A series of peptide-acridine conjugates was desned and synthesized, based on three features of the proposed catalytic mechanism of RNase A: 2'-proton abstraction by proton donation to the leaving 5'-oxygen by His-119, and stabilization of the pentacoordinated phosphorous transition state by Lys-41. The substrate binding capability of RNase A was mimicked by the intercalator, acridine.Lysine served as a linker between acridine and the catalytic tripeptide. Cleavage of target RNA was monitored by agarose gel electrophoresis and by gel-permeation chromatography. The carboxyl-amidated conjugates HGHK(Acr)-NH2, HPHK(Acr)-NH2, and GGHK(Acr)-NH2 (where Acr indicates 2-methyl-9-acridinemethylene) all had similar hydrolytic activity. The catalytic mechanim most likely involved only the abstraction of the 2'-proton and stabilization of the transition state. These RNase mimics utilized rRNA and single-stranded RNA but not double-stranded RNA and tRNA as substrates.The design and synthesis of molecules having catalytic activity and substrate specificity, mimicking natural enzymes, have been a long-standing goal of chemists (1). RNA has become an interesting target for artificial hydrolytic enzymes during the last few years, partly because of possible therapeutic applications. Transition-metal ions (2), transitionmetal complexes (3-5), polypeptides (6, 7), oligoamines (8), and many other compounds (9, 10) have been reported to have RNase activity. We now report a type of compound that is capable of cleaving RNA under physiological conditions. In this compound, the active site of RNase A was mimicked by a tripeptide. The substrate recognition capability was accomplished by the use of an intercalator group, and these two portions were connected by a lysine residue (Fig. 1). Thus, the mimics simultaneously had substrate recognition and catalytic ability.The initial design of the cleaving moiety of our peptide analogs was based on the proposed hydrolytic mechanism of RNase A (11). This mechanism involves (i) protonation ofthe phosphate group at the RNA backbone by His-119, (ii) abstraction of a proton from the 2'-hydroxyl by His-12, (iii) attack of this 2'-oxyanion to form a pentacoordinated phosphorous transition state, which is stabilized by Lys-41, and (iv) proton transfer from phosphate to the leaving 5'-oxygen by Inspection of the structure of the 9-aminoacridine-5-iodocytidylyl(3'-5')guanosine complex (12) suggested that incorporation of the catalytic tetrapeptide at the 9-position of acridine would satisfy the structure requirements for an RNase mimic. The first and the third amino acid residues of our mimics could contact the phosphate and 2'-hydroxyl groups simultaneously, as well as act as an acid and a base catalyst, respectively. An imidazole or primary amino group was put at the first position to imitate the His-119 or Lys-41, and histidine at position three was expected to have similar properties to His-12 at the active site ofRNase A. The second position was a spacer that allowed the reactive groups on ...