When incorporated into DNA, structurally biased nucleoside and nucleotide monomers usually transmit these preferences to influence the overall topology of the oligomer. We have previously studied the structural effects of fluorine substitution in the furanose ring of a nucleoside and how this may affect biological activity, vis-a-vis enzyme binding. As mentioned in the last annual report, we extended this to vicinal 2'.3'- difluorinated nucleosides and have now completed a comprehensive study of the structure of three difluorinated uridine derivatives by NMR, ab initio calculations and X-ray spectroscopy. We found that the F-F gauche effect is operable only when the H6 proton of the uridine base is absent. This proved a previous assertion that there is a strong C3""""""""F-H6 attractive force operating pyrimidines with a C3'-endo disposed fluorine atom. A handful of groups are currently exploiting the use of conformationally """"""""locked"""""""" nucleoside building blocks in the construction of oligonucleotides with distinct helical folds. As outlined in project Z01 BC 06174-15 LMC by Dr. Marquez, his group has been refining procedures for the preparation of conformationally """"""""locked"""""""" [3.1.0] bicyclic systems as templates for both 2'-endo (B DNA-like) and 2'-exo-(A DNA/RNA-like)-puckered nucleotide congeners. We have incorporated these monomers into oligonucleotides at strategic positions to study the effect that base pairs with defined sugar puckers have on the overall structure of a DNA duplex. In particular, we have incorporated A-like monomers into a typical B-like strand of DNA in hopes of disrupting the structure in defined ways. Currently we are studying six different oligomers, based on the Dickerson-Drew dodecamer, by NMR spectroscopy. We have shown that the melt temperatures for these duplexes are highly affected by the incorporation of A-like monomers into the sequence. A reassessment of the duplex stabilities showed that there may be a mixture of duplex and hairpin structures in equilibrium in some constructs. Circular dichroism (CD) spectroscopy has also shown that most of the strands form duplexes at 25oC, however their CD signatures are different than those from the wild type sequences. NMR spectroscopy has shown that protons contained within modified nucleotide base pairs have similar coupling signatures as when they are monomeric, proving the rigidity of the modified base pairs within the duplex. We are currently completing the assignments of six oligonucleotides by NMR spectroscopy and are implementing experiments that will allow us to measure any degree of bending in the modified duplexes.
| Wu, Zhengrong; Maderia, Melissa; Barchi Jr, Joseph J et al. (2005) Changes in DNA bending induced by restricting nucleotide ring pucker studied by weak alignment NMR spectroscopy. Proc Natl Acad Sci U S A 102:24-8 |
| Maier, Martin A; Choi, Yongseok; Gaus, Hans et al. (2004) Synthesis and characterization of oligonucleotides containing conformationally constrained bicyclo[3.1.0]hexane pseudosugar analogs. Nucleic Acids Res 32:3642-50 |
