Target-induced steric hindrance protection of DNAzyme junctions for completely enzyme-free and amplified sensing of transcription factors

Abstract

The interactions between proteins and DNA play critical roles in numerous essential cellular activities. Therefore, sensitive investigation of these interactions is crucial for the study of different biological activities. Hence, a convenient and enzyme-free sensing approach for homogeneous and ultra-sensitive assay of transcription factors was explored based on the steric hindrance-protected Mg2+-DNAzyme amplification strategy. The target proteins can specifically bind the unique sequences of the Y-shaped DNA probes with strong affinity to form the protein/DNA complexes and to protect them from being disrupted by the external invading DNA. The target-protected and intact Mg2+-DNAzymes further hybridize with the fluorescently quenched DNA hairpin substrate sequences and cleave them cyclically to yield substantially enhanced fluorescence response for amplified detection of transcription factors. Using nuclear factor kappa B p50 as the example target, a low detection limit down to 2.8 pM can be achieved. Besides, the approach also exhibits excellent selectivity due to the high specificity of the recognition regions and can be further applied in human serums. This sensing platform thus can expect to be a promising alternative for the sensitive analysis of multiple DNA-binding proteins by changing the corresponding recognition units.