In order to understand further the contribution of the 3'-hydroxyl groups of 2-5A (ppps'(A2'p) ({ }_{n} A) ) to its interaction with RNase (L). we synthesized a series of analogues in which the (3^{\prime}) '-hydroxyl moiety was replaced by fluorine to yield 3 '-fluoro-3'-deoxyadenosine
sponding monophosphates. When these oligomers were evaluated for their ability to activate RNase L from various sources, we found that the replacement of the second from the (5^{\circ}). terminus adenosine residue of (2-5 \mathrm{~A}) with the fluoro analogue caused major reductions in activity. We conclude that the hydroxyl group of this second or middle nucleotide residue of 2-5A trimer maly act as a hydrogen bond donor to an acceptor group in RNase L and that this hydrogen bond may be key to what we presume may be a critical conformational change required for nuclease activity of RNase (\mathrm{L}). We have also found that this substitution of fluorine for hydroxyl in the second or penultimate residue of 2-5A trimer results in an oligomer with a (2^{\prime}, 5^{\prime})-phosphodiesterase sensitivity comparable to that of (2-5) A itself. When viewed in terms of earlier experiments, these results suggest that the role of the (3^{\prime}-\mathrm{OH}) group of the penultimate nucleotide of (2-5) A may be to anchor the substrate to the phosphodiesterase through its action as a hydrogen bond receptor. ( 1993 A Aademic Prew. Inc.