Part:BBa_K5242025
sensorRNA_Chk1s
1. Introduction
This part is to assay the variant of chk1. When the target RNA is present, the sensor RNA is edited by ADAR, enabling the translation process of downstream genes. This part is only used for the detection of splice isomers.
Figure 1.The function of Sensor_Chk1s
2. Design
2.1 Repoters
A schematic of the part is shown in Figure 1. In it, green fluorescent protein and red fluorescent protein are reporter genes, in which green fluorescent protein indicates the edited sensor RNA while red fluorescent protein indicates the total sensor RNA, and the editing efficiency of ADAR can be derived by calculating the ratio of the two fluorescence intensities. In practice, these two reporter genes can be replaced with other genes.
Figure 2.The diagram of Sensor_Chk1
2.2 ogRNA
The sequence of ogRNA is derived from the complementary sequence of the target RNA, which has one of the UGGs changed to a UAG, and 4 MS2 loops used to recruit ADARs gives the sequence of ogRNA. This site triggers an A-C mismatch and is the core of the ogRNA. When working, the ogRNA will be complementary paired with the target RNA, and with MS2 recruitment, the ADARs will mutate the base A at the mismatch site to I, which is regarded as G, allowing downstream translation to proceed normally.
2.3 2A Peptides and GSG linker
The 2A peptides is a small, self-cleaving sequence derived from the foot-and-mouth disease virus (FMDV). The 2A peptide works by causing a ribosome to skip the final glycine of the 2A sequence, leading to a cleavage event that separates the proteins encoded upstream and downstream of the 2A sequence. We use E2A and LV2A to separate two fluorescent proteins and intermediate peptides. The GSG linker is used to link the 2A peptides with upstream peptides.
3. Experimental Characterization
3.1 Application of sensor RNA for splice isomer detection
Knowing that Chk1 and Chk1s are a pair of splice heterodimers, we set up four sets of experiments to examine the potential of ADARs to detect splice heterodimers. In the experiments, we applied the same plasmid importing sensor RNA and target RNA. And we used the TEF1 promoter and 4 MS2 loops. The groupings and predicted results are shown in Table 1.
| Group | the Target of Sensor RNA | the Transcribed RNA | Green Fluorescence |
|---|---|---|---|
| kk | Chk1 | Chk1 | Yes |
| ks | Chk1 | Chk1s | No |
| sk | Chk1 | Chk1s | No |
| ss | Chk1s | Chk1s | Yes |
3.1.1 Plasmid Construction and Yeast Transformation
As before, we used the Gibson Assembly to build our plasmids. The sequencing results of the two plasmids are shown in the Figure 3,Figure 4, Figure 5,Figure 6, proving that the plasmid construction was successful.
Figure 3.The sequencing results
Figure 4.The sequencing results
Figure 5.The sequencing results
Figure 6.The sequencing results
After that, we performed a successful yeast transformation.
3.1.2 Fluoresence Observasion
3.1.2.1 LCFM Analysis
Figures 7 and 8 show the fluorescence microscopy observations. However, contrary to the expected results, for ADAR1, all four groups showed green fluorescence, while for ADAR2, all but the last group showed green fluorescence. We decided to use flow cytometry to count the percentage of each group that showed green fluorescence.
Figure 7.The LCFM results of ADAR1_p150
Figure 8.The LCFM results of ADAR2_MCP
3.1.2.2 FACs Analysis
We got nice data from Flow cytometry analysis, though the results were not exactly as what we expected. The editing efficiency are shown in Figure 9 and Figure 10.
Figure 9.The FACs results of ADAR1_p150
Figure 10.The FACs results of ADAR2_MCP
3.1.3 Conclusions of in vivo dynamic monitoring of splice variant
According to the datas, we came to some conclusions:
①ADAR2_MCP has the higher edit efficiency at about 80-95%, but this causes no evidence of selectivity. So we couldn't evaluate whether our sensor function well or not.
②The extremely high efficiency of TA2 in yeast shows the potential of becoming a powerful gene edit tool in yeast.
③ADAR1_p150 has the edit efficiency >15% according to the datas from our Optimization group, which is significantly higher than the stop codon readthrough or off target effect (edit efficiency is around 5%). So we could infer that TA1 has a strong selective edit effect for pSensor-Chk1 with Target-Chk1. But TA1 only has the edit effect on pSensor-target_Chk1-Chk1, that was beyond our expectations.
④Design of Sensor is important for the selectivity of ADAR. After the experiment, we put the sequences into IntaRNA and predict the binding energy and binding area of sensor and its corresponding target. According to the results differences, we inferred that the sensor might not have more than one potential binding area and the base around the A-C mismatch might be strictly complementary pairing (not pairing like CAU-AUC).
Figure 16.IntaRNA Simulation of Binding Energy of Chk1(s)-Chk1(s)
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1454
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 2210
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 175
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