B2 - Deciphering the inherent molecular features underlying catalytically active DNA sequences
Catalytically active DNA sequences, known as DNAzymes, carry a great therapeutic and biotechnological potential. In general, DNAzymes for different functions have been developed by in vitro selection. Target-selective RNA cleavage is arguably among the most important reactions that can be catalyzed by DNAzymes. However, many aspects of this process, including the fundamental properties that distinguish catalytically active DNA sequences from the large pool of their inactive counterparts, remain elusive. Following our recent success in elucidating the structure of one of the most active DNAzymes in the precatalytic complex with its target RNA, we here propose to carry out a comprehensive characterization of the genuine properties that are the origin of the DNA’s catalytic activity. This will include (i) a comparative analysis of the properties that are already inherent to the single-stranded DNA and the effects induced by DNA-RNA complex formation, (ii) the influence of environmental parameters on the complex formation and the catalytic activity, and (iii) the characterization of the factors that determine DNA-RNA selectivity and catalytic turnover.
Figure 1: a) Schematic DNAzyme representation (red) in the presence (left) and absence (right) of RNA target. Key factors investigated in this project are highlighted, including (I) mutations in the loop that affect activity, (II) the composition of the arm sequence, (III) single-nucleotide mismatches in the arm sequence, and (IV) elongated (full-length) RNA constructs. b)-d) Initial data reporting on effects of complex formation and environmental parameters. b) Percent of sequential (NMR-detected) stacking contacts within the DNAzyme’s arm and loop region in the presence (black) or absence (blue) of RNA target. The data indicate that in the absence of RNA the DNAzyme’s loop region forms considerably more stacking interactions than the arm region. The stacking interactions of the loop further appear to be only moderately changed by the presence of the RNA, pointing to a tendency of the loop sequence to adopt a (partly) predefined structure already in the absence of RNA. c) pH-dependence of the DNAzyme cleavage activity (after 1h). d) NMR-detected pH-induced changes in the chemical environment of the DNAzyme. The so-called fingerprint region (pyrimidine H5-H6 correlations) is shown that clearly separates into signals from the arm and loop region (grey/purple ellipse, respectively). The spectra reveal that, unlike the arm region, the pH has a strong effect on the nucleotides of the loop.