Amino Modifier C6-dA CE-Phosphoramidite
Amino Modifier C6-dA CE-Phosphoramidite
Key featuresShow Hide
- Incorporates primary amine internally to an oligonucleotide, for subsequent conjugation.
- Base labile trifluoroacetate (TFA) group useful where purifcation "handle" is not necessary.
- After deprotection, the primary amine is distanced from the oligonucleotide by a total of 10 atoms and can be labelled or attached to a biomolecule such as an enzyme.
- Note that the linker on the 8-position does cause some destabilisation of the duplex pairing to T (approximately 2 ºC per insertion).
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Incorporation of a primary amine reactive functional group at specific sites within an oligonucleotide allows for subsequent post-synthesis conjugation of the oligo with a number of different affinity, reporter or protein labels, depending on the application. Such labels need to be reactive towards the incorporated functional group: for example, NHS esters or isothiocyanates will react with primary amines. This approach is often necessary where the desired label or tag is either not available as a phosphoramidite, or is sensitive or unstable to the conditions of oligonucleotide synthesis or deprotection. A common example is the attachment of a rhodamine dye using the TAMRA NHS ester. Functionally-derivatised oligos can also be covalently attached to surfaces such as glass slides or gold microspheres for use in various microarray or nanoelectronic applications.
Internal amino-functions, ready for further post-synthetic modification, can be introduced to oligonucleotides by a number of products. The correct choice is dependent primarily on the nucleobase and linker combination you desire, the former simply being chose as per your desired sequence.
In the case of the C6 analogues, after deprotection, the primary amine is distanced from the oligonucleotide by a total of 10 atoms and can be labelled or attached to a biomolecule such as an enzyme. Similarly, in the mdC TEG products, the amine group is attached to a modified cytidine residue via triethyleneglycol linker, making it less likely for the label to interact with the double stranded duplex, but also conferring additional hydrophilicity.
A C2 spacer is more appropriate for applications where the attached label is designed to interact with the oligonucleotide.
The C6-dA product is useful for introducing an amino function at a dA site, although the linker on the 8-position does cause some destabilisation of the duplex pairing to T (approximately 2 ºC per insertion).
Applications of the internal modification technique are varied. For example, when synthesising wavelength shifting FRET probes using FAM/ROX, combine the ROX-modified amino-dT with a FAM modification at the 5'-end.
Physical & Dilution Data
Dilution volumes (in ml) are for 0.1M solutions in dry acetonitrile (LK4050). Adjust accordingly for other concentrations. For µmol pack sizes, products should be diluted as 100µmol/ml to achieve 0.1M, regardless of molecular weight.
Amino Modifiers (LK2071, LK2141, LK2149 & LK2135)
No changes are required from the standard method recommended by the synthesiser manufacturer. Coupling is as per standard nucleoside amidites.
Cleavage & Deoprotection
The trifluoroacetyl (TFA) protecting group on the primary amine is removed during standard ammonium hydroxide solution deprotection. However, a minor side reaction during 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, synthesise using acetyl-protected C and, prior to cleavage and deprotection, wash the column with 10-20% DEA/acetonitrile. Deprotect in AMA at 65°C for 15min.
Thiol Modifier (2191)
BTT or DCI can be used as activators. Do not use ETT. A coupling time of 10min (600s) is recommended. To facilitate AMA deprotection, dmf-G and Ac-C should be used.
Cleavage, Deprotection & Purification
Before carrying out the cleavage and deprotection, wash with a 20% DEA/MeCN solution, then:
- Cleave and deprotect using 100mM TCEP in AMA at room temperature for 2h.
- Desalt using a G25 column.
- Purify if required.
- Dry the oligo.
- Add 100μl of 87mM TCEP in water.
- Leave for 1h at room temperature.
- Pass through a G25 column pre-equilibrated with the conjugation buffer.
To conjugate, add the appropriate maleimide, acetamide or equivalent to the above solution and react as appropriate.
Once the reaction is complete, pass through a G25 column pre-equilibrated with 0.1M TEAA or, if conjugation is on a solid surface, thoroughly with water.
Storage & Stability
Store in a freezer below -10°C. Diluted samples must be freshly prepared for use and used within 24h.