Specificity of site directed psoralen addition to RNA

We describe the attachment of a psoralen derivative (site specific psoralen, SSP) to the 5' end of a DNA oligonucleotide and the hybridization and the photoreaction of this reagent with a complementary target site on an RNA molecule. SSP was coupled to a variety of DNA oligonucleotides to investigate the structural requirements for addition to the RNA. Efficient SSP photoadducts were made on specific uridines by designing an intercalation site at an unpaired nucleotide in the RNA strand within the heteroduplex region. The optimal location for this site was five nucleotides from the oligonucleotide 5' end and just 5' to the target uridine residue. Because the attachment of the SSP to the oligonucleotide is through a disulfide bond, the DNA oligonucleotide can be removed with reduction to leave SSP attached to the RNA strand. The SSP adduct made in this way will be useful for subsequent biochemical and biophysical experiments.