Ultrasensitive Detection of Transcription Factors Using Transcription-Mediated Isothermally Exponential Amplification-Induced Chemiluminescence

Abstract

Transcription factors (TFs) are important cellular components that modulate gene expression, and the malregulation of transcription will lead to a variety of diseases such as cancer and developmental syndromes. However, the conventional methods for transcription factor assay are generally cumbersome and costly with low sensitivity. Here, we develop a label-free strategy for ultrasensitive detection of transcription factors using a cascade signal amplification of RNA transcription, dual isothermally exponential amplification reaction (EXPAR), and G-quadruplex DNAzyme-driven chemiluminescence. Briefly, the specific binding of TF with the detecting probe prevents the cleavage of the detecting probe by exonuclease and subsequently facilitates the conversion of TF signal to abundant RNA triggers in the presence of T7 RNA polymerase. The obtained RNA triggers can initiate the strand displacement amplification to yield abundant DNAzymes and DNA triggers, and the released DNA triggers can further initiate the next rounds of EXPAR reaction. The synergistic operation of dual EXPAR reaction can produce large amounts of DNAzymes, which subsequently catalyze the oxidation of luminol by H₂O₂ to yield an enhanced chemiluminescence signal with the assistance of cofactor hemin. Conversely, in the absence of target TF, the naked detecting probes will be completely digested by exonucleases, leading to neither the transcription-mediated EXPAR nor the DNAzyme-driven chemiluminescence signal. This method has a low detection limit of as low as 6.03 × 10^–15 M and a broad dynamic range from 10 fM to 1 nM and can even measure the NF-κB p50 of crude cell nuclear extracts. Moreover, this method can be used to measure a variety of DNA-binding proteins by simply substituting the target-specific binding sequence in the detecting probes