We constructed a RNA sensor to transform RNA signals in vivo and built RNA SenSor based Analysis system in Yeast(RNAssay), which is mainly composed of an enzyme ADAR and a sensor RNA. In total, 27 new basic parts and 10 new composite parts are used for our project and 31 of them were well characterized(see Parts ). To make our RNA sensor useful and reliable, we made great efforts to carry out a series of experiment on:

1. Plasmid and yeast strain construction

2. Characterization of exogenous ADAR(adenosine deaminases acting on RNA) in Saccharomyces cerevisiae

3. Characterization of RNA sensor performance in Saccharomyces cerevisiae

我们构建了一个RNA传感器用于转换活体RNA信号，并建立了基于RNA传感器的酵母RNA分析系统（RNAssay），其主要由一种ADAR酶和传感RNA组成。总共使用了27个新基础元件和10个新复合元件，其中31个元件得到了充分的表征（参见Parts）。为了使我们的RNA传感器实用且可靠，我们针对以下方面开展了一系列实验：

1. 质粒和酵母菌株的构建

2. 外源ADAR（腺苷脱氨酶）在酿酒酵母的表征

3. RNA传感器在酿酒酵母中的性能表征

By this way, we successfully measured the basic imformation and application potential of our system, including:

1. The expression profile of ADAR1_p150 & ADAR2(DD)E488Q–MCP in S.cerevisiae

2. The subcellular location of ADAR1_p150 & ADAR2(DD)E488Q–MCP in S.cerevisiae

3. The cytotoxicity of ADAR1_p150 & ADAR2(DD)E488Q–MCP in S.cerevisiae

4. How different fluorescence reporting systems influence our RNA sensor

5. How different numbers of MS2 sequences on sensor RNA influence our RNA sensor

6. How expression level of ADAR1_p150 & ADAR2(DD)E488Q–MCP influence our RNA sensor

7. The ability of our RNA sensor to distinguish different splice variants

8. How the quantity of target transcript and A-C mismatch influence our RNA sensor

通过这些实验，我们成功测量了系统的基本信息和应用潜力，包括：

1. ADAR1_p150 和 ADAR2(DD)E488Q–MCP 在酿酒酵母中的表达谱

2. ADAR1_p150 和 ADAR2(DD)E488Q–MCP 在酿酒酵母中的亚细胞定位

3. ADAR1_p150 和 ADAR2(DD)E488Q–MCP 在酿酒酵母中的细胞毒性

4. 不同荧光报告系统对RNA传感器的影响

5. 传感RNA上不同数量的MS2序列对RNA传感器的影响

6. ADAR1_p150 和 ADAR2(DD)E488Q–MCP 的表达水平对RNA传感器的影响

7. RNA传感器区分不同剪接异构体的能力

8. 目标转录本的数量和A-C错配对RNA传感器的影响

Totally, we successfully constructed 26 different plasmids and 28 different yeast strains (details are shown in Results ). For each plasmid or yeast strain, colony PCR of E.coli, gene sequencing and colony PCR of S.cerevisiae were performed to ensure successful construction.

我们总共成功构建了26种不同的质粒和28种不同的酵母菌株（详情见Results）。为了确保每种质粒或酵母菌株构建成功，我们进行了大厂杆菌菌落PCR、基因测序以及酿酒酵母菌落PCR。

We used E.coli as a platform to select and store the plasmid we constructed. To make sure the plasmids are successfully transformed into cells, we use colony PCR and gel electrophoresis to detect whether the cell contains our plasmids. A sequence about 1kbp long on the genes of interest were chosed as markers. And only selected colonies are further prepared for gene sequencing.

我们使用大肠杆菌作为筛选和存储构建质粒的平台。为了确保质粒成功转入细胞，我们使用菌落PCR和凝胶电泳来检测细胞是否含有我们的质粒。选择了目标基因上约1kbp长的序列作为标记。仅选中的菌落才被进一步准备进行基因测序。

Gene sequencing is carried out as the golden standard for a successful constructed plasmid. We provided sequencing primers for genes of interest and Sanger sequencing was carried out by company TsingKe. Only when the sequencing result matches our design will the plasmid be stored for further use.

基因测序是成功构建质粒的黄金标准。我们为目标基因提供了测序引物，测序由公司TsingKe采用Sanger测序法完成。只有当测序结果与设计相符时，质粒才会被存储用于后续实验。

Sequencing Result Group1: pCEV-ADAR1, pCEV-ADAR2-7, pCEV-ADAR2-8, pUC19-ADAR1-C2, pUC19-ADAR2_MCP-C2-1, pUC19-ADAR2_MCP-C2-2, pSensor1_URA_Pre

基因测序第一组：pCEV-ADAR1， pCEV-ADAR2-7， pCEV-ADAR2-8， pUC19-ADAR1-C2， pUC19-ADAR2_MCP-C2-1， pUC19-ADAR2_MCP-C2-2，pSensor1_URA_Pre

Sequencing Result Group2: pSensor2_URA_Pre-1, pSensor2_URA_Pre-2, pSensor3_URA_Pre-1, pSensor3_URA_Pre-2, pSensor4_URA_Pre-1, pSensor4_URA_Pre-2, pLocate-ADAR1

基因测序第二组：pSensor2_URA_Pre-1，pSensor2_URA_Pre-2，pSensor3_URA_Pre-1，pSensor3_URA_Pre-2，pSensor4_URA_Pre-1，pSensor4_URA_Pre-2，pLocate-ADAR1

Sequencing Result Group3: pLocate-ADAR_MCP, TEF-C1, TEF-C2-ADAR1, TEF-C2-ADAR_MCP, pSensor-MS2-0, pSensor-MS2-2, pSensor-MS2-4

基因测序第三组：pLocate-ADAR_MCP，TEF-C1，TEF-C2-ADAR1，TEF-C2-ADAR_MCP，pSensor-MS2-0，pSensor-MS2-2，pSensor-MS2-4

Sequencing Result Group4: Xyl-pSensor-HO-C1, Xyl-pSensor-HO-C2, pSensor-target_Chk1-Chk1, pSensor-target_Chk1-Chk1s, pSensor-target_Chk1s-Chk1, pSensor-target_Chk1s-Chk1s

基因测序第四组：Xyl-pSensor-HO-C1，Xyl-pSensor-HO-C2，pSensor-target_Chk1-Chk1，pSensor-target_Chk1-Chk1s，pSensor-target_Chk1s-Chk1，pSensor-target_Chk1s-Chk1s

For the same reason above, we carried out colony PCR of S.cerevisiae to make sure our plasmids are successfully transformed. Similarly, a sequence about 1kbp long on the genes of interest were chosed as markers. Since almost all the yeast strains we aimed to construct contains two exogenous genes, we usually carried out colony PCR for two genes at once to make the result reliable.

基于同样的理由，我们对酿酒酵母进行了菌落PCR，以确保质粒成功转入。类似地，选择了目标基因上约1kbp长的序列作为标记。由于几乎所有我们要构建的酵母菌株都包含两个外源基因，通常我们同时对两个基因进行菌落PCR，以使结果更可靠。

Since ADAR does not exist naturally in yeast, we have to introduce this enogenous gene into S.cerevisiae. To figure out whether ADAR1_p150 & ADAR2(DD)E488Q–MCP can be expressed well in our yeast strain, we carried out qPCR experiments. The result shows that both ADARs can be expressed by S.cerevisiae, while the transcription level is relatively low. We also compared the expression level of ADARs with different promoters(TEF1/PDC1). Result showed that after switching to a stronger promoter, the expression level of ADARs significantly increased.

由于 ADAR 在自然情况下并不存在于酵母中，我们需要将该外源基因引入 S. cerevisiae。为了确定 ADAR1_p150 和 ADAR2(DD)E488Q–MCP 能否在我们的酵母株中良好表达，我们进行了 qPCR 实验。结果表明，S. cerevisiae 能够表达这两种 ADAR，但转录水平相对较低。我们还比较了使用不同启动子（TEF1/PDC1）时 ADAR 的表达水平。结果显示，在更强的启动子下，ADAR 的表达水平显著提高。

We also wondered whether the translation process goes wrong, so we carried out Western Blot experiment for both ADARs. The tags bound to primary antibodies for both ADAR1 and ADAR2 were FLAG.GADPH (30 kDa) was selected as the intracellular reference protein. ADARs with different promoters(TEF1/PDC1) were all detected. We successfully confirmed the expression of ADAR1_p150 with two promoters but failed to catch ADAR2(DD)E488Q-MCP, which is against to our other experiment result. Some hypothesis to explain the result are proposed in Results

我们还想知道翻译过程是否出错，因此对这两种 ADAR 进行了 Western Blot 实验。ADAR1 和 ADAR2 的一级抗体标记为 FLAG。GAPDH（30 kDa）被选作细胞内参考蛋白。不同启动子（TEF1/PDC1）下的 ADAR 都进行了检测。我们成功验证了使用两种启动子的 ADAR1_p150 的表达，但未能捕捉到 ADAR2(DD)E488Q-MCP，这与我们其他实验结果相悖。关于这个结果，我们提出了一些假设来解释，具体见Results

In our design, to better coordinate with sensor RNA, ADARs are expected to be located in the cytoplasm instead of cytoblast. To figure out the subcellular localization of ADAR1_p150 and ADAR2(DD)E488Q-MCP, we fused our ADAR with a red fluorescence protein: mScarlet, which were then checked under confocal fluorescence microscope. Our results suggested that ADAR1 is predominantly nuclear, with some cytoplasmic distribution while ADAR2_MCP exhibited a clear exclusion of fluorescence from the nuclear region, indicating predominant cytoplasmic localization.

为了更好地与传感 RNA 协同工作，我们希望 ADARs 定位于细胞质，而非细胞核。为了确定 ADAR1_p150 和 ADAR2(DD)E488Q-MCP 的亚细胞定位，我们将 ADAR 与红色荧光蛋白 mScarlet 融合，并在共聚焦荧光显微镜下进行观察。结果表明，ADAR1 主要位于细胞核，但也有少量分布在细胞质中，而 ADAR2_MCP 的荧光则明确排除在核区，表明其主要位于细胞质。

Through many times of culturing, we noticed that ADARs are slightly toxic to S.cerevisiae and we further carried out follow-up experiments to measure the cytotoxicity more precisely. Experiments were carried out with adequate control groups. Results showed that both ADAR1_p150 and ADAR_MCP inhibit yeast growth, but our current experiments cannot fully explain the relationship between ADAR expression intensity and its toxicity. It is necessary to follow our advisor's advice, eliminate strain activity interference from refrigeration, and conduct new experiments to clarify this relationship.

通过多次培养，我们注意到 ADARs 对 S. cerevisiae 略有毒性，因此进一步开展了后续实验以更精确地测量其细胞毒性。实验中使用了充分的对照组。结果表明，ADAR1_p150 和 ADAR_MCP 均抑制了酵母的生长，但我们当前的实验尚不能完全解释 ADAR 表达强度与其毒性之间的关系。我们需要根据导师的建议，排除冷藏干扰菌株活性，开展新实验以阐明这种关系。

We tried different fluorescence reporting system: one with eGFP in the upstream while eBFP in the downstream, and another with mScarlet in the upstream while eGFP in the downstream. By comparing two different reporting system by flow cytometry and confocol results, we found that mScarlet is not only more visible than eBFP under confocol fluorescence microscope but also exhibited better cell clustering under flow cytometry, suggesting mScarlet-eGFP reporting system is better.

我们尝试了两种不同的荧光报告系统：一个在上游有 eGFP，而下游为 eBFP，另一个则是上游 mScarlet， 下游 eGFP。通过流式细胞仪和共聚焦显微镜的结果对比，我们发现 mScarlet 不仅在共聚焦显微镜下比 eBFP 更明显，在流式细胞仪下也展现了更好的细胞分群效果，表明 mScarlet-eGFP 报告系统效果更好。

It is reported that adding MS2 sequences can increase the editing efficiency of ADARs. We wanted to confirm this in our system and select a relevant proper number of MS2 sequence. Therefore, we measured the editing efficiency of ADAR by flow cytometry, which is defined by $\frac{number~of~cells~emiting~two~fluorescence}{number~of~cells~emiting~only~red~fluorescence}$. Results showed that adding MS2 sequences do increased the editing efficiency. But more MS2 sequence is not always better.

据报道，添加 MS2 序列可以提高 ADAR 的编辑效率。我们想在自己的系统中验证这一点，并选择合适的 MS2 序列数量。因此，我们通过流式细胞仪测量了 ADAR 的编辑效率，即$\frac{发射两种荧光的细胞数量}{只发射红色荧光的细胞数量}$。结果显示，添加 MS2 序列确实提高了编辑效率，但更多的 MS2 序列并不总是更好。

As shown in Fig. 45, we also compared the editing efficiency of ADARs with different promoters, namely, with different expression levels. Result showed that despite the significant difference in transcription level, the editing efficiency didn't increase as much, suggesting that the amount of ADAR is not the key to increase the activity of our system.

如图45，我们还比较了使用不同启动子（即不同表达水平）时 ADAR 的编辑效率。结果显示，尽管转录水平有显著差异，但编辑效率并未相应提高，表明 ADAR 的数量并非提高我们系统活性的关键因素。

As key information for application for in vivo monitoring of splice variants, the ability of RNAssay to distinguish different splice variants were measured by four control groups. Chk1-Chk1s means the sensor is designed for Chk1 while used for targeting Chk1s. Results showed that RNAssay do possess the ability to distinguish different splice variants.

作为用于体内监测剪接异构体的关键信息，我们通过四个对照组测量了 RNA 传感器区分不同剪接变体的能力。Chk1-Chk1s 表示该传感器基于Chk1设计，但用于靶向 Chk1s。结果显示，我们的RNA 传感器确实具有区分不同剪接异构体的能力。

We utilized xylose-inducible promoter to express different quantities of target transcript, which is precisely measured through qPCR. qPCR and flow cytometry were carried out at the same time to make sure all samples are treated with xylose for same period. Although no clear relationship was observed between the target transcript concentration due to the unsuccessful inducement, the result demonstrated that unlike the normal promoter-case, under low expression level of the target transcript, little fluorescence signals can be detected. This suggested that the fluorescence signals were activated by the target transcript, which is important for our RNA sensor.

我们利用木糖诱导型启动子来表达不同数量的目标转录本，其浓度通过 qPCR 精确测量。qPCR 和流式细胞术同时进行，以确保所有样品都经过相同时间的木糖处理。尽管由于诱导失败，靶标转录本浓度没有显示出明显的关系，实验结果表明，与正常启动子的情况不同，在靶标转录本表达水平较低的情况下，几乎检测不到荧光信号。这表明荧光信号由靶标转录本激活，这对我们的 RNA 传感器系统来说非常重要。

In pre-experiment, we also tried to analyze the influence of A-C mismatch. In Fig. 49, first two columns represent systems with and without A-C mismatch. However, due to the poor editing efficiency for the system used in pre-experiment, no reliable conclusion can be made.

在预实验中，我们还尝试分析了 A-C 错配的影响。在图48中，前两列分别代表有和没有 A-C 错配的系统。然而，由于预实验中使用的系统编辑效率较低，无法得出可靠的结论。