Cystamine oligonucleotides

Figure 1.72 Cystamine may be used to label phosphate groups, such as at the 5 -end of oligonucleotides, via a carbodiimide reaction using EDC. The resultant phosphoramidate linkage is a common way to modify oligonucleotides at the 5 -end.

Modification of Nucleic Acids and Oligonucleotides with Cystamine... 

Figure 27.6 The 5 -phosphate group of oligonucleotides may be labeled with cystamine using the EDC/imid-azole reaction. This results in the formation of an amine-terminal spacer containing an internal disulfide group. Reduction of the disulfide provides a route to creating a free thiol for further derivatization.

Add to the tube 7.5 pi of RNA or DNA containing a 5 phosphate group. The concentration of the oligonucleotide should be 7.5-15 nmol/pl or total of about 57-115.5 pg. Also immediately add 5 pi of 0.25 M cystamine in 0.1M imidazole, pH 6.0. Because EDC is labile in aqueous solutions, the addition of the oligo and cystamine/imidazole solutions should be done quickly. 

For reduction of the cystamine disulfides, add 20 pi of 1.0 M DTT and incubate at room temperature for 15 minutes. This will release 2-mercaptoethylamine from the cystamine modification site and create the free sulfhydryl on the 5 terminus of the oligonucleotide. 

Figure 27.14 An oligonucleotide modified with cystamine and reduced to generate a free sulfhydryl may be conjugated with an SPDP-modified enzyme, forming a disulfide linkage.

Cystamine Labeled Oligonucleotide (after reduction to sulfhydryl)... 

The piezoelectric quartz crystal resonators modified with oligonucleotide probes were used for detection of HCV in serum by Skladal et al. [66]. The gold electrodes on either rough or smooth surface crystals were modified with a self-assembled monolayer of cystamine. After activation with glutaraldehyde, either avidin or streptavidin were immobilized and used for attachment of biotinylated DNA probes (four different sequences). 

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