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What are Oligonucleotide Modifications?

Oligonucleotide Modifications

Chemically synthesized oligonucleotides allow for the creation of new artificial molecules. These can be tailored to specific needs as needed for a variety of applications.  Synthetic oligonucleotides either natural or modified are heavily used in molecular diagnostics and therapeutics. The demand for synthetic oligonucleotides has resulted in the development of various reagents useful for the introduction of different functional groups into oligonucleotides. Most of the modifications are synthetically incorporated using phosphoramidite based reagents. Since the introduction of functional groups into oligonucleotides changes their physicochemical properties, optimal purification strategies may need to be employed. 

Location of Modifications




5’-terminal modifications


Modification at the bases




3’-terminal modifications

X = O, Y = O

Natural DNA

X = O, Y = S


X = S, Y = S


X = CH3, Y = O


X = NR2, Y = O





Oligonucleotide Modification Nomenclature




A, C, G, T, Cm [for 5-Me-dC]

Phosphorothioated DNA

A*, G*, C*, T*, Cm*


rA, rG, rC, rU

Phosphorothioated RNA

rA*, rG*, rC*, rU*

2'O-Methyl RNA

mA, mG, mC, mU, mCm [for 2'-OMe, 5-Me C]

Phosphorothioated 2'O-Methyl RNA

mA*, mG*, mC*, mU*, mCm*


fA, fG, fC, fU


+A, +C, +G, +T, +rA, +rG, +rC, +rU




Modification Types

(1)  Modification of oligonucleotides at the 5’-end

a.   Phosphorylation. (Δm: HPO3, 80 {79.96633} Da; or H3PO4, 98 Da)
      5’-phosphorylation is needed for DNA ligase substrates. 3’-phosphorylation
      inhibits degradation of oligonucleotides by 3’-exonucleases.
      3’-phophorylation also blocks extension by DNA polymerases.

b.   Amination. Primary amino groups react with a number of other reactive
      molecules such as  isothiocyanates, NHS esters, or activated carboylates.
      These reactions are useful for further functionalization of oligos.

c.   Thiolation. Thiol groups can be used for the attachment of a variety of compounds
      to oligonucleotides such as fluorophores. Oligonucleotides containing oxidized thiol
      groups will need to be reduced prior to use if needed. Dithiothreitol (DTT) or Tris
      (2-carboxyethyl) phosphine (TCEP) are commonly used for the reduction reaction.

d.   Introduction of a spacer. Spacers can be used to increase the distance between
      the oligonucleotide and a functional group. Many spacers use ethylene glycol units
      called polyethylene glycol (PEG) spacers.

e.   Conjugation. A variety of conjugation reactions can be used for the attachment of
      functional groups such as biotins, amino-modifiers, azides, cholesteryl, alkynes, or
      thiols, among others.

f.   Branching phosphoramidites. Branching phosphoramidites are used for the
     generation of fork or comb oligonucleotide structures. Often branching is used to
     create enhanced oligonucleotides for hybridization assays or signal amplification for
     a variety of diagnostic assays.

g.  Blocking of 5’end with 5’-OMedT

(2)  Modifications of oligonucleotides at the 3’-end

a.   3’-phosphate group

b.   3’-amino group

c.   3’-sulphydryl group

d.   Introduction of a spacer arm at the 3’-end

e.   Conjugation

f.    3’-3’ linked oligonucleotides

g.   Modified bases at the 3’-terminus

(3)  Modification of oligonucleotides at the internucleotide linkages

a.   Phosphorothioates

b.   Methylphosphonates

c.   Phosphorodithioates

d.   Oligonucleotides with 3’-3’ and 5’-5’ linkages

(4)  Modification at the 2’-position

a.   2’-O-alkyl-RNA

b.   2’-fluoro-U, 2’-fluoro-C, phosphorothioate-RNA

(5)  Modification at the ribose unit

a.   Deoxyuridine

b.   3’-Deoxyadenosine

c.   Dideoxyadenosine and dideoxycytosine

d.   α-Oligonucleotides

e.   Ara cytidine

(6)   Modification at the nucleobases

a.   Inosine

b.   Deoxynaphtosine

c.   Etheno-adenosine and etheno-cytidine

d.   C-5 modified deoxycytidine and deoxyuridine

e.   Introduction of a primary amino group by modified bases

f.    Introduction of a carboxy group by modified bases

g.   Introduction of biotin via modified bases

h.   Other modified bases

i.    Convertible nucleosides