Arabinonucleic acid (ANA), the 2'-epimer of RNA, was synthesized from arabinonucleoside building blocks by conventional solid-phase phosphoramidite synthesis. In addition, the biochemical and physicochemical properties of ANA strands of mixed base composition were evaluated for the first time. ANA exhibit certain characteristics desirable for use as antisense agents. They form duplexes with complementary RNA, direct RNase H degradation of target RNA molecules, and display resistance to 3'-exonucleases. Since RNA does not elicit RNase H activity, our findings establish that the stereochemistry at C2' (ANA versus RNA) is a key determinant in the activation of the enzyme RNase H. Inversion of stereochemistry at C2' is most likely accompanied by a conformational change in the furanose sugar pucker from C3'-endo (RNA) to C2'-endo ("DNA-like") pucker (ANA) [Noronha and Damha (1998) Nucleic Acids Res. 26, 2665-2671; Venkateswarlu and Ferguson (1999) J. Am. Chem. Soc. 121, 5609-5610]. This produces ANA/RNA hybrids whose CD spectra (i.e., helical conformation) are more similar to the native DNA/RNA substrates than to those of the pure RNA/RNA duplex. These features, combined with the fact that ara-2'OH groups project into the major groove of the helix (where they should not interfere with RNase H binding), help to explain the RNase H activity of ANA/RNA hybrids.