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 approximately 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.