Catalysis in biology is restricted to RNA (ribozymes) and protein enzymes, but synthetic biomolecular catalysts can also be made of DNA (deoxyribozymes) or synthetic genetic polymers. In vitro selection from synthetic random DNA libraries identified DNA catalysts for various chemical reactions beyond RNA backbone cleavage. DNA-catalysed reactions include RNA and DNA ligation in various topologies, hydrolytic cleavage and photorepair of DNA, as well as reactions of peptides and small molecules. In spite of comprehensive biochemical studies of DNA catalysts for two decades, fundamental mechanistic understanding of their function is lacking in the absence of three-dimensional models at atomic resolution. Early attempts to solve the crystal structure of an RNA-cleaving deoxyribozyme resulted in a catalytically irrelevant nucleic acid fold. Here we report the crystal structure of the RNA-ligating deoxyribozyme 9DB1 (ref. 14) at 2.8 Å resolution. The structure captures the ligation reaction in the post-catalytic state, revealing a compact folding unit stabilized by numerous tertiary interactions, and an unanticipated organization of the catalytic centre. Structure-guided mutagenesis provided insights into the basis for regioselectivity of the ligation reaction and allowed remarkable manipulation of substrate recognition and reaction rate. Moreover, the structure highlights how the specific properties of deoxyribose are reflected in the backbone conformation of the DNA catalyst, in support of its intricate three-dimensional organization. The structural principles underlying the catalytic ability of DNA elucidate differences and similarities in DNA versus RNA catalysts, which is relevant for comprehending the privileged position of folded RNA in the prebiotic world and in current organisms.

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We describe a general and simple method to identify catalytically and structurally important nucleotides in functional RNAs. Our approach is based on statistical replacement of each nucleoside with a non-nucleosidic spacer (C3 linker, D), followed by separation of active library variants and readout of interference effects by analysis of enzymatic primer extension reactions.Many examples of natural and artificial functional RNA motifs have been identified, including ribozymes, aptamers, riboswitches, small interfering RNAs, or protein-binding RNAs, that demonstrate the diversity of RNA functions beyond the transmission of genetic information. 1 Elucidating the structure and sequence requirements of non-coding nucleic acids is crucial for understanding their mechanisms of action. Furthermore, such knowledge facilitates design and engineering of RNA for practical applications in biomolecular chemistry and synthetic biology.Numerous biochemical and biophysical methods are used to analyse nucleic acid architectures at various levels of resolution. 2 Recent additions to the traditional repertoire are geared towards high-throughput analyses on massively parallel arrays, or combine mutations and footprinting studies with computational methods. 3,4 Our laboratory has recently demonstrated combinatorial analysis methods (CoMA, 5 dNAIM, 6 NDS 7 ) that allowed the simultaneous assessment of all possible single point mutants of deoxyribozymes, as well as the identification of catalytically important nucleotides and their functional groups. However, due to the method design, the reported approaches are only applicable for studying functional DNAs. They are based on the combinatorial solid-phase synthesis of DNA oligonucleotide libraries, in which the mutations are marked with a chemical tag: the hydroxyl group resembling the 2 0 -OH group of ribonucleotides. Mutations tagged in this way can be easily decoded by alkaline hydrolysis. This is an asset for the analysis of functional DNAs, but, for obvious reasons, the same strategy cannot be applied to RNA.In the present work we established an analogously versatile combinatorial approach for RNA that reveals functionally important nucleotides in a simple set of experiments that do not require sophisticated instrumentation. We used the threecarbon linker D as non-nucleosidic spacer unit to statistically replace (''delete'') standard ribonucleotides within functional nucleic acids (Fig. 1). Following separation of the functionally active and inactive library variants, the spacer substitutions were decoded by primer extension reactions. Analysis of the primer extension pattern allowed identification of the essential and non-essential nucleotides. We demonstrated our new approach