A new method for sensitive detection of microphthalmia-associated transcription factor based on “OFF-state” and “ON-state” equilibrium of a well-designed probe and duplex-specific nuclease signal amplification

• A DSN based amplification strategy for MITF detection was proposed.
• This strategy employed a probe changed in equilibrium of “ON-state” and “OFF-state”.
• The DNA1 is well-designed by modifying with 2-OMe-RNA to prevent meaningless digestion.
• The detection limit of the strategy can be lowered to 1.1 pM.

Herein, we report a new method which the “stage change” of DNA1 and the cleavage feature upon recycled recognition by DSN was used to detect target microphthalmia-associated transcription factor (MITF) in cell nuclear extracts. In this method, we employed a well-designed DNA1 as a recognition element which can converting in two states, “ON-state” and “OFF-state”. Also, the DNA1 is modified with 2-OMe-RNA on hybridization part in the “OFF-state” to prevent meaningless digestion. By taking advantage of the high amplification efficiency of DSN-aided recycling, high sensitivity of MITF is realized with a detection limit as low as 1.1 pM, which is superior or comparable to that of the reported literature. This method is a fast and easy-to-use one-pot method that was carried out in a tube, while being quantitative and applicable to other proteins in the sample without involving complicated procedures or sophisticated instrumentations. It is very simple and fast, needing only mixing of DNA1, DNA2, MITF and DSN enzyme and incubating within 60 min, which is in the homogeneous solution, and not requiring separation and troublesome procedures. Considering the superior sensitivity and specificity, as well as the multiplex and simple-to-implement features, this method holds great promise of becoming a routine tool for simultaneously quantitative analysis of multiple proteins and supplies valuable information for transcription factor-based early stage cancer diagnosis.