During DNA synthesis, each nucleotide is coupled sequentially (from 3’ to 5’) to the growing chain according to the standard ß-cyanoethyl chemical reactions.

• Deblocking: the first nucleotide, attached to the solid support is deprotected by removing the DMT-protecting group. This produces a free 5’ hydroxyl group to react with the next nucleotide.
• Coupling: the second nucleotide is activated then added to the reaction and is covalently attached (i.e. coupled) to the previous nucleotide
• Capping: any of the first nucleotide that failed to react is capped so that it will no longer participate at any subsequent step.
• Oxidation: the bond between the first nucleotide and the successfully coupled second nucleotide is oxidized to stabilize the growing chain.
• Deblocking: the 5’ DMT group is removed from the second nucleotide to prepare it for further cycles.
• At the end of the oligo synthesis, the crude product is cleaved from the solid support (CPG or polystyrene beads) and purified using one of a variety of methods.
• In silico structural analysis for oligos longer than 100 bases (optional).

How are modifications incorporated into an oligonucleotide?

Oligos can be modified in several different ways by utilizing the active groups of the nucleotide or creating nucleotide analogues. Modifications can occur either during or after synthesis.

• Direct incorporation of modified nucleosides during automated DNA synthesis. Using this method, modified bases can be incorporated internally or on the 5’ end. Thymidine analogues (which replace T-bases in the sequence) are frequently used as modified bases. This site-specific method is constrained by the availability of specialized phosphoramidites.
• 3’ end modification using special solid support. Once again, this method relies on the existence of modified CPG columns.
• Modifications using functional groups (via an amino modifier, via the 3’ or 5’ hydroxyl groups, or via the phosphate group). Although the yields are often low, the reaction of functional groups attached to the bases with activated dyes molecules (e.g. N-hydroxysuccinimide esters) is widely used to label oligos either on the ends or internally.

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Leaflets

• Oligonucleotides Catalogue

Product citations

LEGENDRE D. et al., "Engineering a regulatable enzyme for homogeneous immunoassays", Nature Biotechnology, vol. 17, n° 1, p.67-72, 1 January 1999

MOMENI P. et al., "Mutations in a new gene, encoding a zinc-finger protein, cause tricho-rhino-phalangeal syndrome type I", Nature Genetics, vol. 24, p. 71-74, 1 January 2000

PEREL Y. et al., "Galanin and galanin receptor expression in neuroblastic tumours: correlation with their differentiation status", British Journal of Cancer, vol. 86, n° 1, p. 117-122, 7 January 2002

OSTERMANN G. et al., "JAM-1 is a ligand of the 2 integrin LFA-1 involved in transendothelial migration of leukocytes", Nature Immunology, vol. 3, n° 2, p.151-158, 1 February 2002

GOMEZ D. et al., "Interaction of Telomestatin with the Telomeric Single-strand Overhang", Journal of Biological Chemistry, vol. 279, n° 40, p. 41487-41494, October 2004

RZEM et al., "A gene encoding a putative FAD-dependent L-2-hydroxyglutarate dehydrogenase is mutated in L-2-hydroxyglutaric aciduria", PNAS, vol. 101, n° 48, 16849-16854, November 2004

SU T.-J. et al., "DNA bending by M.EcoKI methyltransferase is coupled to nucleotide flipping", Nucleic Acids Research, vol. 33, n° 10, 3235-3244, June 2005

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