On-Column Oligonucleotide Conjugations


Modified oligonucleotides where labels (e.g. reporter, carrier, biomolecule) have been conjugated to the oligonucleotide have a vast array of applications such as diagnostics, capture and therapeutics.1

While the preferred method of conjugation is via solid phase synthesis using phosphoramidite chemistry, there remain many examples where this method is not feasible, either because the label does not exist as a phosphoramidite or solid support, or that the label is not compatible with oligonucleotide synthesis and/or deprotection. In these cases there are two choices:

  1. The label is conjugated on solid phase after oligonucleotide synthesis but prior to cleavage and deprotection.

    In this case the active functional group in the oligonucleotide must be easily deprotected without cleaving the oligonucleotide from the resin and the label must be compatible to the required deprotection conditions.


  2. The label is conjugated in solution phase after cleavage and deprotection.

    In this case the label must have some degree of solubility and must be stable in aqueous solution even if mixed with a co-solvent such as DMSO or DMF.

In either scenario, the oligonucleotide is most commonly functionalised with one reactive group and the label functionalised with a complementary reactive group. The most commonly used pairings are amines/NHS esters and thiols/maleimides, although many others are available e.g. alkynes/azides (click chemistry) and furan/maleimides (Diels-Alder chemistry).

Of these scenarios, solution phase conjugations is the most common but on-column conjugations are particularly useful where the label and the conjugation product are difficult to separate or where the label is not soluble in aqueous phase - hence solution phase coupling is not feasible. On completion of the coupling reaction, excess label is washed from the column prior to cleavage and deprotection leaving only separation of the conjugate, unlabelled oligonucleotide and failure sequences from the reaction mixture. Typical labels include dyes such as ROX and TMR, lipophilic compounds that are not available as synthesis reagents, and amino acids or small peptides.  

Link offers a range of phosphoramidites and solid supports that allow on-column conjugations with amino, thiol, carboxy and aldehyde modified oligonucleotides.

Amino Modified Oligonucleotides

Link offers four amino modifiers that fit into this category; these are shown in Figure 1.

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3'-Amino-C7 CPG (2350) is used to incorporate the amino modifier at the 3’-end of an oligonucleotide. The Fmoc group is easily removed with 20% piperidine in MeCN with no cleavage of the oligonucleotide from the support.2

5'-MMT-Amino-Modifier C6-CE Phosphoramidite (2123), 5'-MMT-Amino-Modifier C12-CE Phosphoramidite (2133) and 5'-MMT-Amino-Modifier-11-CE Phosphoramidite (2193) are all used to incorporate an amino functionality at the 5’-end of an oligonucleotide. The MMT group is removed using an elongated detritylation step. In this case it is recommended that the resin is washed with 20% diethylamine in acetonitrile to ensure the free amine is not in the protonated form.

The label, typically an active ester such as NHS esters, can then be conjugated to the free amine. For small molecules such as fluorescent dyes, this is often carried out in DMF with up to six equivalents of the NHS ester.

Short peptides and amino acid residues are generally added via a typical peptide coupling using a coupling agent (e.g. HATU or DCC) or a crosslinker (such as DSS) where the C-terminus of the peptide is coupled to the amino functionality of the oligonucleotide.

Figure 2 depicts on-column labelling of an oligonucleotide modified with 2350.

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Thiol Modified Oligonucleotides

Link offers two thiol modifiers that fit into this category; these are shown in Figure 3.

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Thioctic Acid NHS Ester (2166) must be used in conjunction with one of the amino-modifiers mentioned previously but thereafter has the ability to be used as a thiol reactive site. However, this is generally used as a means of conjugating oligonucleotides to silver or gold nanoparticles.3

Thiol-Modifier C6 S-S CE Phosphoramidite (2126), once incorporated into an oligonucleotide, introduces the possibility of reducing the disulphide bridge with e.g. TCEP in water or mercaptoethanol followed by conjugation to a maleimide or acetamide active label. See Figure 4.

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Carboxylate Modified Oligonucleotides

Link offers one carboxylate modifier compatible with on-column labelling; 5'-Carboxylate Modifier-CE Phosphoramidite (2057).

In this case, the oligonucleotide is synthesised ‘DMT OFF’ to remove the chlorotrityl group and conjugation of the label carried out prior to cleavage and deprotection. Typically the coupling reaction is carried out using a peptide coupling reagent such as HATU to an amino functionalised label to form a stable amide linkage. This is indicated in Figure 5. Labels in this case are generally amino functionalised dyes such as the near infrared dye Cyanine 7 amine, or amino acids and small peptides where coupling occurs between the N-terminus of the peptide and the 5’-end of the oligonucleotide.

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Aldehyde Modified Oligonucleotides

Link offers one aldehyde modifier compatible with on-column labelling; Formylindole Modifier-CE Phosphoramidite (2056).

Among other functional groups, aldehydes will react with amines to form an imine (Schiff’s base) which is generally followed by a borohydride reduction due to the instability of the imine bond.4  These will also react with a hydrazine to form a hydrazone.5 Solulink HyNicTM conjugation technology6 is derived from this type of coupling. Although semi-carbizide couplings are commonly used to attach oligonucleotides to glass slides, it is feasible the reaction of an aldehyde with a semi-carbizide to form a semi-carbizone7 can be applied to on-column coupling.

These couplings are shown in Figure 6.

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By combining both options, i.e. on-column and solution phase conjugations, it is possible to incorporate the same functional group with orthogonal protection into an oligonucleotide where each position can be labelled in turn. This is illustrated in Figure 8; an oligonucleotide modified at the 3’-end with 2350, the 5’-end with 2193 and internally with Amino-Modifier C6-dT-CE Phosphoramidite (2135) (See Figure 7) allows stepwise conjugation in three positions. The oligonucleotide is synthesised ‘DMT-ON’ and the Fmoc group at the 3’-end is removed and labelled at this position followed by a capping step as per solid phase oligonucleotide synthesis. Detritylation to remove the MMT group from the 5’-end is then carried out which is in turn labelled followed by a capping step. The resin is then treated with 20% DEA in MeCN then cleaved, deprotected and - if necessary - purified. The final conjugation step can now be carried out resulting in an oligonucleotide modified in three positions.

Alternatively, more than one functional group can be incorporated into the oligonucleotide, e.g. 2135 can be replaced with Bz-S-C6-dT-CE Phosphoramidite (2191) (See Figure 7) or 2193 replaced with 2126 (See Figure 3). Here one of the conjugation reactions would become thiol/maleimide or thiol/acetamide.

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While on-column post synthetic conjugations are not the most widely used method of labelling an oligonucleotide, this method opens up the ability to improve on solution phase couplings where the label is either unstable or has poor solubility in aqueous buffers. This also opens up the possibility of carrying out multiple post synthetic coupling reactions on the same oligonucleotide.

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  1. Bioconjugate Techniques, 3rd Edition, 2013, Hermanson, G.T.
  2. US Patent no. 5736626, 1998; Solid Support Reagents for the Direct Synthesis of 3’-Labelled Polynucleotides.
  3. (a) Enhanced oligonucleotide-nanoparticle conjugate stability using thioctic acid modified oligonucleotides, J.A. Dougan, C. Karlsson, W.E. Smith and D. Graham, Nucleic Acids Research, 35, 3668-3675, 2007; (b) Highly sensitive detection of dye-labelled DNA using nanostructured gold surfaces, R.J. Stokes, A. Macaskill, J.A. Dougan, P.G. Hargreaves, H.M. Stanford, W.E. Smith, K. Faulds and D. Graham, Chem. Commun., 2811-2813, 2007.
  4. Zatsepin.T.S, Stetsenko.D.A., Gait.M.J, and Oretskaya.T.S, Use of Carbonyl Group Addition-Elimination Reactions for the Synthesis of Nucleic Acid Conjugates. Bioconjugate Chemistry, 2005, 16, 471-489.
  5. Dirksen.A. and Dawson.P.E, Rapid Oxime and Hydrazone Ligations with Aromatic Aldehydes for Biomolecular Labelling, Bioconjugate Chemistry 2008, 19, 2543-2548.
  6. Abrams.M.J., Juweid.M., TenKate.C.I., Schwartz.D.A, Hauser.M.M., Gaul.F.E, Fuccello.J, Rubin.R.H, Strauss.H.W and Fischman.A.J., Technetium 99m Human Polyclonal IgG Radiolabeled via the Hydrazino Nicotinamide, The Journal of Nuclear Medicine, 1990, 31, 2022-2028.
  7. Podyminogin.M.A, Lukhtanov.E.A and Reed.M.W, Attachment of Benzaldehyde-Modified Oligonucleotide Probes to Semi-Carbazide Coated Glass, Nucleic Acids Research, 2001, 29, 5090-5098.


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