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Oligo modification—Post-synthesis conjugation explained

Did you know IDT can add modifications to your oligos post-synthesis using NHS ester chemistry? We can also add modifications through azide and alkyne groups post-synthesis, using click chemistry. Read about how these reactions are done and get answers to common questions regarding post-synthesis conjugation.

Phosphoramidite synthesis vs. post-synthesis conjugation

Modifications can be incorporated into synthetic oligonucleotides in a variety of ways. Standard DNA bases are synthetically coupled via phosphoramidite 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 phosphoramidite chemistry directly on the synthesis column (Figure 1).

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

Most modifications are attached using phosphoramidite chemistry. However, some modifications, such as NHS esters and click chemistry modifications, are attached post synthesis. The following sections will focus on the 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).

standard NHS ester modification reaction
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).When ordering modified oligos, certain modifications like fluorescent dyes may have to be attached to the oligo via the NHS ester attachment route. For example, 5' MAX™ or 5' JOE™ will be designated as 5' MAX (NHS Ester) or 5' JOE (NHS Ester) when ordered.

amino modifiers used to attach NHS esters
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.

What is click chemistry?

The nature and mechanism of click chemistry was described by Dr K. B. Sharpless at the Scripps Institute in 2001 [1,2]. A click chemistry reaction entails 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 with no side products (Figure 4). There are also copper-free click reactions [3].

basic click chemistry reaction
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 oligo 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 create custom oligo modifications. Alternatively, IDT can do the click conjugation for you by including a reactive alkyne group 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 oligo 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).

alkyne modifiers that can be used in click chemistry
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 custom oligo modifications outside of our normal catalog offerings. Simply contact us to request non-catalog products or if you have questions about oligonucleotide modifications.

Table 1. NHS ester modifications.

Modification name 5' Internal 3'
Alexa Fluor® 488
Alexa Fluor 532
Alexa Fluor 546
Alexa Fluor 594
Alexa Fluor 647
Alexa Fluor 660
Alexa Fluor 750
ATTO™ 488
ATTO 532
ATTO 550
ATTO 565
ATTO Rho101
ATTO 590
ATTO 633
Azide (NHS Ester)
Dy 750™

IRDye® 800

LightCycler® 640
Rhodamine Green-X™
Rhodamine Red-X
Texas Red-X



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

For research use only. Not for use in diagnostic procedures.
Unless otherwise agreed to in writing, IDT does not intend these products to be used in clinical applications and does not warrant their fitness or suitability for any clinical diagnostic use. Purchaser is solely responsible for all decisions regarding the use of these products and any associated regulatory or legal obligations. Doc ID: RUO23-1769_001

Published Sep 12, 2011
Revised/updated Apr 5, 2023