SATA Modification of Amines on Nucleotides

Dissolve the amine-modified oligonucleotide to be thiolated in 250pi of 50mM sodium phosphate, pH 8.0. 

Remove excess reagents from the modified oligo by gel filtration. 

To deprotect the acetylated thiol group, add 100 pi of 50 mM hydroxylamine hydrochloride, 2.5 mM EDTA, pH 7.5. 

The sulfhydryl-containing oligonucleotide may be used immediately to conjugate with a sulfhydryl-reactive label, or it can be purified from excess hydroxylamine by gel filtration. 

Oligonucleotides containing amine groups introduced by enzymatic or chemical means may be modified with SATA (Chapter 1, Section 4.1) to produce protected sulfhydryl derivatives. The NHS ester end of SATA reacts with a primary amine to form a stable amide bond. After modification, the acetyl protecting group can be removed as needed by treatment with hydroxylamine under mildly alkaline conditions (Fig. 401). The result is terminal sulfhydryl groups that can be used for subsequent labeling with thiol-reactive probes or activated-enzyme derivatives (Kumar and Malhotra, 1992). 

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