Ribonuclase L is a key mediator of the antiviral action of interferons. It is specifically activated by certain 2’, 5’-oligoadenylates known as 2-5A which are synthesized form ATP by activation of the double-stranded RNA-dependent 2-5A synthetase. This review summarizes the 2-5A molecular structural features that have been explored in order to define the 2-5A structure-activity relationships of RNase L. These studies have an immediate impact upon therapeutics discovery and development based upon the 2-5A system, as illustrated by the ability of 2-5A- antisense constructs to inhibit the virus and cancer replication.