STRUCTURALLY COMPLEX AND HIGHLY-ACTIVE RNA LIGASES DERIVED FROM RANDOM RNA SEQUENCES

Seven families of RNA ligases, previously isolated from random RNA seq uences, fall into three classes on the basis of secondary structure an d regiospecificity of ligation. Two of the three classes of ribozymes have been engineered to act as true enzymes, catalyzing the multiple-t urnover transformation of substrates into products. The most complex o f these ribozymes has a minimal catalytic domain of 93 nucleotides. An optimized version of this ribozyme has a k(cat), exceeding one per se cond, a value far greater than that of most natural RNA catalysts and approaching that of comparable protein enzymes. The fact that such a l arge and complex ligase emerged from avery limited sampling of sequenc e space implies the existence of a large number of distinct RNA struct ures of equivalent complexity and activity.