SPDP Modification of Amines on Nucleotides

Oligonucleotide probes that have been modified with an amine-terminal spacer arm using any of the methods discussed in Sections 1 and 2, may be thiolated to contain a sulfhydryl residue. Theoretically, any of the amine-reactive thiolation reagents described in Chapter 1, Section 4.1, may be used to convert an amino group on a DNA 

Reduction of the pyridyldisulfide end after SPDP modification releases the pyridine-2-thione leaving group and generates a terminal —SH group. This procedure allows sulfhydryl-reactive derivatives such as maleimide-activated enzymes (Chapter 16, Sec- 

Dissolve the amine-modified oligonucleotide to be thiolated in 250 jxl of 50 mM sodium phosphate, pH 7.5. 

Dissolve SPDP (Pierce) at a concentration of 6.2 mg/ml in DMSO (makes a 20 mM stock solution). Alternatively, LC-SPDP may be used and dissolved at a concentration of 8.5 mg/ml in DMSO (also makes a 20 mM solution). The LC form of the cross-linker provides a longer spacer arm that often results in better probe activity after modification. If the water-soluble Sulfo-LC-SPDP is used, a stock solution in water may be prepared just prior to addition of an aliquot to the thiolation reaction. In this case, prepare a 10 mM solution of Sulfo-LC-SPDP by dissolving 5.2 mg/ml in water. Since an aqueous solution of the cross-linker will degrade by hydrolysis of the sulfo-NHS ester, it should be used quickly to prevent significant loss of activity. 

Add 50 xl of the SPDP (or LC-SPDP) solution to the oligo solution. Add 100 xl of the Sulfo-LC-SPDP solution, if the water-soluble cross-linker is used. Mix. 

Remove excess reagents from the modified oligo by gel filtration on a desalting resin. The modified probe now may be used to conjugate with a sulfhydryl-containing molecule, or it may be reduced to create a thiol for conjugation with sulfhydryl-reactive molecules. 

Purify the thiolated oligonucleotide from excess DTT by dialysis or gel filtration using 50mM sodium phosphate, ImM EDTA, pH 7.2. The modified probe should be used immediately in a conjugation reaction to prevent sulfhydryl oxidation and formation of disulfide crosslinks. 

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