Crystal structure of a DNA catalyst

Abstract

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.

Catalytic Activities of Ribozymes and DNAzymes in Water and Mixed Aqueous Media

“…Our results exhibited that many other organic compounds also have an ability to enhance the activity of this DNAzyme, although the activity was completely abolished at the low MgCl 2 concentration of 1 mM. Although the detailed mechanism and structure of the R3C ribozyme have not been elucidated, the crystal structure of 9DB1 DNAzyme has been reported, and it shows the presence of tertiary interactions in the catalytic domain; however, the role of metal ions in the reaction remains unclear [56]. The mixed solutions enhanced the catalytic activities of both R3C(T) and 9DB1, implying a similar mechanism by which the environmental factors influence the activity of these enzymes, whereas correlation analyses suggested that changes in the activity of these enzymes was not determined by a single environmental factor.…”

Catalytic DNA: Scope, Applications, and Biochemistry of Deoxyribozymes

“…In early 2016, a research team led by Höbartner and Pena reported the first deoxyribozyme X-ray crystal structure [114], that of the RNA-ligating 9DB1 deoxyribozyme [115]. This structure reveals several important facets of catalysis by DNA, including the fundamental confirmation that deoxyribozymes can have well-defined tertiary structures that are responsible for their catalysis.…”

“…Now that the first crystal structure has been reported [114], surely others will follow. Höbartner’s elegant combinatorial minimization methods, which were applied to 9DB1 before its crystallization [121], will likely be invaluable for enabling crystallization of unrelated deoxyribozymes.…”

“…For example, many studies have addressed functional roles of metal ions and investigated kinetics, but not to the extent where we truly understand mechanisms of DNA catalysis. With the first deoxyribozyme crystal structure now reported [114] and hopefully more on the way, the prospects are strong for understanding the detailed mechanisms by which DNA enzymes catalyze many reactions. A reasonable expectation is that deoxyribozymes and ribozymes will share common contributions to catalysis, such as acid-base chemistry involving the nucleobases [123].…”

DNA-Catalyzed Amide Hydrolysis

“…Another hypothesis, not yet evaluated empirically, is that any catalytically relevant HO dU 5-(hydroxymethyl) group can be replaced by a combination of the dT 5-methyl group and a water molecule, efficiently for AmideHy1 and much less well for AmideHy2; this would be analogous to abasic and other rescue experiments for ribozymes 13 and similar rescue studies for protein mutants. 14 Further experiments, likely involving high-resolution structural characterization, 15 are needed to explain the role of HO dU modifications in DNA-catalyzed amide hydrolysis.…”