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Rigid spin-labeled nucleoside Ç, an analog of deoxycytidine that base-pairs with deoxyguanosine, was incorporated into DNA oligomers by chemical synthesis. Thermal denaturation experiments and circular dichroism (CD) measurements showed that Ç has a negligible effect on DNA duplex stability and conformation. Nucleoside Ç was incorporated into several positions within single-stranded DNA oligomers that can adopt two hairpin conformations of similar energy, each of which contains a four-base loop. The relative mobility of nucleotides in the alternating C/G hairpin loops, 5′-d(GCGC) and 5′-d(CGCG), was determined by electron paramagnetic resonance (EPR) spectroscopy. The most mobile nucleotide in the loop is the second one from the 5′-end, followed by the third, first and fourth nucleotides, consistent with previous NMR studies of DNA hairpin loops of different sequences. The EPR hairpin data were also corroborated by fluorescence spectroscopy using oligomers containing reduced Ç (Çf), which is fluorescent. Furthermore, EPR spectra of duplex DNAs that contained Ç at the end of the helix showed features that indicated dipolar coupling between two spins. These data are consistent with end-to-end duplex stacking in solution, which was only observed when G was paired to Ç, but not when Ç was paired with A, C or T.

A nucleoside carrying a perfluorinated tert-butyl group (4) was prepared by a Sonogashira coupling of 5-iodo-2′-deoxyuridine with 4,4,4-trifluoro-3,3-is(trifluoromethyl)-1-butyne in nearly quantitative yield and subsequently incorporated into DNA oligomers. Thermal denaturation studies showed that 4 had a negligible effect on duplex stabilty when compared to thymidine. Transition from single strand to duplex was monitored by 19 F NMR spectroscopy at micromolar concentrations of oligomers, demonstrating the sensitivity of 4 as an NMR reporter nucleoside.Fluorine has many advantages as a probe for NMR spectroscopy of nucleic acids and other biopolymers.

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