A dual-mode strategy based on β-galactosidase and target-induced DNA polymerase protection for transcription factor detection using colorimetry and a glucose meter.

Abstract

In this work, we report a novel dual-mode method for the highly specific and sensitive detection of transcription factors (TFs) via the integration of Klenow polymerase protection induced by target-specific recognition, cascade-signal amplification using the hybridization chain reaction (HCR) and CRISPR/Cas12a system, and dual-signal transduction mediated by β-galactosidase (β-gal) and two substrates. A dual-mode signal-sensing interface was constructed by immobilizing the oligo DNA probe (P1) tethered β-gal in a 96-well plate. A hairpin H1 with the ability to initiate HCRs was designed to contain the TF binding site. The binding between the TF and H1 protected the H1 from being extended by the Klenow fragment. After thermal denaturation, the reserved H1 launched the HCR and the HCR products activated CRISPR/Cas12a to cleave P1 and reduce the β-gal on the sensing interface, and thus the contents of the TFs and the corresponding signals mediated by the catalysis of β-gal showed a correlation. This work was the first attempt at utilizing β-gal for dual-signal transduction. It is a pioneering study to utilize the HCR-CRISPR/Cas12a system for dual-mode TF sensors. It revealed that DNA polymerase protection through the binding of TF and DNA could be applied as a new pattern to develop TF sensors.