Long-range cooperativity in molecular recognition of RNA by oligodeoxynucleotides, with multiple C5-(1-propynyl) pyrimidines

A heptamer composed of C5-(1-propynyl) pyrimidines (Y-p's) is a potent and specific antisense agent against the mRNA of SV40 large T antigen (Wagner, R. W.; Matteucci, M. D,; Grant, D.; Huang, T.; Froehler, B. C, Nat. Biotechnol. 1996, 14, 840-844). To characterize the role of the propynyl groups in molecular recognition, thermodynamic increments associated with substitutions in DNA:RNA duplexes, such as 5 ' -dCCUCCUU-3 ' :3 ' -r (GA) over bar GGAGGAA (AU) over bar -5 ', have been measured by UV melting experiments. For nucleotides tested. an unpaired dangling end stabilizes unmodified and propynylated duplexes similarly: except that addition of a 5 ' unpaired rA is 1.4 kcal/mol more stabilizing on the propynylated, PODN:RNA, duplex than on the DNA:RNA duplex. Free energy increments for addition of single propynyl groups range from 0 to -4.0 kcal/mol, depending on the final number and locations of substitutions. A preliminary model for predicting the stabilities of Y-p-containing hybrid duplexes is presented. Eliminating one amino group, and therefore a hydrogen bond, by substituting inosine (I) for guanosine (G), to give 5 ' -dC(p)C(p)U(p)C(p)C(p)U(p)U(p)-3 ' :3 ' =r (GA) over bar GIAGGAAAU-5 ' , destabilizes the duplex by 3.9 kcal/mol, compared to 1.7 kcal/mol for the same change within the unpropynylated duplex, This 2.2 kcal/mol difference is eliminated by removing a single propynyl group three base pairs away. CD spectra suggest that single propynyl deletions, within the PODN:RNA duplex have position-dependent effects on helix geometry. The results suggest long-range cooperativity between propynyl groups and provide insights for rationally programming oligonucleotides with enhanced binding and specificity. This can be exploited in developing technologies that are dependent upon nucleic acid-based molecular recognition.