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

Abstract

Deoxyribozymes (DNA enzymes) are DNA catalysts for a variety of chemical reactions that typically involve nucleic acid substrates. 1 Our laboratory has focused on the in vitro selection of DNA enzymes for RNA ligation. 2 A highly challenging goal has been deoxyribozymes that synthesize native 3′-5′ RNA linkages rapidly and in high yield for a wide variety of RNA sequences, rather than for only a limited set of substrates. 2f We recently described a selection strategy that favors native RNA ligation by incorporating a stringently 3′-5′-selective step into the selection rounds. 2h We have now applied this strategy to identify two RNA ligase deoxyribozymes that rapidly form high yields of 3′-5′ linkages with modest sequence requirements for the two RNA substrates, thereby fulfilling all requirements for useful RNA ligase reagents. Because RNA ligation by protein enzymes 3 does not always provide acceptable yields, 4,5 the identification of general DNA enzymes for 3′-5′ RNA ligation enables alternative synthetic routes that will be useful for practical biochemistry.Our recently described selection methodology 2a was used to identify deoxyribozymes that join a 2′,3′-diol to a 5′-triphosphate (Figure 1). Previously, such ligations led to 2′,5′-branched RNA by reaction of an internal 2′-hydroxyl group, 2b,e or they led to linear 3′-5′ RNA but with restrictive and impractical sequence requirements. 2f Here, 3′-5′ selectivity during ligation was enforced by incorporating the RNA-cleaving 8-17 deoxyribozyme 6 into the selection procedure, 2h starting at either round 2 (for selections using 40 mM Mg 2+ ) or round 5 (for selections using 1 mM Zn 2+ ). In both cases, >95% of the ligation products from each uncloned selection pool had 3′-5′ linkages ( Figure S1). When the ligation activities had stopped increasing, individual deoxyribozymes were cloned. On the basis of a preliminary survey of activities, two clones, named 9DB1 (from round 9 of the Mg 2+ selection) and 7DE5 (from round 7 of the Zn 2+ selection) were examined further. By cleaving the ligation products from each of the two new deoxyribozymes with 8-17, which is highly selective for 3′-5′ RNA linkages, 2a both 9DB1 and 7DE5 were verified to create 3′-5′ linkages ( Figure S1).The 9DB1 and 7DE5 deoxyribozymes ligate RNA under practical in vitro incubation conditions. As shown in Figure 2, 9DB1 provides 60-70% yield of ligated RNA with k obs ∼ 0.04 min -1 (t 1/2 ∼ 15 min) at 40 mM Mg 2+ , pH 9.0, and 37°C. Similarly, 7DE5 has 40-50% yield of ligated RNA with k obs ∼ 0.02 min -1 (t 1/2 ∼ 30 min) at 1 mM Zn 2+ , pH 7.5, and 23°C. The 9DB1 deoxyribozyme is also effective at pH 7.5, where k obs is ∼0.2 h -1 (t 1/2 ∼ 4 h; data not shown). Incubation at the lower pH value of 7.5 instead of 9.0 should be useful for synthesis of larger RNAs that may experience more nonspecific degradation during an overnight incubation period, particularly at higher pH.In the selection design that led to 9DB1 and 7DE5, only one RNA nucleotide of each substrate was not base-paired with ...

Efficient RNA 5′-adenylation by T4 DNA ligase to facilitate practical applications

“…Our own in vitro selection efforts using 59-triphosphate RNA substrates (Wang and Silverman 2003a;Purtha et al 2005) can now be extended to RNAs for which 59-triphosphates are difficult to prepare and 59-adenylates serve as close functional surrogates (Hager and Szostak 1997;Silverman 2004, 2005). Because 59-adenylated RNA should be resistant to exonuclease degradation from the 59-terminus, 59-capping of RNA by adenylation should be a useful RNA modification, perhaps for in vivo applications.…”

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

“…R3C(H) was used for the analysis of singleturnover rate, and R3C(T) was used to analyze the turnover rate. We also investigated 9DB1 shown in Figure 3a, derived from the 9DB1 DNAzyme obtained through in vitro selection [47]. This DNAzyme catalyzes the formation of a 3′,5′-phosphodiester bond of two substrate RNAs in the presence of Mg 2+ , and it was designed to catalyze the ligation of the same substrates as the ligase ribozymes.…”

Structure determination and dynamics of protein–RNA complexes by NMR spectroscopy

“…RNA ligation can be performed by T4 RNA or T4 DNA ligase [135][136][137] or by using a deoxyribozyme that catalyzes RNA ligation [138].…”