Phosphorylation at 5' end of guanosine stretches inhibits dimerization of G-quadruplexes and formation of a G-quadruplex interferes with the enzymatic activities of DNA enzymes

During an analysis of DNA enzymes by gel electrophoresis, we found that some DNA enzymes can adopt more than one conformation. The DNA enzyme Dz31 that formed more than one conformer contained a stretch of G residues. Further investigations, involving kinetic analysis and measurements of circular dichroism, indicated that this DNA enzyme and its derivatives formed G-quadruplexes. Moreover, we found that some derivative oligomers were capable of forming dimeric G-quadruplexes. We also compared the catalytic activities of Dz31 and its mutant derivatives. The present findings suggest that DNA enzymes with five or more continuous G residues are less favorable than those without G₅ in the association step in the enzymatic reaction and, thus, the choice of targets that contain a continuous stretch of C residues downstream of the cleavage site should be avoided. In addition, we found that negative charge–charge repulsion disrupted the dimerization of G-quadruplexes when a phosphate group was added directly to the 5′-terminal G of oligomers with continuous guanosine residues. In the case of 5′-phosphorylated G₅CTA, direct attachment of a phosphate group to the continuous G₅ sequence inhibited dimerization of G-quadruplexes, at least during electrophoresis on a denaturing gel.