The ability of oligonucleotides to interact selectively with their targets is an important consideration in the design of antisense oligonucleotides. This is especially important in the case of antisense oligomers, such as psoralen-derivatized oligomers, which can irreversibly bind to their targets. We have studied the interactions of a series of psoralen-derivatized antisense oligonucleoside methylphosphonates with the mRNAs of vesicular stomatitis virus (VSV), mRNAs that have a high degree of sequence homology. Cross-linking reactions were carried out under conditions of low ionic strength in order to reduce mRNA secondary structure. A 12-mer, whose sequence was complementary to VSV M -mRNA and partially complementary to sequences found in N, NS, and G mRNA cross-linked extensively to N-message. On the other hand, 16-mers whose sequences were uniquely complementary to binding sites on N- or M-mRNA specifically and efficiently cross-linked to their targeted mRNAs over the temperature range 0° to 37°C. A reverse transcriptase-catalyzed primer extension assay was used to show that one of the N-specific oligomers cross-linked at the expected site on N-mRNA and to estimate the extent of cross-linking. The results demonstrate that psoralen-derivatized oligonucleoside methylphosphonates can cross-link in a sequence-specific manner if the sequences of these oligomers are chosen carefully so as to avoid extensive partial complementarity with other mRNA sequences.
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