Sequence-function relationships provide new insight into the cleavage site selectivity of the 8-17 RNA-cleaving deoxyribozyme

Abstract

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′ rN18-N1.1 3′) cleavage site: (i) positions 2.1 and 12 represent a primary determinant of the selectivity at the 3′ position (N1.1) of the cleavage site; (ii) positions 15 and 15.0 represent a primary determinant of the selectivity at the 5′ position (rN18) 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.

Tandem DNAzymes for mRNA cleavage: Choice of enzyme, metal ions and the antisense effect

“…The efficiency for cleaving different junctions by the 8-17 DNAzyme has been systematically studied. 21 We show here that the 10-23 DNAzyme also cleaves different junctions quite differently. If we accept the notion that both DNAzymes belong to the same family, and each junction may have its own optimal substrate junction sequence for cleavage, these two classic DNAzymes are just two examples of this DNAzyme family.…”

“…19 At the same time, it is also studied as a model DNAzyme due to its recurrence in many different in vitro selections and high catalytic efficiency. [20][21][22][23][24][25] Examples of using the 8-17 DNAzyme for gene silencing are also known. 18,26 While many examples of DNAzyme-based intracellular mRNA cleavage are reported, 13 concerns have also been raised that the 10-23 DNAzyme is basically inactive with physiological concentrations of free Mg 2+ .…”

“…However, after extensive selection and mutation work, the Li group discovered that the 8-17 DNAzyme can cleave essentially all the 16 possible dinucleotide junctions, although the rate varies over 5 orders of magnitude for these junctions. 21 By introducing mutations in the enzyme, the 8-17 DNAzyme can be optimized for each type of junction. It was recently proposed that the 8-17 and 10-23 DNAzymes might be the same enzyme.…”

“…For example, when an rAC junction was used, barely any cleavage was observed even with 500 mM Mg 2+ ( Figure S1, ESI). Therefore, it is also true for the [10][11][12][13][14][15][16][17][18][19][20][21][22][23] DNAzyme that its cleavage activity for every junction is not the same.…”

“…These DNAzymes target the GG dinucleotide junctions to achieve a high cleavage activity. 21 Since the PCR primers cover the whole region, any cleavage should result in the signal suppression. To avoid artifacts due to DNAzyme concentration, all the samples contained the same amount of total DNAzyme (e.g.…”