key: cord-0296861-8vg8myd4 authors: Zhang, Ziheng; Sun, Zhe; Liu, Ji-Long title: Detecting apoptosis via DODO date: 2022-05-14 journal: bioRxiv DOI: 10.1101/2022.05.14.491923 sha: 347b925c30ee226f4f8ef1c14d84d0a0482eb962 doc_id: 296861 cord_uid: 8vg8myd4 The real-time detection of intracellular biological processes by coded sensors has broad application prospects. Here we develop a degron based modular reporting system: the Device of Death Operation (DODO), which can be used to detect a series of biological processes. The DODO system consists of “reporter”, “sensor” and degron. After protease activation and cleavage, the degron will be released from the fluorescent protein and eventually lead to the stabilization of the fluorescent protein. By replacing different “sensors” and “reporters”, a series of biological processes can be reported through different signals. The system can effectively report the existence of TEV. To prove this concept, we successfully apply the DODO system to report apoptosis. In addition, the reporter based on degron will help to design protease reporters other than caspase. The DODO system consists of "reporter", "sensor" and degron. After 23 protease activation and cleavage, the degron will be released from the 24 fluorescent protein and eventually lead to the stabilization of the 25 fluorescent protein. By replacing different "sensors" and "reporters", a 26 series of biological processes can be reported through different signals. 27 The system can effectively report the existence of TEV. To prove this 28 concept, we successfully apply the DODO system to report apoptosis. In 29 addition, the reporter based on degron will help to design protease 30 reporters other than caspase. Signals of many intracellular events are transmitted through the 53 activation of zymogens. A classic case based on zymogen activation is 54 apoptosis, which is performed by caspase 4-5 . Apoptosis has always been 55 the focus of attention because it is related to developmental events, 56 cancer treatment and many diseases [5] [6] . In the past many years, people 57 have developed a series of methods to detect apoptosis. Mainly by 58 detecting the activation of key signaling molecules, such as caspase3. The activation of zymogen plays a fundamental role in many 60 intracellular pathways, especially the report of zymogen activation is 61 very important. Recently, it has been reported that a series of degrons are mainly used 64 to artificially regulate protein degradation 7-9 . A famous example is AID, 65 a powerful tool developed from auxin receptors in in plants 7 . F-box 66 transport inhibitor reaction 1 (TIR1) is a receptor for auxin. By binding to 67 auxin, chimeric E3 ligase (SCF-TIR1) recruits and ubiquitinates the 68 degron 10-11 . This is a natural and rapid method of regulating substrate 69 proteins. Fortunately, the orthologs of TIR1 is only found in plants, which In addition, many proteins have natural unstable motifs at the N-terminal 80 or C-terminal, which may be composed of a few amino acids. Large 81 scale N-segment screening or C-terminal screening can be used to 82 characterize these unstable motifs 14-15 . These results have been 83 confirmed by some natural proteins and can be used to develop a series 84 of tools [16] [17] . This is a suitable element for muting the reporting signal in 85 the control unit. In this report, we introduce the concept of modularity, 86 which combines a reporter, an inducer and a degron into a complete Strategies for developing DODO 96 97 DODO, a modular self-degradation reporting system, consists of three 98 components, a reporter, an inductor and a degron. Among them, the 99 reporter is used to report signals; the inductor is used to monitor specific 100 signals and the degron is used to trigger its own degradation. When 101 there is no target signal in the cell, it will trigger the degradation of 102 reporter gene and make the cell negative. Otherwise, the sensor will cut 103 off the degron, stabilize the reporter and accumulate in the cell, and 104 finally be detected (Fig. 1a ). In this system, the reporter can be 105 determined according to needs, such as fluorescent protein, luciferase 106 or some labels; The inductor depends on the target signal. For example, 107 the apoptosis inductor we used in this study is caspase3 cleavage 108 sequence (DEVDG); For degron, we choose C-degron (RRRG), which 109 has strong degradability 16 . Background noise is usually an important factor that makes it impossible 112 to accurately determine the authenticity of the target signal. Therefore, 113 we first determined the degradation ability of degron. We use 114 fluorescent protein to achieve this. By adding degron to the C-terminal To test whether the DODO system can respond to specific signals, we 129 introduce a TEV cleavage site between the reporter and degron as an 130 inductor, and detect whether the presence of TEV can be reported. TEV 131 has categoric sites and high specificity, and has been widely used in a 132 series of applications including protein purification. The presence of TEV 133 was detected and reported by introducing the cleavage sequence of 134 TEV between the reporter and degron (Fig. 2a) . In order to ensure the consistency of data, we constructed a stable cell cluster. Over time, the cells located in the cell cluster will undergo 171 apoptosis and finally form a cavity (Fig. 4a) . This process is mainly divided into two stages. One is the proliferation can report apoptotic cells in apoptosis stage. We took images of MCF-10A cell clusters on day 6 of 3D culture (Fig. 4b) . 188 environment, and it also shows that the reporting system will not 192 misreport in the stage of cell proliferation. We also imaged MCF-10A cells in the apoptotic state (Fig. 4c) The codable tools for reporting cellular biological processes are of 202 great significance for people to identify the physiological state of cells. In particular, modular tools can provide researchers with a variety of 204 options and reduce application restrictions. In this report, we describe a new degron-based modular reporting provides researchers with wider applicability, which can be assembled 225 into appropriate tools according to their needs and replaced simply. In conclusion, we develop DODO as a codable and degron-based 228 modular protease reporting system in this study. By changing the 229 components of the system, different protease signals can be reported 230 in a variety of reporting ways. 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