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.

Sep 12, 2011

Revised/updated Dec 14, 2016

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).PostSynth Conj_Fig 1

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).PostSynth Conj_Fig 2

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).PostSynth Conj_Fig 3

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].PostSynth Conj_Fig 4

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).PostSynth Conj_Fig 5

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 to request non-catalog products.

Contact Technical Support at with any additional questions you have about oligonucleotide 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
ATTO™ 647N
Azide (NHS Ester)
Dy 750™

IRDye® 800

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

Table 1. NHS ester modifications


  1. Kolb HC, Finn MG, Sharpless KB (2001) Click Chemistry: Diverse ChemicalFunction 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’ Cycloadditionand 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 invivo imaging. Proc Natl Acad Sci, 104(43):16793–16797.