Part:BBa_K5242000
ADAR1_p150
1. Biology and Usage
A-to-I editing exists in many metazoan organisms, and ADAR (Double-Stranded RNA-Specific Adenosine Deaminase) is the crucial enzyme that catalyzes the process. The three human ADAR genes give rise to four known isoforms: ADAR1_p150, ADAR1_p110, ADAR2 and ADAR3. Among these isoforms, ADAR1_p150 can shuttle in and out of the nucleus, while ADAR1_p110 and ADAR2 exist in the nucleus. Because mature RNAs exist in the cytoplasm, we choose ADAR_p150 and engineered MCP-ADAR2 to be introduced to yeast cells.
This part is a codon-optimized wild-type hADAR1_p150 gene for yeast expression, with a Flag-tag added at the N-terminal for Western blot. The ADAR1_p150 protein is composed of 3 dsRNA binding domains,a deaminase domain and 2 Z-DNA binding domains. ADAR_p150 can recognize dsRNA and edit base A to base I, which will be recognized as base G. Usually, ADAR1_p150 doesn't work when the double-stranded RNA is well matched, while this deamination process can be accelerated by secondary structure elements like terminal loops, internal loops, bulges and mismatches. Especially, the ADARs can edit the A-C mismatch point in Double-stranded RNA with high specificity, which can be used for RNA editing and RNA sensing. In our design, ADAR_p150 is an exogenous protein introduced into yeast cells to edit our sensor, which contains an A-C mismatch site.The design is shown in Figure 1.
Figure 1.The design of our senser RNA.
2. Experimental Characterization
2.1 Preliminary Experiment
2.1.1 Gibson Assembly and Yeast transformation
Initially, given that ADAR_p150 is an exogenous protein, we selected weaker promoters PDC1 (for homologous recombination) and HXT7 (for free plasmid expression), along with the HHF2 terminator and the ADH1 terminator, respectively, to construct the expression cassette for ADAR_p150.
We used Gibson Assembly to build our plasmid and we cultured transformed E. coli to get more plasmids. We simultaneously tried two methods to express our proteins: Homologous Recombination (pUC) and Free Plasmid Expression (pCEV). Then, we sent the 2 plasmids to the company for sequencing. The result is correct. It indicates that our assembly was successful.
Figure 2 . The sequencing results indicated that our plasmid construction for Free Plasmid Expression was successful.
Figure 3 . The sequencing results indicated that our plasmid construction for Homologous Recombination was successful.
Yeast transformation was done subsequently, and the result of colony PCR is shown below. After that, we preserved our strains on our culture medium.
Figure 4: The yeast Colony PCR result of ADARs. (H: Homologous Recombination, I: Free Plasmid Expression, A1: ADAR1_p150, A2: ADAR2_MCP, P1-P4: pSensor1-4)
2.1.2 Gene expression
After transformation, qPCR and Western blot were done to check whether the transcription and translation processes worked well. Because ADARs may be toxic to yeast cells, we found that the yeast strains with free plasmid expression grew quite slowly. We only conducted the experiments on the strains of Homologous Recombination.
2.1.2.1 qPCR
We used ACT1 as an internal reference for the qPCR experiment. What's more, the transformation of 4 sensor plasmids is done before qPCR. The result is shown below. The mRNA level of ADAR1 is a little lower than ACT1. Because ACT1 is expressed in yeast cells at a low level, we need to use a strong promoter in our future improvement.
Figure 5: The qPCR rusult of ADAR1_p150. After that ,we used the TEF1 promoter to replace the PDC1 promoter to enhance the transcription level. The Relative Expression Level is shown below. The result shown that the TEF1 promoter is effective.
2.1.2.2 Western blot
Figure 6: The Western Blot rusult of ADAR1_p150,which showed that the codon optimized ADAR1_p150 can indeed be normally expressed in yeast.
To verify the expression of ADAR1_p150, we designed and performed Western Blot experiments. We added a Flag-tag to the N-terminus of the protein to facilitate antibody binding, and the results of the experiment are shown in the figure below. A clear band appeared around 170 kDa, which is consistent with the molecular weight of ADAR1_p150, and the successful expression of ADAR1_p150 was verified. The experiment shows that the codon optimized ADAR1_p150 can indeed be normally expressed in yeast.
2.1.3 Edition efficiency analysis
Because our sensor RNAs contain eGFP and eBFP, we introduced our sensor plasmids into transformed cells to indicate editing efficiency.
Our sensor RNA is composed of eGFP, E2A peptide and eBFP. A termination codon is at the end of eGFP. If the sensor RNA is edited by ADAR, the termination codon UAG will be transformed to UGG, which can be translated into tryptophan. E2A is used to cut the long peptide chain into two fluorescent proteins, so that the two fluorescent proteins would not interfere with each other. By measuring the fluorescence intensity and calculating eBFP/eGFP, we can quantify the editing efficiency of ADARs. Our gRNA can complementarily pair with the sensor RNA and become an ADAR-editable A-C mismatch at the termination codon of the sensor RNA.
To make sure our sensor RNA expresses with gRNA at the same time, we loaded them into the same plasmid, which is called pSensor1. Also, we set another 2 groups as negative control and 1 group as positive control. In pSensor2, gRNA is mutated; it can bind with sensor RNA perfectly, without a A-C mismatch. We set up this control group to verify the effect of A-C mismatch on ADAR editing. In sensor 3, gRNA is replaced by another gRNA, which will not form dsRNA with our sensor RNA. We set up this control group to verify the effect of dsRNA on ADAR editing. In pSensor4, sensor RNA is mutated; the terminal codon UAG was already edited to UGG. We set this control group to check if the 2A peptide and downstream eBFP could work normally.
According to our experimental design, the predicted results are as follows: The pSensor1 group, which serves as the experimental group, is expected to exhibit blue fluorescence; the pSensor2 group will not display blue fluorescence because there are no A-C mismatch sites available for editing; the pSensor3 group will not show blue fluorescence due to the lack of double-stranded RNA formation; and the pSensor4 group, because the UAG codon has been edited to UGG, will display blue fluorescence with the highest intensity.
2.1.3.1 LSCM (laser scanning confocal microscope) Analysis
After going through the procedures of transformation, incubation and sample preparation, we put the four groups of samples under the microscope and observed the results as shown in Fig7.
We observed blue fluorescence in both pSensor1 and pSensor4. The result indicated that both our ADAR1_p150 and our sensor RNA could work in yeast cells. However, the blue fluorescent in pSensor1 is much weaker than pSensor4. Therefore, we need to improve our sensing system to enhance its editing efficiency.
Figure 7: The LSCM rusult of ADAR1_p150.
2.1.3.2 Flow cytometry analysis
The Figure.11 has shownd how we processed our raw data, and Figure.12 has shown the result. Unfortunately, for both ADAR1_p150 and ADAR2_MCP, between pSensor1, psensor2 and pSensor3, we did not observe significant differences. While the eBFP/eGFP value of pSensor4 is close to 100%. The result showed that we need to improve the editing efficiency.
Figure 8: The process how we processed our raw data.
Figure 9: The result of Flow cytometry analysis
2.1.4 Subcellular Localization of ADAR1_p150
The dsRNA is mainly formed in the cytoplasm, and the subcellular location of ADARs may have a negative impact on the editing process. Therefore, we set out to investigate the subcellular location of ADARs. We used the GGGGS linker to fuse mScarlet to the C-terminus of ADARs, allowing us to observe the subcellular localization of ADARs under a microscope.
The image revealed that several cells displayed one or more “red bright spots,” likely corresponding to the yeast nucleus, with some fluorescence scattered throughout the cytoplasm. This indicates that ADAR1 is primarily localized to the nucleus, with a minor distribution in the cytoplasm, which could potentially explain the reduced editing efficiency observed in our preliminary experiments.
Figure 10: The Subcellular Localization of ADAR1_p150
2.1.5 ADAR toxicity verification
During pre-experimental cultures, we observed that yeast introduced with ADARs grew more slowly than normal yeast. We suspected that this might be due to the RNA editing activity of ADARs that allowed the modification of key RNAs during yeast growth resulting in the inhibition of yeast growth.To better control the copy number of our genes, we utilized homologous recombination to express two ADAR proteins in yeast using two promoters of different strengths. The groupings in the experiment are shown in Table 1.
| Group | ADAR | Promoter | Expression intensity |
|---|---|---|---|
| B | \ | \ | \ |
| A1 | ADAR1_p150 | PDC1 | Low |
| A2 | ADAR2_MCP | PDC1 | Low |
| T1 | ADAR1_p150 | TEF1 | High |
| T2 | ADAR2_MCP | TEF1 | High |
To determine the optimal initial concentration , we conducted multiple sets of preliminary experiments. The final determined initial concentration was OD600=0.001.The results of the incubation of OD600=0.001 in the pre-experiment are shown in Figure 11, and the final experimental results are shown in Figure 12.
Figure 11.The results of the incubation of OD600=0.001 in the pre-experiment.
Figure 12.The final experimental results.
The both of results showed that the yeast strains with ADAR grew more slowly than the control, that was what we expected. Interestingly, both of results showed that ADAR1_p150 strains with a strong promoter grew faster than those with a weak promoter, We propose two possible explanations:
① The results were accurate, and the relationship between ADAR1_p150 expression intensity and toxicity is complex, requiring further exploration of its metabolic impact in yeast cells. ② After consulting our advisor, it was suggested that long-term storage at -80°C reduces glycerol stock activity, necessitating a longer activation time. To mitigate this, we plan to plate the glycerol stocks on solid medium for activation and select single colonies to repeat the experiment, thereby minimizing interference from refrigeration.
2.1.6 Conclusion of Preliminary Experiment
In summary, the preliminary experiment basically proved that our RNAssay is feasible. ADAR1_p150 is correctly expressed in yeast and play certain functions, but the editing efficiency is relatively low. Subcellular localization experiments show that ADAR1 is mostly located in the nucleus. The toxicity test of ADAR1_p150 proves that ADAR1_p150 does have certain cytotoxicity. In formal experiments, we had a total of two groups involved in testing ADAR1_p150 itself, with one group testing how the editing efficiency of ADAR_p150 could be improved, and the other group testing the specificity of ADAR1_p150 in recognizing similar sequences.
2.2 Editing System Optimation
After obtaining preliminary experimental results, we realized that the editing efficiency of ADAR was relatively low. Therefore, we attempted the following two improvements: 1) replacing the promoter with the stronger TEF1 promoter, and 2) increasing the number of MS2 sites around the binding region, with quantities of 0, 2, and 4 respectively. After optimizing the ADAR editing efficiency, we designed a plasmid induced by xylose expression. By measuring the editing efficiency and the expression level of the target transcript, we can semi-quantitatively detect the expression level of the target transcript.
2.2.1 Promoter Replacement
2.2.1.1 Plasmind Construction and Yeast Transformation
The plasmids are basically the same as pUC19-C1and pUC19-ADAR1-C2. We only replaced the PDC1 promoter with the TEF1 promoter. As before, we used the Gibson Assembly to build our plasmids. The sequencing results of the two plasmids are shown in the Figure13, proving that the plasmid construction was successful.
Figure 13.The Sequencing results
After that, we performed a successful yeast transformation.
To reduce the experimental burden, the effect of the ADAR promoter on the fluorescence intensity will be characterized later together with the pair fluorescence intensity of MS2. This section only shows the results of qPCR.
2.2.1.2 qPCR
To verify whether there was an increase in transcription after switching to a different promoter, we performed qPCR experiments, the results of which are shown in Figure 14. The relative transcript level of ADAR_p150 relative to ACT1 increased significantly.
Figure 14. The relative transcript level of ADAR_p150 relative to ACT1 increased significantly.
2.2.2 Add MS2
2.2.2.1 Plasmid Construction
Literature suggests that adding MS2 sequences on both sides of the binding region can improve ADAR editing efficiency. Therefore, we designed three types of sensors with 0, 2, and 4 MS2 sequences around the binding region. As before, we used the Gibson Assembly to build our plasmids. The sequencing results of the two plasmids are shown in the Figure15, proving that the plasmid construction was successful.
Figure 15.The Sequencing results
After that, we performed a successful yeast transformation.
2.2.2.2 Edition efficiency analysis
We replaced the fluorescent protein due to the relatively weak intensity of the blue fluorescence.We placed the red fluorescence before the stop codon and the green fluorescence after the stop codon. In this way, when sensor RNA is edited, there will be green fluorescence.
We planed to include a side-by-side comparison of the two ADAR proteins at the same time. In our experimental design, we introduced two parts into yeast.The first part is the ADAR protein expression, with four gene types: PDC promoter + ADAR1, PDC promoter + ADAR_MCP, TEF promoter + ADAR1, and TEF promoter + ADAR_MCP. The second part is the sensor, with three types containing 0, 2, or 4 MS2 sequences. The overall design is shown in Table 2. This method allows us to determine the combination with the highest editing efficiency. After construction, we observed these 12 engineered yeast strains using confocal microscopy and flow cytometry to calculate ADAR editing efficiency.
| Group | Promoter | ADARs | the Number of MS2 |
|---|---|---|---|
| 1 | TEF1 | ADAR1_p150 | 0 |
| 2 | TEF1 | ADAR1_p150 | 2 |
| 3 | TEF1 | ADAR1_p150 | 4 |
| 4 | TEF1 | ADAR2_MCP | 0 |
| 5 | TEF1 | ADAR2_MCP | 2 |
| 6 | TEF1 | ADAR2_MCP | 4 |
| 7 | PDC1 | ADAR1_p150 | 0 |
| 8 | PDC1 | ADAR1_p150 | 2 |
| 9 | PDC1 | ADAR1_p150 | 4 |
| 10 | PDC1 | ADAR2_MCP | 0 |
| 11 | PDC1 | ADAR2_MCP | 2 |
| 12 | PDC1 | ADAR2_MCP | 4 |
Figures 16 and 17 clearly show that the addition of MS2 sequences effectively enhances the editing efficiency of the entire system. However, an unexpected observation is that increasing the number of MS2 sequences leads to a decrease in editing efficiency for both TEF-ADAR1 and TEF-ADAR2. This outcome contrasts with the results observed for PDC-ADAR2. We hypothesize that the MS2 sequences may interfere with the transcription process, potentially causing premature termination of the mScarlet-EGFP fusion protein. When ADAR1/2 is driven by the PDC promoter, the number of MS2 sequences correlates with increased binding affinity, leading to enhanced editing efficiency. In contrast, under the control of the TEF promoter, the high expression levels of ADAR1/2 may lead to saturation, making the concentration of sensor RNA a limiting factor. Given that MS2 sequences might increase the likelihood of abortive transcription, a higher number of MS2 sequences could reduce the available concentration of sensor RNA, thereby decreasing editing efficiency.
Figure 16.Confocal Results of Editing Systems Containing Different MS2 sequences
Figure 17.FACs Results of Editing Systems Containing Different MS2 sequences
Furthermore, Figures 18 and 19 highlight that TEF-ADAR2 exhibits the highest editing efficiency, likely attributable to its elevated expression level.
Figure 18.Confocal Results of Editing Systems Containing Different ADAR Types and Promoters
Figure 19.FACs Results of Editing Systems Containing Different ADAR Types and Promoters
2.2.3 Conclusions of System Optimization
Through the above experiments, we demonstrated that switching promoters did increase the expression of the two ADAR proteins, however, although for ADAR2_MCP there was a significant increase in the editing efficiency after switching promoters, for ADAR1_p150 the increase in the editing efficiency by switching promoters was not significant. Therefore, we abandoned the idea of using ADAR1_p150 during the subsequent determination of the change curve of fluorescence intensity with RNA level. In addition, although the addition of the MS2 loop did improve the editing efficiency of the two ADARs, the effect of the amount of MS2 on the editing efficiency was more complex, which may imply that there is still an unknown mechanism in the process of ADAR and RNA action.
2.3 Application for in vivo dynamic monitoring of splice variant
Due to the presence of half identical sequences in the vicinity of the splice heterodimer's junction, a proof-of-concept for the splice heterodimer is somehow equivalent to a characterization of ADAR1_p150's ability to recognize and edit similar sequences.
Knowing that Chk1 and Chk1s are a pair of splice heterodimers, we set up four sets of experiments to examine the potential of ADAR1_p150 to detect splice heterodimers. In the experiments, we applied the same plasmid importing sensorRNA and targetRNA. And we used the TEF1 promoter and 4 MS2 loops. The groupings and pridicted results are shown in Table 3.
As before, we have also included in this section a side-by-side comparison of the two ADAR proteins
| 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 |
2.3.1 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 20,Figure 21, Figure 22 ,Figure 23, proving that the plasmid construction was successful.
Figure 20.The sequencing results
Figure 21.The sequencing results
Figure 22.The sequencing results
Figure 23.The sequencing results
After that, we performed a successful yeast transformation.
2.3.2 Fluoresence Observasion
2.3.2.1 LCFM Analysis
Figures 24 and 25 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 24.The LCFM results of ADAR1_p150
Figure 25.The LCFM results of ADAR2_MCP
2.3.2.2 FACs Analysis
We got nice datas from Flow cytometry analysis, though the results were not exactly as what we expected. The editing efficiency are shown in Figure 26 and Figure 27.
Figure 26.The FACs results of ADAR1_p150
Figure 27.The FACs results of ADAR2_MCP
2.3.3 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 28.IntaRNA Simulation of Binding Energy of Chk1(s)-Chk1(s)
Both ADAR1 and ADAR_MCP demonstrated editing ability in yeast, but the underlying mechanisms are complex. We observed some positive results regarding selective ability, though the outcomes were somewhat unexpected.More experiments are needed. We are also eager to find out more potential of this monitoring system.
3. Reference
Jiang, K. et al. Programmable eukaryotic protein synthesis with RNA sensors by harnessing ADAR. Nature Biotechnology 41, 698-707 (2023).
Savva, Y. A., Rieder, L. E. & Reenan, R. A. The ADAR protein family. Genome Biology 13, 252 (2012).
Sun, J. et al. Cloning and characterization of a panel of constitutive promoters for applications in pathway engineering in Saccharomyces cerevisiae. Biotechnology and bioengineering 109, 2082-2092 (2012).
Martin Mann, Patrick R. Wright, and Rolf Backofen IntaRNA 2.0: enhanced and customizable prediction of RNA–RNA interactions Nucleic Acids Research, 2017, 45 (W1), W435–W439.
Patrick R. Wright, Jens Georg, Martin Mann, Dragos A. Sorescu, Andreas S. Richter, Steffen Lott, Robert Kleinkauf, Wolfgang R. Hess, and Rolf Backofen CopraRNA and IntaRNA: predicting small RNA targets, networks and interaction domains Nucleic Acids Research, 2014, 42 (W1), W119-W123.
Anke Busch, Andreas S. Richter, and Rolf Backofen IntaRNA: efficient prediction of bacterial sRNA targets incorporating target site accessibility and seed regions Bioinformatics, 2008, 24 (24), 2849-56.
Martin Raden, Syed M Ali, Omer S Alkhnbashi, Anke Busch, Fabrizio Costa, Jason A Davis, Florian Eggenhofer, Rick Gelhausen, Jens Georg, Steffen Heyne, Michael Hiller, Kousik Kundu, Robert Kleinkauf, Steffen C Lott, Mostafa M Mohamed, Alexander Mattheis, Milad Miladi, Andreas S Richter, Sebastian Will, Joachim Wolff, Patrick R Wright, and Rolf Backofen Freiburg RNA tools: a central online resource for RNA-focused research and teaching Nucleic Acids Research, 46(W1), W25-W29, 2018.
4. Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1927
Illegal XbaI site found at 2779
Illegal XbaI site found at 2992 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1927
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Illegal BglII site found at 382
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Illegal BglII site found at 2636
Illegal BglII site found at 3152 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1927
Illegal XbaI site found at 2779
Illegal XbaI site found at 2992 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1927
Illegal XbaI site found at 2779
Illegal XbaI site found at 2992
Illegal AgeI site found at 2542 - 1000COMPATIBLE WITH RFC[1000]
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