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 alpha- or beta-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 alpha-globin mRNA specifically cross-linked to alpha-globin mRNA upon irradiation of a solution of the oligomer and rabbit globin mRNA at 4 degrees 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.(ABSTRACT TRUNCATED AT 250 WORDS)

The affinity cleavage reagent methidiumpropyl-EDTA (MPE) [Hertzberg, R. P., & Dervan, P. B. (1982) J. Am. Chem. Soc. 104, 313-315] intercalates between base pairs in helical DNA and, when complexed with Fe(II), cleaves the DNA by oxidative degradation of the deoxyribose. We find that this reagent is useful for mapping structure in some RNA molecules. The reagent binds to poly(A)-poly(U) with the same or slightly lower affinity as the related ethidium intercalator, selectively binds double-helical in preference to single-stranded RNA, and when complexed with Fe(II) readily cleaves the RNA backbone. The reagent binds to three or four helical locations in tRNAPhe with an affinity of 10(5)-10(6) M-1 (0.1 M Na+, pH 7.6, 37 degrees C). With a 345-base RNA fragment covering the S8/S15 protein binding region of Escherichia coli 16S ribosomal RNA, MPE-Fe(II) intercalates strongly at two helical sites: one is located at or near a single base bulge and the other at the end of a helix. Intense cutting is also seen in a region that is not part of a Watson-Crick helix. Ethidium bromide binds at these sites with high affinity (about 10(7) M-1 at 0.1 M Na+, pH 7.6, 37 degrees C). The sites are all clustered in a region of the RNA thought to bind S15. Tertiary folding of the RNA may distort helices in the molecule to create sites with particularly high affinities for intercalators; such sites may have functional significance in protein recognition or RNA-RNA interactions.

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Antisense oligonucleoside methylphosphonates complementary to the 12 nucleotides found at the intron/exon junction of the splice acceptor site of herpes simplex virus type 1 (HSV-1) immediate early mRNAs 4 and 5 were synthesized. The methylphosphonate oligomers contained either 2'-deoxyribose nucleosides, d-OMPs, or 2'O-methylribose nucleosides, mr-OMPs. At 37 degrees C, the affinity of the mr-OMP for a complementary 12-mer RNA target was approximately four times higher than that of the corresponding d-OMP as measured by a constant activity gel electrophoresis mobility shift assay. An mr-OMP whose sequence contained two mismatched bases did not bind to the RNA target under these conditions. The mr-OMP also showed improved ability to inhibit HSV-1 replication in HSV-1 infected Vero cells in culture. Thus the IC50 of the mr-OMP was five times less than that of the d-OMP. No inhibition was observed by the mismatched mr-OMP, and no inhibition of herpes simplex virus type 2 (HSV-2) replication was observed with any of the oligomers. These results demonstrate a direct correlation between oligomer binding affinity and antisense activity in cell culture and suggest that oligo-2'-O-methylribonucleoside methylphosphonates are promising candidates for development of effective antisense reagents.

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