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Long-Range Cooperativity in Molecular Recognition of RNA by Oligodeoxynucleotides with Multiple C5-(1-Propynyl) Pyrimidines

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journal contribution
posted on 11.04.2001, 00:00 by Thomas W. Barnes, Douglas H. Turner
A heptamer composed of C5-(1-propynyl) pyrimidines (Yp'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‘-rGAGGAGGAAAU-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 Yp-containing hybrid duplexes is presented. Eliminating one amino group, and therefore a hydrogen bond, by substituting inosine (I) for guanosine (G), to give 5‘-dCpCpUpCpCpUpUp-3‘:3‘-rGAGIAGGAAAU-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.