Many sequence variations of the 8–17 RNA-cleaving deoxyribozyme have been isolated through in vitro selection. In an effort to understand how these sequence variations affect cleavage site selectivity, we systematically mutated the catalytic core of 8–17 and measured the cleavage activity of each mutant deoxyribozyme against all 16 possible chimeric (RNA/DNA) dinucleotide junctions. We observed sequence-function relationships that suggest how the following non-conserved positions in the catalytic core influence selectivity at the dinucleotide (5’ rN 18 -N 1.1 3’) cleavage site: (i) positions 2.1 and 12 represent a primary determinant of the selectivity at the 3’ position (N 1.1 ) of the cleavage site; (ii) positions 15 and 15.0 represent a primary determinant of the selectivity at the 5’ position (rN 18 ) of the cleavage site and (iii) the sequence of the 3-bp intramolecular stem has relatively little influence on cleavage site selectivity. Furthermore, we report for the first time that 8–17 variants have the collective ability to cleave all dinucleotide junctions with rate enhancements of at least 1000- fold over background. Three optimal 8–17 variants, identified from »75 different sequences that were examined, can collectively cleave 10 of 16 junctions with useful rates of ̧0.1 min
1 , and exhibit an overall hierarchy of reactivity towards groups of related junctions according to the order NG ` NA ` NC ` NT.