Inhibition of G-quadruplex assembling by DNA ligation: A versatile and non-covalent labeling strategy for bioanalysis

• DNA ligation was coupled to split G-quadruplex probes.
• Highly selective detection of small biomolecules (ATP and NAD+) was realized.
• The effect of single-base mismatch position on the ligation efficiency was also validated.
• The high selectivity is attributed to cofactor/sequence-dependent activity of DNA ligase.
• The non-covalent labeling strategy is simple, cost-effective and versatile.

Through tuning relative thermodynamic stabilities (I, II and III), DNA ligation was coupled to split G-quadruplex probes and a versatile, non-covalent labelling and fluorescent strategy was constructed based on inhibition of template-directed G-quadruplex assembling by ligation reaction. The non-covalent complex between G-quadruplex and fluorescent probe was employed as signalling label and thus covalent modification of DNA probes with fluorescent probes was avoided. Selective detection of small biomolecules (ATP and NAD+) in the nanomolar range was realized due to the cofactor-dependent activity of DNA ligases (T4 and Escherichia coli DNA ligase). By virtue of the simple strategy, the effect of mismatch position of single-base mismatched template DNA on the ligation efficiency was validated. Meanwhile, highly mismatch-influenced ligation efficiency of ligase endows the cost-effective strategy great potential for single-nucleotide polymorphism (SNP) analysis. The non-covalent labeling strategy provides a versatile and cost-effective platform for monitor of DNA ligation, cofactor detection, SNP analysis and other ligase-based assays.