Sequence Modifiers are designed for use in automated synthesis. The carboxy-dT is hydrolyzed during deprotection and can be coupled directly to a molecule containing a primary amino group by a standard peptide coupling or via the intermediate N-hydroxysuccinimide (NHS) ester. Amino-Modifier dA, Amino-Modifier dC, N2-Amino-Modifier dG and both Amino-Modifier dT products can be added in place of a dA, dC, dG and dT residue, respectively, during oligonucleotide synthesis. Corresponding Amino-Modifier supports can replace their respective deoxynucleoside supports. After deprotection, the primary amine on the C6 analogues is separated from the oligonucleotide by a spacer arm with a total of 7 -10 atoms and can be labelled or attached to an enzyme. The C2 analogue is more suitable for the attachment of molecules designed to react with the oligonucleotide.
Coupling: Amino-Modifier C6 dT reacts in a manner identical to normal phosphoramidites. To prevent side reactions, synthesize using acetyl-protected dC. See Deprotection for futher details.
Deprotection: No changes needed from standard method recommended by synthesizer manufacturer. The TFA protecting group is removed during standard ammonium hydroxide deprotection. A minor side reaction during ammonia deprotection can lead to irreversibly capping 2-5% of the amine. This could be significant if multiple additions of the modifier are made. To prevent the reaction, synthesize using acetyl-protected dC and deprotect in 30% ammonia/40% methylamine 1:1 (AMA) at 65°C for 15 minutes.
The extinction coefficient of DNA at 260nm is around 10,000/mole or 10/Âµmole. The extinction coefficient of Amino-Modifier C6 dT and C2 dT is around 8.7. The synthesis of these monomers does not pass through the deoxynucleoside and this figure is estimated by comparison to dT-CE Phosphoramidite.Note: dA=15.4, dC=7.4, dG=11.5, T=8.7