Oligonucleotides can be modified by direct incorporation during the synthesis or by post-synthesis labelling.

Direct incorporation


3’ modifications 

Since automated oligonucleotide synthesis is realised from 3’ to 5’, these modifications are only possible if the corresponding solid support (CPG column) is available and if the modification is compatible with the chemistries used during the synthesis. Typical examples are 3’-phosphate, 3’ Biotin, 3’ FAM, 3’ DDQ I, 3’ BHQ-1R…


5’ and internal modifications

Many modifications can be directly introduced at the 5’end or at internal positions of the oligonucleotides using the phosphoramidites. However these modifications need to support the somewhat harsh cleavage-deprotection conditions including a strong basic pH. Typical examples are 5’ Biotin, 5’ Phosphate, 5’ Cholesterol, 5’ FAM, 8-Oxo-dA,Biotin-dT, DABCYL-dT…

Post-synthesis incorporation

Post-synthesis modifications may influence the yield of the reaction. A lower yield may result from poly-modifications and/or strong secondary structures. Two major post-synthesis reactions are used to introduce sensitive dyes or compounds that do not exist as phosphoramidites. In the first case the label is conjugated to an amino-modified oligonucleotide (3’, 5’ or on a dT) using its amino-reactive version (N-hydroxysuccinimide (NHS) ester in most cases). The second possibility (originally also used for synthesis of molecular beacons) is the addition of a maleimide-modified label to a thiol-modified oligonucleotide. 

Label conjugated to an amino-modified oligonucleotide
Label conjugated to an amino-modified oligonucleotide

Maleimide-modified label to a thiol-modified oligonucleotide.
Maleimide-modified label to a thiol-modified oligonucleotide.

Oligonucleotides modification classification
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• Post-synthesis modifications are highlighted in blue.
• Some modifications can be inserted after or during the synthesis and are in red. 
• To know the best purification according to each modification, consult the oligonucleotides purification table



Discover the Dyes compatibility tables

Modifications

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