Target binding protection mediated rolling circle amplification for sensitive detection of transcription factors

Abstract

Transcription factors (TFs) play central roles in the regulation of gene expression by binding with specific DNA sequences. As a potential diagnostic marker, sensitive detection of TFs is essential for pharmacological research development and clinical disease diagnosis. Here, a new fluorescent method based on target binding protection mediated rolling circle amplification (RCA) was developed for TFs detection. A hairpin probe with recognition site for target binding, cleavage site for Nt.BbvCI digestion and two hanging DNA strands with part of G-quadruplex complementary sequences for signal output was designed. Moreover, the hairpin probe could serve as template of RCA after being ligated. Firstly, TFs bound with hairpin probes and protected signal complementary sequences against cleavage by Nt.BbvCI due to space hindrance effect, while the excess hairpin probes were effectively digested to avoid false positive signal. Then, TFs and Nt.BbvCI were dissociated from hairpin probes by heating, complete hairpin probes being preserved. Next, protected hairpin probes were specifically connected to dumbbell templates under the action of T4 DNA ligase. Subsequently, dumbbell templates hybridized with primer to initiate the RCA reaction, obtaining numerous G-quadruplex sequences. Finally, N-methyl-mesoporphyrin IX (NMM) bound with G-quadruplex to generate enhanced fluorescence signal. The proposed assay system achieved excellent specificity and sensitivity toward TATA-binding protein (TBP) with a detection limit as low as 88pM, and with a linear range from 100pM to 40nM. The strategy proposed here was looking forward to offer a powerful tool for TFs related bioanalysis and disease diagnostics.