Protein binding-protected DNA three-way junction-mediated rolling circle amplification for sensitive and specific detection of transcription factors

Abstract

Transcription factors (TFs) are DNA-binding proteins that regulate gene transcription and their expression levels are closely associated with disease development. Sensitive and specific detection of TFs is significant to clinical diagnostics and drug development. Herein, a label-free fluorescent strategy for the sensitive and specific detection of TFs was developed based on protein binding-protected DNA three-way junction (TWJ)-mediated rolling circle amplification (RCA). A trifunctional TWJ was designed including a target binding site, AlwI recognition site and sutured primer of RCA. Firstly, TFs bound with a target binding site, protecting the four and five bases downstream from the AlwI recognition site against cleavage by AlwI. Next, the sutured primer in the protected TWJ hybridized with the padlock probe, initiating RCA. Finally, the sutured primer was extended with multiple G-quadruplex sequences, binding with N-methyl-mesoporphyrin IX (NMM) to yield an enhanced fluorescence response. Residual TWJs were digested by AlwI, effectively blocking the RCA reaction, thus suppressing nonspecific amplification. Taking NF-κB p50 as a model target of TFs, high sensitivity was achieved with a low detection limit of 6.8 pM and a broad linear range from 8 pM to 15 nM. We successfully measured NF-κB p50 in HeLa cell nuclear extracts with a low detection limit of 0.34 ng μL⁻¹. The strategy was also effectively used to assay the inhibition effect of a model inhibitor of NF-κB, oridonin. The results indicated the proposed strategy holds great promise for studying TFs in disease diagnostics and drug developments.