We designed three trigger sequences that can complement each other and pair to form an new open sequence, as well as a switch sequence that can be combined with the open part of trigger sequence (Figure 1). Under normal circumstances, the switch sequence, due to its high proportion of palindromic sequences, forms a spiral ring that prevents downstream gene transcription (Figure 2); The trigger open sequence can bind to the switch and open the spiral ring, thereby promoting transcription (Figure 2). Through this structure, we can ensure that anti-tumor proteins can be synthesized only when the three trigger sequences are simultaneously expressed, that is, they can meet all three tumor microenvironment conditions simultaneously.

Subsequently, we identified low oxygen, low pH, and high lactate tumor microenvironment conditions as control factors and selected corresponding promoters (lactate (pLldR), pH (pCadc), and hydrogen (pPepT)), which were constructed at the front end of three trigger sequences as gate system control elements. At the same time, the target protein iRGD was constructed after the switch sequence as an expression element in the gate circuit system. (Figure 3).

We connected the LUC reporter gene downstream of three promoters to verify their expression ability. We transformed the constructed expression vector into DH5 α Strain used to amplify LUC reporter genes. Subsequently, we conducted a luciferase reporter experiment to characterize the effects of the three promoters in vitro. Figure 4 shows the normalized fluorescence intensity under lactate, pH and hypoxia induction. According to Figure 4A, we can clearly see that with the continuous increase of pH value from 5.6 to 7.6, the fluorescence intensity shows a state of first increasing and then decreasing, and reaches its peak (145.5) at pH around 6.5. The pH range of this peak is very similar to the pH value of the malignant tumor microenvironment (6.5-6.9). Meanwhile, in Figure 4B, it can be seen that the concentration of lactic acid is almost proportional to the fluorescence intensity. Even, the fluorescence intensity at a concentration of 10mM of lactic acid is about a thousand times higher than that at a concentration of 0mM. Finally, for the hypoxic experiment, we established two groups, as shown in Figure 4C. One group represents the oxygen concentration in the normal environment (21%), and the other group represents the simulated oxygen concentration in the tumor microenvironment (approximately 1.3%). The results showed that in an hypoxic environment, the fluorescence intensity was 193.5, which is about twice that of a normal environment (79.5). All the above results indicate that the three promoters have the ability to correspond to the tumor microenvironment and initiate expression programs.

To verify the in vitro secretion ability of CDD-iRGD protein, we added a 6-his label before the switch sequence and CDD-iRGD sequence. The expression of CDD-iRGD protein was determined by Western blot assay.

To confirm that the gate circuit expression system we constructed can function in real tumor environments, we transformed the trigger and switch plasmids into DH5 α In cells and co cultured with tumor cells. After co culturing for a period of time, the survival rate of tumor cells and the destruction of tumor spheres were tested.