Characterization of long RNA-cleaving deoxyribozymes with short catalytic cores: the effect of excess sequence elements on the outcome of in vitro selection

Abstract

We previously conducted an in vitro selection experiment for RNA-cleaving deoxyribozymes, using a combinatorial DNA library containing 80 random nucleotides. Ultimately, 110 different sequence classes were isolated, but the vast majority contained a short14–15 nt catalytic DNA motif commonly known as 8–17. Herein, we report extensive truncation experiments conducted on multiple sequence classes to confirm the suspected catalytic role played by 8–17 and to determine the effect of excess sequence elements on the activity of this motif and the outcome of selection. Although we observed beneficial, detrimental and neutral consequences for activity, the magnitude of the effect rarely exceeded 2-fold. These deoxyribozymes appear to have survived increasing selection pressure despite the presence of additional sequence elements, rather than because of them. A new deoxyribozyme with comparable activity, called G15–30, was ∼2.5-fold larger and experienced a ∼4-fold greater inhibitory effect from excess sequence elements than the average 8–17 motif. Our results suggest that 8–17 may be less susceptible to the potential inhibitory effects of excess arbitrary sequence than larger motifs, which represents a previously unappreciated selective advantage that may contribute to its widespread recurrence.