Journal of Molecular Biology
Optimization and Generality of a Small Deoxyribozyme that Ligates RNA
Introduction
Site-specifically modified RNAs are valuable for studies of RNA structure, folding, and catalysis.1., 2., 3., 4., 5., 6., 7., 8., 9., 10., 11., 12., 13., 14., 15., 16., 17., 18., 19. Modifications are most readily incorporated by solid-phase methods using synthetic ribonucleoside phosphoramidites.20., 21., 22., 23. Because many interesting RNAs are larger than can be prepared directly by solid-phase synthesis, new approaches for RNA ligation are in demand. We have recently begun to develop deoxyribozymes (DNA enzymes)24., 25., 26., 27. for RNA ligation. At least two general strategies may be employed to identify deoxyribozymes that ligate RNA. First, in vitro selection may be performed using a random DNA pool as the starting point. Second, a deoxyribozyme that is known to cleave RNA may be evolved to operate “in reverse” as an RNA ligase. In our hands, both approaches have been successful.28., 29., 30. In particular, the latter strategy provided a family of deoxyribozymes (Figure 1(a)) that were evolved from the 8–17 RNA-cleaving DNA enzyme31 (Figure 1(b)). One of the new RNA ligase deoxyribozymes, designated 7Q10, provides ∼30% yield of ligated RNA in 12 hours at pH 7.5 and 40 mM Mg2+ with a particular set of RNA substrates.29 The ligated RNA product was shown to be joined by a non-native 2′–5′ phosphodiester linkage, which should be tolerable or even advantageous under many circumstances.28., 29. Here, we report the systematic optimization of 7Q10 as a general 2′–5′ RNA ligase that can be applied to join a wide range of RNA substrates. Additionally, 7Q10 offers the potential for future structural and mechanistic investigations that should expand our understanding of how nucleic acids can mediate chemical reactions.
Section snippets
Results
In Figure 1(a) are shown four deoxyribozymes that ligate RNA. These DNA enzymes were previously identified by in vitro evolution from the 8–17 DNA enzyme that cleaves RNA.29 Here, we used the highest-yielding of these RNA ligase deoxyribozymes, 7Q10, as the starting point for optimizing the RNA ligation reaction and for establishing its generality with a wide range of RNA substrate sequences.
Discussion
Our major goals in this study were to optimize the 7Q10 deoxyribozyme for RNA ligation and to explore the extent of its generality for joining various RNA substrate sequences. By determining the activity of 7Q10 and 30 related deoxyribozymes as shown in Figure 2, Figure 3, we determined that the parent 7Q10 sequence itself is optimal in terms of RNA ligation yield. We further established that outside of a very limited region of the RNA substrates surrounding the ligation site (UA↓G), the 7Q10
Kinetics assays
All of the kinetics assays used the trimolecular format shown in Figure 1(c). The 32P-radiolabeled left-hand RNA substrate L was the limiting reagent relative to the right-hand substrate R and deoxyribozyme E (the ratio L:E:R was ∼1:3:6 to 1:10:30, with the concentration of E equal to ∼0.5–3 μM). Increasing the concentration of E or R (or both) did not significantly change the observed kinetics or yields, indicating that the observed yields were not limited by availability of E or R. See our
Acknowledgements
This research was supported by the Burroughs Wellcome Fund (New Investigator Award in the Basic Pharmacological Sciences to S.K.S.), the March of Dimes Birth Defects Foundation (Research grant no. 5-FY02-271 to S.K.S.), the National Institutes of Health (GM-65966 to S.K.S.), and the UIUC Department of Chemistry. Acknowledgement is made to the donors of The Petroleum Research Fund, administered by the American Chemical Society, for partial support of this research (38803-G4 to S.K.S.). We thank
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