A Deoxyribozyme that Forms a Three-Helx-Junction Complex with its RNA Substrates and has General RNA Substrates and has General RNA Branch-Forming Activity.

We recently used in vitro selection to identify 7511, a deoxyribozyme that synthesizes 2',5'- branched AMA. The 7511 DNA enzyme mediates the nucleophilic attack of an adenosine 2'-hydroxyl group at a 5'-triphosphate, forming 2',5'-branched AMA in a reaction that resembles the first step of in vivo AMA splicing. Here, we describe 7S1 i characterization experiments that have two important implications for nucleic acid chemistry and biochemistry. First, on the basis of a comprehensive analysis of its substrate sequence requirements, 7S1 1 is shown to be generally applicable for the synthesis of a wide range of 2',5'-branched RNAs. Strict substrate sequence requirements are found at the two AMA nucleotides that directly form the branched linkage, and these requirements correspond to those nucleotides found most commonly at these two positions in natural spliced RNA5. Outside of these two nucleotides, most substrate sequences are tolerated with useful ligation activity, although rates and yields vary. Because chemical synthesis approaches to branched AMA are extremely limited in scope, the deoxyribozyme-based route using 7511 will enable many experiments that require branched RNA. Second, comprehensive nucleotide covariation experiments demonstrate that 7S11 and its AMA substrates adopt a three-helix-junction structure in which the branch-site nucleotide is located at the intersection of the three helices. Because many natural ribozymes have multi-helix junctions, 7S11 is an interesting model system for catalytic nucleic acids. [ABSTRACT FROM AUTHOR]