An analytical test based on detection of 2,4-dinitrophenyl (DNP) labelled oligonucleotides with anti-DNP antibodies has been proposed. We have chosen the branched triethylene glycol (TEG) spacer in our version of DNP phosphoramidite since it can be added once or several times to the 3' or 5' terminus.
Coupling: 12-15 minute coupling time. To maintain label yield, carry out the synthesis DMT-on.
Deprotection: To maintain label yield as a 5' modifier, cleave and deprotect as required by nucleobases and then remove the DMT group. Alternatively, treat column with 10% diethylamine in acetonitrile for 2 minutes at room temperature (2x) to remove cyanoethyl protecting groups and rinse with acetonitrile. At this point, the DMT group may be safely removed without loss of label during deprotection.
Modification reagents based on a 1,2-diol, e.g., BioTEG, DNP phosphoramidites, or a 1,3-diol, e.g., fluorescein, biotin phosphoramidites, can be added several times at the 5'-terminus since they contain an alcohol group capable of further addition with phosphoramidites. Can this alcohol also be a substrate for T4 polynucleotide kinase for 32P labelling of these modified oligonucleotides? Surprisingly, the answer is yes. Teoule and coworkers have shown(1) that oligos labelled at the 5'-terminus are substrates for kinase. Interestingly, the oligos modified with reagents based on 1,2-diols are labelled to 50%, indicating that only one diastereomer is labelled, while those modified with 1,3-diol reagents are labelled to 100%.||REFERENCE(S): (1) M.L. Fontanel, H. Bazin, and R. Teoule, Analytical Biochemistry, 1993, 214, 338-340. ||