Abstract:

In vitro evolution was previously used to identify a small deoxyribozyme, 7Q10, that ligates RNA with formation of a 2′-5′ phosphodiester linkage from a 2′, 3′-cyclic phosphate and a 5′-hydroxyl group. Ligation occurs in a convenient "binding arms" format analogous to that of the well-known 10-23 and 8-17 RNA-cleaving deoxyribozymes. Here, we report the optimization and generality of 7Q10 as a 2′-5′ RNA ligase. By comprehensive mutagenesis of its 16-nucleotide enzyme region, the parent 7Q10 sequence is shown to be optimal for RNA ligation yield, although several mutations are capable of increasing the ligation rate approximately fivefold at the expense of yield. The 7Q10 deoxyribozyme ligates any RNA substrates that form the sequence motif UA↓GR (arrowhead=ligation site and R=purine), providing at least 30% yield of ligated RNA in ∼1-2 hours at 37°C and pH 9.0. Comparable yields are obtained in ∼12-24 hours at pH 7.5, which may be more suitable for larger RNAs that are more sensitive to non-specific degradation. For RNA substrates that form the related ligation junction UA↓GY (Y=pyrimidine), somewhat lower yields are obtained, but significant ligation activity is still observed. These data establish that 7Q10 is a generally applicable RNA ligase. A plot of log(kobs) versus pH from pH 6.9 to 9.0 has a slope of just under 1, suggesting that a single deprotonation occurs during the rate-determining reaction step. The compact 7Q10 deoxyribozyme has both practical utility and the potential for increasing our structural and mechanistic understanding of how nucleic acids can mediate chemical reactions.