Oligodeoxyribonucleoside methylphosphonates derivatized at the 5' end with 4'-(amino-alkyl)-4,5',8-trimethylpsoralen were prepared. The interaction of these psoralen-derivatized methylphosphonate oligomers with synthetic single-stranded DNAs 35 nucleotides in length was studied. Irradiation of a solution containing the 35-mer and its complementary methylphosphonate oligomer at 365 nm gave a cross-linked duplex produced by cycloaddition between the psoralen pyrone ring of the derivatized methylphosphonate oligomer and a thymine base of the DNA. Photoadduct formation could be reversed by irradiation at 254 nm. The rate and extent of cross-linking were dependent upon the length of the aminoalkyl linker between the trimethylpsoralen group and the 5' end of the methylphosphonate oligomer. Methylphosphonate oligomers derivatized with 4'-[[N-(2-aminoethyl)amino]methyl]- 4,5',8-trimethylpsoralen gave between 70% and 85% cross-linked product when irradiated for 20 min at 4 degrees C. Further irradiation did not increase cross-linking, and preirradiation of the psoralen-derivatized methylphosphonate oligomer at 365 nm reduced or prevented cross-linking. These results suggest that the methylphosphonate oligomers undergo both cross-linking and deactivation reactions when irradiated at 365 nm. The extent of cross-linking increased up to 10 microM oligomer concentration and dramatically decreased at temperatures above the estimated Tm of the methylphosphonate oligomer-DNA duplex. The cross-linking reaction was dependent upon the fidelity of base-pairing interactions between the methylphosphonate oligomers and the single-stranded DNA. Noncomplementary oligomers did not cross-link, and the extent of cross-linking of oligomers containing varying numbers of noncomplementary bases was greatly diminished or eliminated.(ABSTRACT TRUNCATED AT 250 WORDS)

Oligodeoxyribonucleoside methylphosphonates which have sequences complementary to the initiation codon regions of N, NS, and G vesicular stomatitis virus (VSV) mRNAs were tested for their ability to inhibit translation of VSV mRNA in a cell-free system and in VSV-infected mouse L cells. In a rabbit reticulocyte lysate cell-free system, the oligomers complementary to N (oligomer I) and NS (oligomer II) mRNAs inhibited translation of VSV N and NS mRNAs whereas oligomer III had only a slight inhibitory effect on N protein synthesis. At 100 and 150 microM, oligomer I specifically inhibited N protein synthesis in the lysate. In contrast, at 150 microM, oligomer II inhibited both N and NS protein synthesis. This reduced specificity of inhibition may be due to the formation of partial duplexes between oligomer II and VSV N mRNA. The oligomers had little or no inhibitory effects on the synthesis of globin mRNA in the same lysate system. Oligomers I-III specifically inhibited the synthesis of all five viral proteins in VSV-infected cells in a concentration-dependent manner. The oligomers had no effects on cellular protein synthesis in uninfected cells nor on cell growth. An oligothymidylate which forms only weak duplexes with poly(rA) had just a slight effect on VSV protein synthesis and yield of virus. Oligomers I-III have extensive partial complementarity with the coding regions of L mRNA. The nonspecific inhibition of viral protein synthesis in infected cells may reflect the role of N, NS, and/or L proteins in the replication and transcription of viral RNA or result from duplex formation between the oligomers and complementary, plus-strand viral RNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Oligodeoxyribonucleoside methylphosphonates which are complementary to the 5' end, the initiation codon regions, or the coding regions of rabbit globin mRNA were synthesized. These oligomers were shown to interact with their complementary mRNA binding sites by their ability to serve as primers for reverse transcriptase. In several cases, the priming efficiency of the oligomers was enhanced when the oligomer was preannealed with the mRNA. This behavior correlates with the predicted secondary structure of the mRNA and suggests that some oligomer binding sites occur in hydrogen-bonded stem regions of the mRNA. Methylphosphonate oligomers inhibit translation of globin mRNA in reticulocyte lysates. Inhibition is due to the interaction of the oligomers with mRNA. The extent of inhibition is affected by the sequence and chain length of the oligomer, the location of the oligomer binding site on the mRNA, and the secondary structure of the binding site. Oligomers which bind to the 5' end and initiation codon regions of beta-globin mRNA inhibit both alpha- and beta-globin synthesis whereas oligomers which bind to the coding region of alpha-globin mRNA or the coding region of beta-globin mRNA inhibit translation of their target mRNA in a specific manner. Oligodeoxyribonucleoside methylphosphonates inhibit globin synthesis in rabbit reticulocytes. The effects of various oligomers on cellular globin synthesis are similar to those in the lysate system and suggest that the conformation of globin mRNA is the same in both systems during translation.

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)

Oligodeoxyribonucleotides 8-12 nucleotides in length whose sequences are complementary to the 5' end, the initiation codon regions, or the coding regions of rabbit globin mRNA were tested for their ability to inhibit translation in a rabbit reticulocyte lysate and in a wheat germ extract. The oligomers interact specifically with their target mRNAs as shown by their ability to serve as primers with reverse transcriptase. In the reticulocyte lysate, oligomers complementary to the 5' end or the initiation codon regions inhibit translation of both alpha- and beta-globin mRNA, whereas oligomers complementary to the coding regions have little or no effect. This suggests that reticulocyte ribosomes are able to displace the oligomers from the mRNA during the elongation but not the initiation step of translation. In the wheat germ system, translation was effectively inhibited by all oligomers regardless of their binding site on the message. In contrast to their behavior in the reticulocyte system, the oligomers inhibited alpha- and beta-globin synthesis in a specific manner. This observation suggests that control of alpha- and beta-globin mRNA translation is coordinated in the reticulocyte lysate system but not in the wheat germ extract. The results of our studies indicate that oligodeoxyribonucleotides may be useful probes for studying control of mRNA translation in cell-free systems.