General Deoxyribozyme-Catalyzed Synthesis of Native 3‘−5‘ RNA Linkages

Department of Chemistry, University of Illinois, Urbana-Champaign, Urbana, Illinois, United States
Journal of the American Chemical Society (Impact Factor: 12.11). 10/2005; 127(38):13124-5. DOI: 10.1021/ja0533702
Source: PubMed


An elusive goal for nucleic acid enzymology has been deoxyribozymes that ligate RNA rapidly, sequence-generally, with formation of native 3'-5' linkages, and in preparatively useful yield. Using in vitro selection, we have identified Mg2+- and Zn2+-dependent deoxyribozymes that simultaneously fulfill all four of these criteria. The new deoxyribozymes operate under practical incubation conditions and have modest RNA substrate sequence requirements, specifically D downward arrowRA for 9DB1 and A downward arrowR for 7DE5 (D = A, G, or U; R = A or G). These requirements are comparable to those of deoxyribozymes such as 10-23 and 8-17, which are already widely used as biochemical tools for RNA cleavage. We anticipate that the 9DB1 and 7DE5 deoxyribozymes will find immediate practical application for RNA ligation.

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    • "Deoxyribozymes are DNA enzymes that typically catalyze reactions involving nucleic acid substrates (Höbartner and Silverman 2007). Silverman and co-workers engineered two RNA ligase deoxyribozymes that generate 3 0 -5 0 linkages rapidly and in high yield (Fig. 6c) (Purtha et al. 2005). The deoxyribozyme has modest sequence requirements for Fig. 7 Scheme for preparation of segmentally labeled RNAs from a single template. "
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    • "Even small amount of the ligated product can be detected by fluorescent scanners, and detection of fluorescence from both dyes can be used to identify the correct band. 4 (b) Deoxyribozymes (see S. Silverman, Chapter 5, this volume) that are capable of efficient ligation of RNA fragments have recently been evolved in the Silverman laboratory (Purtha et al., 2005). These deoxyribozymes catalyze the formation of the natural 3 0 –5 0 linkages and have reasonably broad sequence requirements at the site of cleavage, so they have a large potential as alternatives to protein ligases. "
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    • "In several other studies, we also found many deoxyribozymes that create native 3′–5′ RNA linkages, using either the 2′,3′-cyclic phosphate substrate combination of Fig. 2A (Purtha et al., 2005;Kost et al., 2008) or the 5′-triphosphate substrate combination of Fig. 2B (Wang and Silverman, 2005a). In all such cases, however, we either know or suspect that these DNA enzymes require enough particular RNA nucleotides so that their sequence generality is poor. "
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