Investigation of intramolecular split G-quadruplex-hemin systems and development of DNAzyme-based probes

G-quadruplex DNAzymes, known for their peroxidase-like activity and ease of rational design, have emerged as promising tools in biosensing applications. Despite the fundamental importance of structural topology, investigations into how the split mode of intramolecular split G-quadruplex (IntrSG)-based DNAzymes modulates their catalytic properties, as well as their implications for the development of DNAzyme-based probes, remain scarce. In this work, eleven IntrSG strands of different split modes were engineered with a classical parallel G-quadruplex sequence (PW17). The catalytic activity of IntrSG-hemin mixtures was systematically investigated via various techniques.

Most IntrSG strands incubated with hemin exhibited apparent catalytic activity, which was influenced by split modes and hybridization of inserted spacer. Interestingly, the mixture of hemin and IntrSG of split mode 5:7 (IntrSG(5:7)) exhibited drastic catalytic activity response after hybridization between its spacer and target, and the underlying mechanism was disclosed, which was hybridization-facilitated G-quadruplex assembling and catalytic enhancement. The IntrSG(5:7)-hemin system can be utilized as an effective "signal-on" signal transducer, owing to its simpleness and universality. Subsequently, influence of sequence context changes on the catalytic response was explored by extending and truncating the target sequence. Ignorable effect of protruding DNA was validated, and robust hybridization was critical for catalytic responses, which were unrelated to hybridization location. Finally, an IntrSG(5:7)-based catalytic probe was employed for quantitative analysis of miRNA (Let-7a), and favorable performance was achieved.

Taken together, a mechanistic framework for topological control of G-quadruplex DNAzyme catalysis was established, highlighting the potential of IntrSG as a versatile platform for biosensing and molecular diagnostics.