Abstract
Deoxyribozymes (DNAzymes) are small, synthetic, single-stranded DNAs capable of catalyzing chemical reactions, including RNA ligation. Herein, we report a novel class of RNA ligase deoxyribozymes that utilize 5′-adenylated RNA (5′-AppRNA) as the donor substrate, mimicking the activated intermediates of protein-catalyzed RNA ligation. Four new DNAzymes were identified by in vitro selection from an N40 random DNA library and were shown to catalyze the intermolecular linear RNA-RNA ligation via the formation of a native 3′-5′-phosphodiester linkage. The catalytic activity is distinct from previously described RNA-ligating deoxyribozymes. Kinetic analyses revealed the optimal incubation conditions for high ligation yields and demonstrated a broad RNA substrate scope. Together with the smooth synthetic accessibility of 5′-adenylated RNAs, the new DNA enzymes are promising tools for the protein-free synthesis of long RNAs, for example containing precious modified nucleotides or fluorescent labels for biochemical and biophysical investigations.
Highlights
DNA is best known in the form of the famous Watson-Crick double helix of two antiparallel phosphodiester single strands held together by hydrogen bonding and stacking interactions of complementary A–T and G–C base pairs
It is well established that the information stored in defined sequences of single-stranded DNA can be exploited for the formation of active-site architectures that catalyze chemical transformations [1,2,3]. Such “enzymes made of DNA” are known as deoxyribozymes, and they are generated in the laboratory by in vitro selection from random libraries of DNA
DNA is not permitted as the acceptor substrate, and the ligation of two DNA substrates cannot be achieved with the SC9 deoxyribozyme. These results demonstrate the high specificity of the new Mn2+ -dependent DNA catalyst for RNA ligation substrates
Summary
DNA is best known in the form of the famous Watson-Crick double helix of two antiparallel phosphodiester single strands held together by hydrogen bonding and stacking interactions of complementary A–T and G–C base pairs. It is well established that the information stored in defined sequences of single-stranded DNA can be exploited for the formation of active-site architectures that catalyze chemical transformations [1,2,3]. Such “enzymes made of DNA” are known as deoxyribozymes (or DNAzymes), and they are generated in the laboratory by in vitro selection from random libraries of DNA. RNA-cleaving DNA enzymes have entered various fields of fundamental and applied research [5,6,7]
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