Know your oligo mod: amino linkers

An amino linker is perhaps the most versatile of all the modifications you can add to an oligonucleotide. Its primary amino group can attach a wide range of modifying chemical groups or immobilise the oligo on a solid surface. In this Know Your Oligo Mod blog post, we take a look at the diverse applications of amino linkers in research, diagnostics and therapeutics. 

A primary amino group on the oligo creates the opportunity to link with additional chemical groups that carry an N-hydroxy succinimidyl (NHS) ester. This allows users to add modifications that can’t simply be included during the solid-support synthesis of the oligo using modified CPGs or phosphoramidites. 

Figure 1. Conjugation of an NHS ester with an amino group attached to an oligo. 

This post-synthetic chemical modification requires HPLC purification and results in lower yields than introducing mods during synthesis. However, it offers the opportunity to incorporate a wide array of modifications, including: 

• biotin,

• digoxigenin,

• some fluorescent dyes,

• magnetic beads,

• enzymes, such as alkaline phosphatase. 

Amino linkers have been used to label oligos since the 1980s.¹ This included cross-linking alkaline phosphatase to oligos that are complementary to DNA of pathogens such as E. coli or hepatitis B virus for creating diagnostic tests.²  

Today, amino-conjugated modifications can be used to tag oligos as probes for in situ hybridisation analysis.³ These probes can measure and localise specific sequences in a section of tissue for studies or diagnosis. 

Amino linkers for your custom oligo 

Incorporating the amino modification during custom oligo synthesis allows it to be positioned accurately. Carbon chain spacers of differing lengths can be included at internal sites or at the 3ʹ or 5ʹ ends, offering a high degree of customisation. Alternatively, the amino group can be conjugated via a ten-atom linker to a modified thymidine, which can participate in hybridisation. 

Figure 2. Amino group conjugated via a ten-atom linker to a modified thymidine.

An alternative approach is to add the amino conjugated via a polyethylene glycol (PEG) linker to a modified cytidine, which may participate in hybridisation at the 3ʹ end of the oligo. The PEG linker is less hydrophobic than the alkane chains, improving solubility in aqueous solvents. 

Figure 3. Amino group conjugated via a polyethylene glycol (PEG) linker to a modified cytidine at the 3ʹ end.

Immobilising oligos on surfaces for microarrays 

Amino linkers have been heavily used in creating DNA microarrays. By attaching the oligos to the surface of the chip, researchers can identify the presence of target sequences in the sample being washed over the chip. A DNA microarray can immobilise a large number of different sequences, making it a valuable tool for high-density screening. 

Amino groups covalently bond with surfaces containing carboxyl, aldehyde, epoxy or isothiocyanate groups.⁴ This provides a way to permanently fix the oligo to the surface with high efficiency, stability and specificity.  

Alternatively, the amino can be used to attach other groups used for immobilisation. Biotin, for instance, can be attached to an amino group and non-covalently bind to streptavidin-coated surfaces. Reacting an amino-conjugated oligo with a thioctic acid NHS ester introduces a dithiol function, which can attach the oligo to gold and silver surfaces. 

Immobilisation with amino linkers is also used to create biosensors. Aptamers specific for particular proteins, for example ones relevant to cancer diagnosis, can be covalently linked to a silicon dioxide surface coated with a layer of epoxide groups.⁵

Protein conjugation for therapeutics and analysis

An amino linker also allows scientists to conjugate the oligo to proteins, such as antibodies.⁶ A linker such as BCN-PEG6-PFP can join the amino group of the oligo with an amino group of a lysine residue on the protein.  

This protein conjugation can be a powerful way to target the delivery of a therapeutic oligo to the desired tissue.^7,8 Oligo–antibody conjugation can also be used to detect multiple different proteins in single cell proteomics studies experiments.^9,10

^{Find the right linker for your purposes}

There’s a wide range of reasons for incorporating an amino linker group into a custom oligo: it increases the variety of possible modifications, it can immobilise the oligo to a surface for microarray or biosensor development, and it can conjugate proteins for use in therapeutics and proteomics. 

With the various options for amino linkers available, there are plenty of choices to optimise oligos for your research and development needs.

Know your oligo mod series 

• 5-Nitroindole – a universal base oligo modification
• BHQ® (Black Hole Quencher®) non-fluorescent quenchers
• Thio C6 linker
• 3' Spacer C3
• Phosphorothioate bonds
• 2'-O-methoxyethyl (2'-MOE)
• Locked nucleic acids

References

1. Ruth JL, Morgan C and Pasko A (1985) Linker arm nucleotide analogue useful in oligonucleotide synthesis. 4:93
2. Jablonski E et al. (1986) Preparation of oligodeoxynucleotide-alkaline phosphatase conjugates and their use as hybridization probes. Nucleic Acids Res. 14:6115–6128. doi: 1093/nar/14.15.6115
3. Basu R et al. (2014) Using amino-labeled nucleotide probes for simultaneous single molecule RNA-DNA FISH. PLoS One. 9(9):e107425. doi: 1371/journal.pone.0107425
4. Nimse SB et al. (2014) Immobilization Techniques for Microarray: Challenges and Applications. Sensors. 14(12):22208–22229. doi: 3390/s141222208
5. Jeddi I and Saiz L (2021) Computational design of single‑stranded DNA hairpin aptamers immobilized on a biosensor substrate. Scientific Reports. 11:10984 doi: 1038/s41598-021-88796-2
6. Rady T et al. (2024) Protocol to generate, purify, and analyze antibody-oligonucleotide conjugates from off-the-shelf antibodies. STAR Protocols. 5(4):103329 doi: 1016/j.xpro.2024.103329
7. Sugo T et al. (2016) Development of antibody-siRNA conjugate targeted to cardiac and skeletal muscles. Journal of Controlled Release. 237:1-13 doi: 1016/j.jconrel.2016.06.036
8. Zavoiura O et al. (2021) Nanobody–siRNA Conjugates for Targeted Delivery of siRNA to Cancer Cells. Molecular Pharmaceutics. 18(3):1048–1060 doi: 1021/acs.molpharmaceut.0c01001
9. Gong H et al. (2015) Simple Method to Prepare Oligonucleotide-Conjugated Antibodies and its Application in Multiplex Protein Detection in Single Cells. Bioconjugate Chem. 27(1):217–225 doi: 1021/acs.bioconjchem.5b00613
10. Wiener J et al. (2020) Preparation of single- and double-oligonucleotide antibody conjugates and their application for protein analytics. Scientific Reports. 10: 1457 doi: 1038/s41598-020-58238-6