Dodecaadenylic acid containing a single 2',5'-linkage at a defined position was formed by the coupling of two hexamers on a poly(U) template at 20. The rate of hydrolysis of this dodecamer was compared with that of a dodecamer that contained only the natural 3'-'-linkages. At 400, in 1 M aqueous ethylenediamine at pH 8 in the absence of poly(U), both dodecamers hydrolyzed at comparable rates, but the addition of two equivalents of poly(U) caused a 7-fold increase in the initial rate of hydrolysis of the oligomer containing the 2',5'-bond, and a 5-fold decrease in the initial rate of hydrolysis of the natural oligomer. When the oligomers are fully constrained in helical form, the ratio of the rates of cleavage of one 2',5'-bond to one 3',5'-bond under these conditions is probably about 900:1. The use of the 3',5'-bond, in combination with a right-handed helix, appears to have had a large selective advantage over the use of the 2',5'-bond for the storage of genetic information. Naturally occurring RNA appears to contain solely the 3',5'-internucleotide linkage. By contrast, successful attempts to demonstrate the template-directed non-enzymatic polymerization of ribonucleotides, which are of interest as models of prebiotic synthesis, have almost invariably resulted-in the production of a large excess of the unnatural 2',5'-isomer (1). Thus, coupling of adenosine 2',5'-cyclic phosphate (A>p) on a poly(U) template is catalyzed by aqueous ethylenediamine, but the resulting dimer contains about 97% of the 2',5'-linkage (2). It has been suggested (3) that these two observations may be related: that the constraint of the helix forces the formation of the 2',5'-bond from the cyclic phosphate, and also stabilizes a 3',5'-bond against hydrolysis in mildly basic solution. As a corollary, the 2',5'-bond is still labile when the constituent nucleotides are part of a right-handed helix, and thus storage of "genetic" information is better accomplished by use of the 3',5'-bond. Previous evidence for the stability of the 3',5'-bond in helical conformation comes from the isolation of oligo(purines) from the base-catalyzed partial hydrolysis of single-stranded RNA. Purine-rich regions have a higher helical content than pyrimidine-rich regions, and better resist alkaline hydrolysis (4, 5).We have made an unambiguous test of the ideas expressed above by measuring the rate of hydrolysis at pH 8 of two dodecaadenylates, one (I) that is entirely 3',5'-linked, the other (II) that contains a single 2',5'-bond at the central position. The measurements have been made both in the presence and in the absence of poly(U). The preparation of the synthetic dodecamer (II) could not be achieved by the Abbreviations: BAP, bacterial alkaline phosphatase; HPLC, high pressure liquid chromatography; A>p, adenosine 2',3'-cyclic phosphate; DP, degree of polymerization; percentages are given as % of total nucleoside units present; molar absorptivities are given per nucleoside unit. * corder. Baseline separation of (A)5A'p from (A)sA>p, and (A)ujA...
