dyes are also much more cost-effective while offering comparable or superior performance for many biological applications. TF3 is much brighter and more photostable on oligonucleotides than Cy3^®, Alexa Fluor^® 555, and DyLight™ 555, although TF3 has almost identical spectra to these three dyes.

3' end labeling of RNA oligos

A chemical approach to 3' end-labeling of an RNA oligo employs a two-step process (Reines and Cantor, 1974; Zearfoss and Ryder, 2012). Sodium periodate is used to oxidize the 3' terminal ribose sugar, forming a reactive dialdehyde. Next, the oxidized sugar is conjugated to an aldehyde-reactive molecular probe tag (Table 6), such as fluorescein 5-thiosemicarbazide. Since periodate oxidation requires vicinal hydroxyl residues, the reaction is highly specific for RNA and only modifies the 3' terminal ribose. The plethora of aldehyde-reactive molecular probes support conjugation of a wide variety of fluorophores to the 3' end. In addition, oligos may be conjugated to biotin, using reagents like (+)-biotin amidohexanoic acid hydrazide (BACH). The cited labeling strategy can readily be performed at the laboratory bench, requiring no specialized equipment.

Dye CPGs for Modifying Oligos

Fluorophore-Chemical Phosphorylation Reagent (CPR) supports can be used to incorporate dye labels at the 3'-terminus of oligos. CPGs are derived from dye carboxylic acids and are attached via an amide linkage, giving an oligo product that is much easier to purify by HPLC. The use of dye CPGs in oligo synthesis proceeds in a manner analogous to using a standard nucleoside support with some necessary modifications. Different fluorophore-CPGs may require different cleavage methods. The cleavage of oligos from FAM and Tide Quenchers™ (TQs) supports is similar to the standard ammonium hydroxide cleavage, while TAMRA CPG must be deprotected under very mild conditions to safeguard the base-labile TAMRA fluorophore. In the latter instance, it is advised to use UltraMild monomers with potassium carbonate in methanol for deprotection. An alternative procedure employing t-butylamine/methanol/water (1:1:2) should allow the use of regular monomers. Tide Quencher™ CPGs are especially useful for making FRET probes, using conventional fluorophores or Tide Fluor™ probes.

Brief Highlight of Applications

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Some of the major applications for modified oligos include Dot, Northern, and Southern blotting, RNA and DNA in situ hybridization (ISH), multicolor fluorescence in situ hybridization (mFISH), comparative genome hybridization (CGH), PCR, Real-Time PCR, DNA sequencing, site-directed mutagenesis, single-nucleotide polymorphism (SNP) assays, and microarray analysis. Fluorophore-, quencher- and hapten- labeled oligos are essential tools in both biochemical and cellular studies. Fluorescent oligos are used extensively in fluorescence fluorimetry, fluorescence microscopy, fluorescence polarization spectroscopy, time-resolved fluorescence (TRF), and fluorescence resonance energy transfer (FRET). FRET oligonucleotides are widely used for diagnosing infectious diseases based upon the molecular beacon and other technologies. FRET oligonucleotides have also been used for cell analysis via fluorescence-associated cell sorting (FACS) either in vivo or in vitro for research and diagnostic purposes. The most important distinguishing features associated with fluorescent oligos correspond to high detection sensitivity without the complications associated with handling radioactivity.

For certain applications, such as DNA sequencing and in situ hybridization (ISH), oligos are usually required to be singly labeled. Subsequent detection and analysis depend upon the fluorescence properties of the dye itself. However, for other biological applications, e.g., probes for real-time PCR quantification of DNA and RNA and allele discrimination (Molecular Beacons™), oligos are required to be doubly labeled. With doubly labeled oligos, one dye serves as the fluorophore, while the other is a quencher. With dual-labeled probes that are not bound to a target sequence, the light fluorophore emission is undetectable, as the quencher dye absorbs it via a process referred to as Fluorescent Resonance Energy Transfer (FRET). Because FRET is a distance-dependent interaction between the excited state of the donor and acceptor dye molecules, their eventual separation through hybridization to target in the detection event allows the fluorescence to be detected. To maximize the FRET efficiency, the FRET pairs need to be carefully selected based upon the consideration of fluorescence lifetime and the spectral overlap of donor emission with acceptor excitation. A variety of FRET building blocks for labeling oligos are commercially available, including classic dyes and optimized Tide Fluor™ (donors) and Tide Quenchers™ (acceptors).

Summary

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Radioactively labeled oligos provide high detection sensitivity, and the probes can readily be added to an oligo post-synthesis. However, their use can be problematic due to the hazardous nature of radioisotopes, the regulatory burden associated with their laboratory use and disposal, and the limited half-lives of the probes. Molecular probes, such as fluorescent dyes and small molecule haptens, are suitable alternatives to radioactive labeling, providing abundant sensitivity for most applications. Additionally, the modification of oligos with fluorophores broadens the range of experimental procedures that can be performed with oligos. For instance, assays based upon fluorescence resonance energy transfer (FRET) and fluorescence polarization (FP) depend upon the physicochemical properties of fluorescent dyes and cannot be undertaken using radioisotope labels.
Similarly, biotin-conjugated oligos can be noncovalently attached to streptavidin-conjugated resins, facilitating retrieval and purification of specific target molecules. The majority of molecular biology techniques employed in modern biomedical research laboratories require chemically synthesized DNA or RNA oligos. Oligo conjugates are widely employed in various biotechnology applications (ISH, microarray analysis, CGH), in research, as diagnostics tools, and for nucleic acid-based therapeutics.

References

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