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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    ABSTRACT: The known biological functions of RNA have expanded in recent years and now include gene regulation, maintenance of sub-cellular structure, and catalysis, in addition to propagation of genetic information. As for proteins, RNA function is tightly correlated with structure. Unlike proteins, structural information for larger, biologically functional RNAs is relatively limited. NMR signal degeneracy, relaxation problems, and a paucity of long-range (1)H-(1)H dipolar contacts have limited the utility of traditional NMR approaches. Selective isotope labeling, including nucleotide-specific and segmental labeling strategies, may provide the best opportunities for obtaining structural information by NMR. Here we review methods that have been developed for preparing and purifying isotopically labeled RNAs, as well as NMR strategies that have been employed for signal assignment and structure determination.
    Journal of Biomolecular NMR 09/2009; 46(1):113-25. DOI:10.1007/s10858-009-9375-2 · 3.14 Impact Factor
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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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    ABSTRACT: Single molecule fluorescence techniques offer unique insights into mechanisms of conformational changes of RNA. Knowing how to make fluorescently labeled RNA molecules and understanding potential limitations of different labeling strategies is essential for successful implementation of single molecule fluorescence techniques. This chapter offers a step by step overview of the process of obtaining RNA constructs ready for single molecule measurements. Several alternative methods are described for each step, and ways of troubleshooting the most common problems, in particular, splinted RNA ligation, are suggested.
    Methods in enzymology 01/2009; 469:47-68. DOI:10.1016/S0076-6879(09)69003-0 · 2.09 Impact Factor
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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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    ABSTRACT: Since their first identification by in vitro selection in 1994, deoxyribozymes have been developed to catalyze a variety of chemical reactions. The first DNA-catalyzed reaction was cleavage of a ribonucleotide linkage within an oligonucleotide substrate. In subsequent years, growing collections of deoxyribozymes have been developed for several reactions that have practical utility for RNA research. These deoxyribozymes are useful for site-specific RNA cleavage as well as ligation to form linear, branched, and lariat RNA products. An application related to RNA ligation is deoxyribozyme-catalyzed labeling of RNA (DECAL), which is used to attach a biophysical tag to a desired RNA sequence at a specific position. With current achievements and likely future developments, deoxyribozymes are a useful contributor to the toolbox of RNA research methods.
    Methods in enzymology 01/2009; 469:95-117. DOI:10.1016/S0076-6879(09)69005-4 · 2.09 Impact Factor
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