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Core Concepts
Scientific Fundamentals Explained

Oligo Modification—Post-Synthesis Conjugation Explained

Phosphoroamidite Synthesis vs. Post-Synthesis Conjugation

Modifications can be incorporated into synthetic oligonucleotides in a variety of ways. Standard DNA bases are synthetically coupled via phosphoroamidite chemistry. The reaction proceeds in the 3’ to 5’ direction where the 5’ hydroxyl group of each base attaches to the 3’ phosphate group of the next base. Many modifications, such as 6-FAM, standard biotin, and internal Cy3, can be attached via phosphoroamidite chemistry directly on the synthesis column (Figure 1).

Figure 1. Internal Cy3 Modification. Standard phosphoroamidite chemistry attaches the modification through the hydroxyl group (arrow H) and the phosphate group (arrow P) to neighboring bases.

Most modifications are attached via phosphoroamidite chemistry. However, some modifications, such as NHS esters and click modifications, are attached post synthesis. The following sections will focus on the most common post-synthesis conjugations performed at IDT.

NHS Ester Modifications

NHS ester modifications contain an NHS (N-hydroxysuccinimide) group that reacts with an amine group to form an amide (Figure 2).

Figure 2. Standard NHS Ester Modification Reaction

Typically, 5’ NHS esters are attached through an Amino Modifier C6 group, internal NHS esters through an Amino Modifier C6 dT, and 3’ NHS esters via an amino group linked to the controlled pore glass (CPG) beads used as the synthesis support (Figure 3). To identify NHS ester modifications, look for (NHS ester) after the modification name such as MAX (NHS ester).

Figure 3. Amino Modifiers Used to Attach NHS Esters. The 3’ Amino Modifier (not shown) is similar in structure to the 5’ Amino Modifier, but is attached to the solid support (controlled pore glass, or CPG) on which the oligos are synthesized.

Click Chemistry

The nature and mechanism of click chemistry was described by Dr. K. B. Sharpless at the Scripps Institute in 2001 [1, 2]. These click chemistry reactions entail coupling azide and alkyne groups through a copper-catalyzed reaction, forming a 1,2,3-triazole. This reaction is thermodynamically favorable, resulting in an irreversible reaction, and it has no side products (Figure 4). There are also copper-free click reactions [3].

Figure 4. Basic Click Chemistry Reaction. The copper catalyzed reaction between an azide group and an alkyne group produces a 1,2,3-triazole. IDT attaches a variety of modifications post synthesis using this click chemistry reaction.

IDT offers a variety of modifications that leave a free azide or alkyne group available for further click conjugation, giving researchers the freedom to conjugate molecules of their choice to their oligonucleotides. Alternatively, IDT can do the click conjugation for you. A reactive alkyne group is included in the oligonucleotide during synthesis. After synthesis, deprotection, and initial purification, the alkyne group is reacted with a modification containing an azide functional group. All 5’ click modifications conjugate through a 5’ hexynyl group (Figure 5A) while internal click modifications conjugate through an internal alkyne (Figure 5B). The internal alkyne group is attached to dT, meaning any internal modification attached via click chemistry will incorporate an additional T base into the oligonucleotide sequence. To identify modifications attached via click chemistry, look for (azide) after the modification name, such as 6-FAM (azide). IDT can also provide a 3’ Alkyne Modifier as a non-catalog request (Figure 5C).
Figure 5. Alkyne Modifiers. These molecules are used to introduce an alkyne group within the oligonucleotide that can be used in a click reaction to conjugate the modification of one’s choice.

IDT NHS Ester and Click Chemistry Modifications

Table 1 shows current IDT catalog offerings of NHS ester and click chemistry modifications. Don’t see the modification you need in our catalog? No worries. IDT routinely accepts requests for modifications outside of our normal catalog offerings. Simply contact custcare@idtdna.com to request non-catalog products.

Contact Technical Support at techsupport@idtdna.com with any additional questions you have about oligonucleotide modifications.

Table 1. IDT NHS Ester and Click Chemistry Products.

Answers to Your Questions About Modifications

Why Choose Post-Synthesis Conjugation Over Phosphoroamidite Chemistry? 

A variety of post-synthesis modifications are not commercially available as amidites, such as the Alexa Fluor® dye family modifiers. Also, some commercially available post-synthesis modifications are not compatible with or can be degraded by the deprotection conditions used in oligonucleotide manufacture. FAM and TAMRA are two common dyes available as amidites, NHS esters, or azides (for click chemistry attachment). Some researchers opt for NHS ester or modifications attached via click chemistry for demanding applications to avoid the possible risk of dye fluorescence reduction caused by deprotection, or to meet specific protocol requirements. However, stable modifications that are available as both amidites and post-synthesis modifications are functionally the same.

Can I Order an Oligo with Both a Free Amino Modifier and an NHS Ester Modification?

Because NHS esters react with amino modifiers, any free amino modifiers at the time of NHS ester conjugation are susceptible to reaction. IDT recommends the researcher look for an alternative method of attaching the needed modification, such as through amidite or click chemistry, which would not react with a free amine.

Can I Order 2 Different NHS Esters on the Same Oligo?

IDT does not employ any method to direct NHS esters to specific free amino groups. Thus it is impossible to ensure the location of the distinct NHS ester modifications in the oligo. Each NHS ester will react with any free amine group, resulting in a product mixture. In this situation it is again best to opt for another form of attachment chemistry if a free amine is desired.


  1. Kolb HC, Finn MG, Sharpless KB (2001) Click Chemistry: Diverse Chemical Function from a Few Good Reactions. Agnew Chem Int Ed Engl, 40(11):2004–2021.
  2. Evans RA (2007) The Rise of Azide–Alkyne 1,3-Dipolar ‘Click’ Cycloaddition and its Application to Polymer Science and Surface Modification. Aus J Chem, 60(6):384–395.
  3. Baskin JM, Prescher JA, et al. (2007) Copper-free click chemistry for dynamic in vivo imaging. Proc Natl Acad Sci, 104(43):16793–16797.

Author: Aftan Vander Zwaag, BS is a Technical Service Representative at IDT