The first structure of a 2'-deoxy-2'-fluoro-D-arabinose nucleic acid (2'F-ANA)/RNA duplex is presented. We report the structural characterization by NMR spectroscopy of a small hybrid hairpin, r(GGAC)-d(TTCG)2 'F-a(GTCC), containing a 2 'F-ANA/RNA stem and a four-residue DNA loop. Complete H-1,C-13, F-19. and P-31 resonance assignments, scalar coupling constants, and NOE constraints were obtained from homonuclear and heteronuclear 2D spectra. In the chimeric duplex, the RNA strand adopts a classic A-form structure having C3 ' endo sugar puckers. The 2'F-ANA strand is neither A-form nor B-form and contains O4 ' endo sugar puckers. This contrasts strongly with the dynamic sugar conformations previously observed in the DNA strands of DNA/RNA hybrid duplexes. Structural parameters for the duplex, such as minor groove width, x-displacement, and inclination, were intermediate between those of A-form and B-form duplexes and similar to those of DNA/RNA duplexes. These results rationalize the enhanced stability of 2 'F-ANA/RNA duplexes and their ability to elicit RNase H activity. The results are relevant for the design of new antisense drugs based on sugar-modified nucleic acids.