To gain insight into the origins of the large binding affinity of RNA toward target duplexes, 2'-deoxy-2'-fluororibonucleic acid (2'F-RNA) and 2'-deoxy-2'-fluoroarabinonucleic acid (2'F-ANA) were tested for their ability to recognize duplex DNA, duplex RNA, and RNA-DNA hybrids. 2'F-RNA, 2'F-ANA, and the corresponding control single-stranded (ss) DNA strands were shown to form triple-helical complexes only with duplex DNA and hybrid DNA (Pu)-RNA (Py), but not with duplex RNA and hybrid RNA (Pu)-DNA (Py). In contrast, an RNA third strand recognized all four possible duplexes (DD, DR, RD, and RR) as previously demonstrated by Roberts and Crothers [(1992) Science 258, 1463-1466]. The 2'F-RNA (C3'-endo) strand exhibited significantly reduced affinity for duplexes compared to an unmodified RNA (C3'-endo) strand. These findings are consistent with the intermolecular 2'-OH-phosphate contact mechanism proposed by Escudé et al. [(1993) Nucleic Acids Res. 24, 5547-5553], as a ribo 2'-F atom should not interact with a negatively charged phosphate. In addition, they emphasize the role of the 2'-OH ribose as a general recognition and binding determinant of RNA. The 2'-F arabino modification (2'F-ANA, C2'-endo) led to a considerable increase in the binding affinity for duplex DNA, as compared to those of DNA and 2'F-RNA third strands. This is likely to be the result of a greater population of C2'-endo pucker of the 2'F-ANA compared to DNA. The enhancement observed for 2'F-ANA strands toward duplex DNA is comparable to that observed with 2'-OMe RNA. Since 2'F-ANA has been shown to be more resistant to nuclease degradation than DNA, these results are likely to stimulate experimental work on arabinose derivatives in laboratories concerned with targeting DNA sequences in vivo ("antigene" strategy).