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).
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).
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).
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].
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).
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 Customer Support to request non-catalog products.
Contact Technical Support at techsupport@idtdna.com with any additional questions you have about oligonucleotide modifications.
Modification Name | 5' | Internal | 3' |
6-FAM | • | • | |
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) | • | • | • |
Digoxigenin | • | • | |
Dy 750™ | • | ||
IRDye® 800 | • | ||
JOE™ | • | • | |
LightCycler® 640 | • | • | |
MAX | • | • | |
Rhodamine Green-X™ | • | • | |
Rhodamine Red-X | • | • | |
ROX | • | • | |
TAMRA | • | • | • |
Texas Red-X | • | • |
Table 1. NHS ester modifications