Deoxyribozymes That Prepare Branched and Linear RNA by Ligation

Abstract

An ongoing challenge for deoxyribozyme-mediated RNA ligations has been synthesis of native 3'-5' phosphodiester linkages. Previously, reactions of 5'-triphosphate RNA have provided mostly 2',5'-branched RNA. Here we report two new in vitro selection strategies designed to favor formation of the native linkage. The first strategy controls the regioselectivity by placing the incipient linkage within a DNA:RNA duplex region, such that 3'-5' is favored over 2'-5' both thermodynamically and kinetically. The second strategy makes use of a known deoxyribozyme that reacts only with 3'-5' RNA linkages. Both strategies were successful in making the native 3'-5' RNA linkage. Two deoxyribozymes, 9DB1 and 7DE5, from the second strategy have very few sequence requirements at the ligation junction. These deoxyribozymes will be useful as biochemical tools to study catalytic nucleic acids.