Photochemical Cross-Linking of Psoralen-Derivatized Oligonucleoside Methylphosphonates to Rabbit Globin Messenger RNA

Antisense oligodeoxyribonucleoside methylphosphonates targeted against various regions of mRNA or precursor mRNA are selective inhibitors of mRNA expression both in cell-free systems and in cells in culture. The efficiency with which methylphosphonate oligomers interact with mRNA, and thus inhibit translation, can be considerably increased by introducing photoactivatable psoralen derivatives capable of cross-linking with the mRNA. Oligonucleoside methylphosphonates complementary to coding regions of rabbit a- or β-globin mRNA were derivatized with 4'-(aminoalkyl)-4,5',8-trimethylpsoralens by attaching the psoralen group to the 5’ end of the oligomer via a nuclease-resistant phosphoramidate linkage. The distance between the psoralen group and the 5’ end of the oligomer can be adjusted by changing the number of methylene groups in the aminoalkyl linker arm. The psoralen-derivatized oligomers specifically cross-link to their complementary sequences on the targeted mRNA. For example, an oligomer complementary to nucleotides 56–67 of a-globin mRNA specifically cross-linked to a-globin mRNA upon irradiation of a solution of the oligomer and rabbit globin mRNA at 4 °C. Oligomers derivatized with 4’-[[N-(2-amino-ethyl)amino] methyl] -4,5’,8-trimethylpsoralen gave the highest extent of cross-linking to mRNA. The extent of cross-linking was also determined by the chain length of the oligomer and the structure of the oligomer binding site. Oligomers complementary to regions of mRNA that are sensitive to hydrolysis by single-strand-specific nucleases cross-linked to an approximately 10-30-fold greater extent than oligomers complementary to regions that are insensitive to nuclease hydrolysis. Oligomers in which the psoralen group is opposite a uracil residue in the mRNA cross-linked to approximately a 10-fold higher extent than those with the psoralen group opposite a cytosine residue. Cross-linking of 5–20 μM psoralen-derivatized oligomers to globin mRNA results in specific inhibition of translation of the targeted mRNA to the extent of 40-60%, which is consistent with the extent of cross-linking of the oligomer. The ability of psoralen-derivatized oligonucleoside methylphosphonates to specifically cross-link with single-stranded regions of mRNA, the inhibitory effect of cross-linked oligomers on mRNA translation, and the relatively long half-lives of these oligomers in serum-containing media suggest that these nucleic acid analogues could be used to specifically control mRNA expression in living cells.