Large Scale Mutational and Kinetic Analysis of a Self-Hydrolyzing Deoxyribozyme

Deoxyribozymes
are catalytic DNA sequences whose atomic structures
are generally difficult to elucidate. Mutational analysis remains
a principal approach for understanding and engineering deoxyribozymes
with diverse catalytic activities. However, laborious preparation
and biochemical characterization of individual sequences severely
limit the number of mutants that can be studied biochemically. Here,
we applied deep sequencing to directly measure the activities of self-hydrolyzing
deoxyribozyme sequences in high throughput. First, all single and
double mutants within the 15-base catalytic core of the deoxyribozyme
I-R3 were assayed to unambiguously determine the tolerated and untolerated
mutations at each position. Subsequently, 4096 deoxyribozyme variants
with tolerated base substitutions at seven positions were kinetically
assayed in parallel. We identified 533 active mutants whose first-order
rate constants and activation energies were determined. The results
indicate an isolated and narrow peak in the deoxyribozyme sequence
space and provide a quantitative view of the effects of multiple mutations
on the deoxyribozyme activity for the first time.