AAT Bioquest

Enzymatic and Chemical Labeling Strategies for Oligonucleotides


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.



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

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.

Table 1. Phosphorothiolate-reactive maleimides potentially suitable for 5’-end labeling of RNA or DNA oligos.

Superior Alternative to
Excitation Max (nm)
Emission Max (nm)
Tide Fluor™ 1 Maleimide [TF1 Maleimide]EDANS341448
Tide Fluor™ 2 Maleimide [TF2 Maleimide]Fluoresceins (FAM and FITC)503525
Tide Fluor™ 2WS Maleimide [TF2WS Maleimide]Fluoresceins (FAM and FITC)503525
Tide Fluor™ 3 Maleimide [TF3 Maleimide]Cy3®554578
Tide Fluor™ 3WS Maleimide [TF3WS Maleimide]Cy3®551563
Tide Fluor™ 4 Maleimide [TF4 Maleimide]ROX/Texas Red®578602
Tide Fluor™ 5WS Maleimide [TF5WS Maleimide]Cy5®649664
Tide Fluor™ 6WS Maleimide [TF6WS Maleimide]Cy5.5682701
Tide Fluor™ 7WS Maleimide [TF7WS Maleimide]Cy7®756780
Tide Fluor™ 8WS Maleimide [TF8WS Maleimide]IRDye® 800785801
  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).

Table 2. Ethylenediamine-reactive succinimidyl esters potentially suitable for labeling of DNA oligos.

Superior Alternative to
Excitation Max (nm)
Emission Max (nm)
Tide Fluor™ 1 Succinimidyl Ester [TF1 SE]EDANS341448
Tide Fluor™ 2 Succinimidyl Ester [TF2 SE]Fluoresceins (FAM and FITC)503525
Tide Fluor™ 2WS Succinimidyl Ester [TF2WS SE]Fluoresceins (FAM and FITC)503525
Tide Fluor™ 3 Succinimidyl Ester [TF3 SE]Cy3®554578
Tide Fluor™ 3WS Succinimidyl Ester [TF3WS SE]Cy3®551563
Tide Fluor™ 4 Succinimidyl Ester [TF4 SE]ROX/Texas Red®578602
Tide Fluor™ 5WS Succinimidyl Ester [TF5WS SE]Cy5®649664
Tide Fluor™ 6WS Succinimidyl Ester [TF6WS SE]Cy5.5682701
Tide Fluor™ 7WS Succinimidyl Ester [TF7WS SE]Cy7®756780
Tide Fluor™ 8WS Succinimidyl Ester [TF8WS SE]IRDye® 800785801
  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)

Table 3. Probe-labeled nucleotides potentially suitable for 3’-end labeling of RNA or DNA oligos.

Unit Size
Cat No.
2-Aminoethoxypropargyl ddATP1 µmoles17084
2-Aminoethoxypropargyl ddCTP1 µmoles17080
2-Aminoethoxypropargyl ddGTP1 µmoles17086
2-Aminoethoxypropargyl ddTTP1 µmoles17082
5-Propargylamino-3'-azidomethyl-dCTP50 nmoles17091
5-Propargylamino-3'-azidomethyl-dUTP50 nmoles17093
7-Deaza-7-Propargylamino-3'-azidomethyl-dATP50 nmoles17090
7-Deaza-7-Propargylamino-3'-azidomethyl-dGTP50 nmoles17092
AA-dUTP [Aminoallyl dUTP sodium salt] *4 mM in Tris Buffer (pH 7.5)* *CAS 936327-10-5*1 µmole17004
AA-dUTP [Aminoallyl dUTP sodium salt] *4 mM in Tris Buffer (pH 7.5)* *CAS 936327-10-5*2.5 µmole17005
AA-UTP [Aminoallyl UTP sodium salt] *4 mM in TE buffer* *CAS 75221-88-4*250 µL17021
Aminopropargyl dATP [7-Deaza-7-Propargylamino-2'-deoxyadenosine-5'-triphosphate]10 µmoles17056
Aminopropargyl dCTP [5-Propargylamino-2'-deoxycytidine-5'-triphosphate]10 µmoles17050
Aminopropargyl ddATP [7-Deaza-7-Propargylamino-2',3'-dideoxyadenosine-5'-triphosphate]10 µmoles17074
Aminopropargyl ddCTP [5-Propargylamino-2',3'-dideoxycytidine-5'-triphosphate]10 µmoles17070
Aminopropargyl ddGTP [7-Deaza-7-Propargylamino-2',3'-dideoxyguanosine-5'-triphosphate]10 µmoles17076
Aminopropargyl ddTTP [5-Propargylamino-2',3'-dideoxyuridine-5'-triphosphate]10 µmoles17072
Aminopropargyl dGTP [5-Propargylamino-2'-deoxyguanosine-5'-triphosphate]10 µmoles17059
Aminopropargyl dUTP [5-Propargylamino-2'-deoxyuridine-5'-triphosphate]10 µmoles17053
Biotin-11-dATP25 nmoles17014
Biotin-11-dGTP25 nmoles17015
Biotin-11-dUTP *1 mM in Tris Buffer (pH 7.5)* *CAS 86303-25-5*25 nmoles17016
Biotin-14-dCTP *1 mM in Tris Buffer (pH 7.5)*25 nmoles17019
Biotin-16-dUTP *1 mM in Tris Buffer (pH 7.5)* *CAS 136632-31-0*25 nmoles17017
Biotin-20-dUTP *1 mM in Tris Buffer (pH 7.5)*25 nmoles17018
Cyanine 5-dATP [Cy5-dATP]25 nmoles17038
Cyanine-3- dUTP [Cy3-dUTP]  *1 mM in Tris Buffer (pH 7.5)*25 nmoles17025
Cyanine-5- dUTP [Cy5-dUTP]  *1 mM in Tris Buffer (pH 7.5)*25 nmoles17026
ddATP [2',3'-Dideoxyadenosine-5'-triphosphate]1 µmole17209
ddCTP [2',3'-Dideoxycytidine-5'-triphosphate]1 µmole17207
ddGTP [2',3'-Dideoxyguanosine-5'-triphosphate]1 µmole17210
ddTTP [2',3'-Dideoxythymidine-5'-triphosphate]1 µmole17208
DEAC-dUTP *1 mM in Tris Buffer (pH 7.5)*25 nmoles17024
Digoxigenin-11-dUTP *1 mM solution in water*25 nmoles17012
Fluorescein-12-dUTP (Perkin-Elmer) *1 mM in Tris Buffer (pH 7.5)*25 nmoles17027
Fluorescein-12-dUTP *1 mM in Tris Buffer (pH 7.5)*25 nmoles17028
Fluorescein-12-dUTP *1 mM in Tris Buffer (pH 7.5)* *CAS 214154-36-6*25 nmoles17022
iFluor® 440-dUTP *1 mM in Tris Buffer (pH 7.5)*25 nmoles17029
iFluor®488-dUTP *1 mM in Tris Buffer (pH 7.5)*25 nmoles17039
MagaDye™ 535-ddGTP5 nmoles17063
MagaDye™ 535-ddGTP50 nmoles17067
MagaDye™ 561-ddATP5 nmoles17062
MagaDye™ 561-ddATP50 nmoles17066
MagaDye™ 588-ddTTP5 nmoles17061
MagaDye™ 588-ddTTP50 nmoles17065
MagaDye™ 613-ddCTP5 nmoles17060
MagaDye™ 613-ddCTP50 nmoles17064
mFluor™ Violet 450-dUTP *1 mM in Tris Buffer (pH 7.5)*25 nmoles17011
Tetramethylrhodamine-dUTP *1 mM in Tris Buffer (pH 7.5)*25 nmoles17023
TF1-dUTP *1 mM in Tris Buffer (pH 7.5)*25 nmoles17006
TF2-dUTP *1 mM in Tris Buffer (pH 7.5)*25 nmoles17007
TF3-dUTP *1 mM in Tris Buffer (pH 7.5)*25 nmoles17008
TF4-dUTP *1 mM in Tris Buffer (pH 7.5)*25 nmoles17009
TF5-dUTP *1 mM in Tris Buffer (pH 7.5)*25 nmoles17010
XFD™488-dUTP *1 mM in Tris Buffer (pH 7.5)*25 nmoles17040


Chemical Conjugation Methods

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.

Table 4. Dye Azides potentially suitable for labeling of DNA oligos by Click Chemistry.

Superior Alternative to
Excitation Max (nm)
Emission Max (nm)
Unit Size
Cat No.
Tide Fluor™ 1 azide [TF1 azide]EDANS3414485 mg2236
Tide Fluor™ 2 azide [TF2 azide]Fluoresceins (FAM and FITC)5035251 mg2252
Tide Fluor™ 3 azide [TF3 azide]Cy3®5545781 mg2254
Tide Fluor™ 4 azide [TF4 azide]ROX/Texas Red®5786021 mg2300
Tide Fluor™ 5WS azide [TF5WS azide]Cy5®6496641 mg2275
Tide Fluor™ 6WS azide [TF6WS azide]Cy5.56827011 mg2302
Tide Fluor™ 7WS azide [TF7WS azide]Cy7®7567801 mg2304
Tide Fluor™ 8WS azide [TF8WS azide] *Near Infrared Emission*IRDye® 8007858011 mg2306

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.

Table 5. Fluorophore phosphoramidites potentially suitable for 3’-end labeling of DNA oligos.

Ex (nm)
Em (nm)
Unit Size
Cat No.
5'-DABCYL C6 phosphoramidite  1 g6009
6-FAM phosphoramidite [5'-Fluorescein phosphoramidite] *CAS 204697-37-0*493517100 µmoles6016
6-FAM phosphoramidite [5'-Fluorescein phosphoramidite] *CAS 204697-37-0*49351710 x 100 µmoles6017
6-Fluorescein phosphoramidite498517100 µmoles6018
6-Fluorescein phosphoramidite49851710 x 100 µmoles6019
6-HEX phosphoramidite [5'-Hexachlorofluorescein phosphoramidite]533559100 µmoles6026
6-HEX phosphoramidite [5'-Hexachlorofluorescein phosphoramidite]53355910 x 100 µmoles6024
6-TET phosphoramidite [5'-Tetrachlorofluorescein phosphoramidite]52154210 x 100 µmoles6025
6-TET phosphoramidite [5'-Tetrachlorofluorescein phosphoramidite] *CAS#: 877049-90-6*52154250 µmoles6021
6-TET phosphoramidite [5'-Tetrachlorofluorescein phosphoramidite] *CAS#: 877049-90-6*521542100 µmoles6027
FAM-xtra™ Phosphoramidite49351750 µmoles6037
FAM-xtra™ Phosphoramidite493517100 µmoles6038
FAM-xtra™ Phosphoramidite49351710 x 100 µmoles6039
Tide Fluor™ 3 phosphoramidite [TF3 CEP] *Superior replacement to Cy3  phosphoramidite*560580100 µmoles2274
Tide Quencher™ 1 phosphoramidite [TQ1 phosphoramidite]  100 µmoles2198
Tide Quencher™ 2 phosphoramidite [TQ2 phosphoramidite]  100 µmoles2208
Tide Quencher™ 3 phosphoramidite [TQ3 phosphoramidite]  100 µmoles2228
6-VIC phosphoramidite52654350 µmoles6086
6-VIC phosphoramidite526543100 µmoles6087
6-VIC phosphoramidite5265431 g6088

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.

Table 6. Aldehyde-reactive molecular probes potentially suitable for 3’-end labeling of RNA oligos.

Ex (nm)
Em (nm)
Unit Size
Cat No.
Biocytin hydrazide *CAS 102743-85-1*  25 mg3086
Biotin hydrazide *CAS 66640-86-6*  25 mg3007
Cyanine 3 hydrazide [equivalent to Cy3® hydrazide]5555691 mg146
Cyanine 5 hydrazide [equivalent to Cy5® hydrazide]6516701 mg156
Cyanine 5.5 hydrazide [equivalent to Cy5.5® hydrazide]6837031 mg177
Cyanine 7 hydrazide [equivalent to Cy7® hydrazide]7567791 mg166
ICG hydrazide7898141 mg987
iFluor™ 350 hydrazide3454501 mg1080
iFluor™ 405 hydrazide4034271 mg1081
iFluor™ 488 hydrazide4915161 mg1082
iFluor™ 555 hydrazide5575701 mg1083
iFluor™ 647 hydrazide6566701 mg1085
iFluor™ 680 hydrazide6847011 mg1086
iFluor™ 700 hydrazide6907131 mg1087
iFluor™ 750 hydrazide7577791 mg1088
iFluor™ 790 hydrazide7878121 mg1364
ReadiView™ biotin hydrazide  5 mg3055
Texas Red® hydrazide *Single Isomer*5866035 mg481

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.


Table 7. Fluorophore-CPG probes potentially suitable for 3’-end labeling of oligos.

Abs/Ex (nm)
Em (nm)
Unit Size
Cat No.
3'-(6-Fluorescein) CPG *1000 Å*4985171 g6014
3'-DABCYL CPG *1000 Å*454 1 g6008
6-TAMRA CPG *1000 Å*5525781 g6051
BXQ-1 CPG (1000 A)522 100 mg2410
BXQ-1 CPG (500 A)522 100 mg2408
BXQ-2 CPG (1000 A)554 100 mg2430
BXQ-2 CPG (500 A)554 100 mg2428
CDPI3-CPG [Minor Groove Binder CPG] *1000A*  100 mg6902
CDPI3-CPG [Minor Groove Binder CPG] *500A*  100 mg6900
Tide Fluor™ 1 CPG [TF1 CPG] *1000 Å* *Superior replacement for EDANS*341448100 mg2241
Tide Fluor™ 1 CPG [TF1 CPG] *500 Å* *Superior replacement for EDANS*341448100 mg2240
Tide Quencher™ 1 CPG [TQ1 CPG] *1000 Å*492 100 mg2194
Tide Quencher™ 1 CPG [TQ1 CPG] *500 Å*492 100 mg2193
Tide Quencher™ 2 CPG [TQ2 CPG] *1000 Å*516 100 mg2204
Tide Quencher™ 2 CPG [TQ2 CPG] *500 Å*516 100 mg2203
Tide Quencher™ 3 CPG [TQ3 CPG] *1000 Å*573 100 mg2224
Tide Quencher™ 3 CPG [TQ3 CPG] *500 Å*573 100 mg2223
Tide Quencher™ 4 CPG [TQ4 CPG] *1000 Å*603 100 mg2063
Tide Quencher™ 4 CPG [TQ4 CPG] *500 Å*603 100 mg2062
Tide Quencher™ 5 CPG [TQ5 CPG] *1000 Å*661 100 mg2078
Tide Quencher™ 5 CPG [TQ5 CPG] *500 Å*661 100 mg2077


Brief Highlight of Applications

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).



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.



  1. Beaucage, S.L.; Iyer, R.P. "The functionalization of oligonucleotides via phosphoramidite derivatives." Tetrahedron 1993, 49, 1925–1963
  2. Czworkowski J, Odom OW, Hardesty B. "Fluorescence study of the topology of messenger RNA bound to the 30S ribosomal subunit of Escherichia coli". Biochemistry. 1991; 30:4821–4830
  3. Guerra CE. "Analysis of oligonucleotide microarrays by 3' end labeling using fluorescent nucleotides and terminal transferase." Biotechniques. 2006 Jul;41(1):53-6
  4. Mergny, J.-L.; Boutorine, A. S.; Garestier, T.; Belloc, F.; Rougée, M.; Bulychev, N. V.; Koshkin, A. A.; Bourson, J.; Lebedev, A. V.; Valeur, B.; Thuong, N. T.; Hélène, C. 'Fluorescence energy transfer as a probe for nucleic acid structures and sequences.' Nucleic Acids Res. 1994, 22, 920-928
  5. Reines SA, Cantor CR. "New fluorescent hydrazide reagents for the oxidized 3'-terminus of RNA". Nucleic Acids Res. 1974; 1:767–786
  6. Rostovtsev, V.V., Green, L.G., Fokin, V. V.,Sharpless, K. B. "A stepwise huisgen cycloaddition process: copper(I)-catalyzed regioselective "ligation" of azides and terminal alkynes." Angew Chem Int Ed Engl, 2002. 41(14): 2596-9
  7. Sarac I, Hollenstein M. "Terminal deoxynucleotidyl transferase in the synthesis and modification of nucleic acids." Chembiochem. 2019 Apr 1;20(7):860-871
  8. Thomas C, Rusanov T, Hoang T, Augustin T, Kent T, Gaspar I, Pomerantz RT. "One-step enzymatic modification of RNA 3' termini using polymerase ?." Nucleic Acids Res. 2019 Apr 23;47(7):3272-3283
  9. Wojczewski, C., Stolze, K. Engels, J. W. "Fluorescent oligonucleotides – Versatile tools as probes and primers for DNA and RNA analysis." Synlett 1999, 10, 1667–1678
  10. Zearfoss NR, Ryder SP. "End-labeling oligonucleotides with chemical tags after synthesis." Methods Mol Biol. 2012; 941: 181–193