Optimisation of the 10-23 DNAzyme-substrate pairing interactions enhanced RNA cleavage activity at purine-cytosine target sites

Abstract

The 10-23 RNA cleaving DNAzyme has been shown to cleave any purine-pyrimidine (RY) junction under simulated physiological conditions. In this study, we systematically examine the DNAzymes relative activity against different RY combinations in order to determine the hierarchy of substrate core dinucleotide sequence susceptibility. The reactivity of each substrate dinucleotide compared in the same background sequence with the appropriately matched DNAzyme was found to follow the scheme AU = GU >> or = GC >> AC. The relatively poor activity of the DNAzyme against AC and GC containing substrates was found to be improved substantially by modifications to the binding domain which subtly weaken its interaction with the substrate core. The most effective modification resulting in rate enhancement of up to 200-fold, was achieved by substitution of deoxyguanine with deoxyinosine such that the base pair interaction with the RNA substrates core C is reduced from three hydrogen bonds to two. The increased cleavage activity generated by this modification could be important for application of the 10-23 DNAzyme particularly when the target site core is an AC dinucleotide.

Figure 1

Secondary structure of the 10–23 and 8–17 DNAzyme–substrate complexes. The 10–23 DNAzyme (top) consists of two variable binding domains, designated arm I and arm II, which flank a conserved 15 base unpaired motif that forms the catalytic domain. The only requirement of the RNA polynucleotide substrate is for a core sequence containing an RY junction. The 8–17 DNAzyme (bottom) has a similar configuration to the 10–23 DNAzyme except that it has a 12 base catalytic motif and a core substrate requirement for AG in which the A is unpaired and the G forms a wobble pair.

Figure 2

Reaction progress for each RY substrate (S1 series) and their matched DNAzymes. Reaction progress at each time point was determined by densitometry of cleavage band intensity. Data points represented by squares, circles, triangles and rhombi correspond to the GU, AU, GC and AC substrates, respectively. Curves were fitted to the data by regression using the formula described in Materials and Methods. The average rates of these reactions were also recorded in Table 1.

Figure 3

Base pair structures showing hydrogen bond interactions.

Figure 4

Reaction progress curves comparing the activity of rC-dG and rC-dI core DNAzyme–substrate pairs for the substrate series S1. The average rates of these reactions were also recorded in Table 1.