Initially regarded as the intermediary between DNA and proteins, RNA is not only the messenger of genetic information(mRNA, tRNA), but also the catalyzer of specific reactions(ribozyme), the inducer of gene disruption(siRNA, miRNA), and the regulator of important biological process(snoRNA, snRNA .etc). Furthermore, hypothesis has been made that RNA might be the primordial molecule of life that built the great RNA world, highlighting the significance of RNA$^1$. So conclusions can be made that RNA not only carries the information of genes, but also the fate of cells, the state of organisms, and even the origin of life. Understanding RNA is an inevitable step for understanding life, and many groups have dived into the research of spatiotemporal transcriptomics$^2$.

RNA起初被视为DNA和蛋白质之间的桥梁，它不仅是遗传信息的信使（mRNA，tRNA），还是特定反应的催化剂（核酶），基因干扰的诱导者（siRNA，miRNA），以及重要生物过程的调节者（snoRNA，snRNA等）。此外，有假说认为RNA可能是构建了RNA World的原始生命分子，这些都凸显了RNA的重要性$^1$。因此，我们可以说：RNA不仅承载着基因的信息，还承载着细胞的命运、生物体的状态，甚至生命的起源。理解RNA是理解生命的必经之路，许多课题组已经开始关注spatiotemporal transcriptomics的相关研究$^2$。

However, DNAs are stable enough to be sequenced, and proteins have many functions or properties that could be observed, only RNAs，which are easy to degrade and no properties more than sequence information, remained hard to detect until now. Therefore, RNA-level sensing provides a significant window into the black-box process from genotype to phenotype in organisms. Developing molecular sensing tools at the RNA level to monitor gene expression in space and time helps understand the complex regulatory mechanisms within cells and is fundamental for the design and control of engineered life systems.

我们知道，DNA足够稳定，可以进行测序；蛋白质也有许多可观察的功能或特性。但直到现在，RNA仍然难以检测的。因为它们容易降解，除了序列信息外没有其他特性。因此，RNA水平的检测为从基因型到表型的黑箱过程提供了一个重要的观察窗口。开发RNA水平的分子检测工具，以在空间和时间上监测基因表达，有助于理解细胞内复杂的调控机制，并且对于设计和控制工程生命系统至关重要。

Currently, available RNA detection methods include qPCR, RNA-seq, fluorescence in situ hybridization (FISH), and type III-E CRISPR (Table 1). They all have their own advantages and disadvantages. Among these, qPCR has strong specificity but requires RNA extraction, which is a cumbersome process with long detection times$^3$, and it necessitates cell lysis, which prevents in situ detection; RNA-seq targets the entire transcriptome for sequencing, which lacks specificity, leading to higher detection costs and subsequent data processing expenses$^4$; FISH offers strong specificity and high spatial resolution, but probe design is complex and lacks genetic programmability, making it difficult to regulate after RNA detection$^5$; type III-E CRISPR needs Cas protein, which is too large to be a perfect tool.

目前，可用的RNA检测方法包括qPCR、RNA-seq、荧光原位杂交(FISH)和III-E型CRISPR（表1）。它们各有优缺点。其中，qPCR具有强大的特异性，但需要RNA提取，这是一个繁琐且耗时的过程，而且它需要细胞裂解，这就无法进行原位检测；RNA-seq针对整个转录组进行测序，缺乏特异性，导致更高的检测成本和后续数据处理费用；FISH提供强大的特异性和高空间分辨率，但探针设计复杂且缺乏遗传可编程性，使得RNA检测后难以进行调控；III-E型CRISPR需要Cas蛋白，而这种蛋白体积过大，不能成为完美的工具。

So, how to detect RNA signals in a way which has strong specificity, low costs, short time and broader applications? Then it will come to our project RNAssay.

After reading through many scientific papers and discussing for a long time, we finally come to a promising RNA recognition and editing system based on deaminases. We based our research on two back-to-back articles: Modular, programmable RNA sensing using ADAR editing in living cells and Programmable eukaryotic protein synthesis with RNA sensors by harnessing ADAR$^{6,7}$.

经过阅读大量科学论文和长时间的讨论，我们终于得出了一个基于脱氨酶的有前景的RNA识别和编辑系统。我们的研究基于两篇连续的文章：Modular, programmable RNA sensing using ADAR editing in living cells 和 Programmable eukaryotic protein synthesis with RNA sensors by harnessing ADAR$^{6,7}$.

ADAR (Adenosine Deaminase Acting on RNA), which is widely present in mammals, can specifically recognize A-C mismatched base pairs on double-stranded RNA and achieve high-precision A to I (adenine to inosine) editing. Using this feature, artificial RNA can be designed to target any endogenous RNA in cells and form hybrid double-stranded RNA regions. By introducing a UAG stop codon on the artificial RNA and an ACC codon on the endogenous RNA to form a single base mismatch, ADAR enzymes are recruited to initiate their editing activity. The resulting UIG codon is usually recognized by the ribosome as UGG (non-stop codon), thereby initiating the translation of downstream transcripts. And using downstream transcripts that produce a signal we could detect will show information of the target RNA we want to know. Utilizing this concept, highly programmable RNA sensing and regulation can be achieved (Figure 1). This design concept has already been validated in mammals.

ADAR（作用于RNA的腺苷脱氨酶）在哺乳动物中广泛存在，能够特异性识别双链RNA上的A-C错配碱基对，并实现高精度的A到I（腺嘌呤到肌苷）编辑。利用这一特性，可以设计人工RNA靶向细胞中的任何内源RNA，并形成杂交双链RNA区域。通过在人工RNA上引入UAG终止密码子，并在内源RNA上引入ACC密码子形成单碱基错配，ADAR酶被招募并对碱基进行编辑。产生的 UIG 密码子通常被核糖体识别为 UGG (非终止密码子)，从而启动下游转录本的翻译。通过使用产生我们可以检测的信号的下游转录本，将显示我们想要了解的目标RNA的信息。利用这一概念，可以实现高度可编程的RNA感应和调控（图1）。目前，这一设计概念已在哺乳动物中得到验证。

Simply put, this system could detect a specific RNA, and then produce a signal we want to see. When we want to detect a specific RNA(target RNA) in cells, we will transfect a plasmid with designed sensor into the cells. Only the gene in the upstream of the sensor could express normally. If the target exsists, the mRNA of sensor transcript will pair with the target. In the middle of the sensor, a stop codon UAG pairing to codon ACC in target, and this will make a A-C mismatch. ADAR will be recruited when the dsRNA formed and ADAR specifically edit the mismatched A-C to I-C . Stop codon UAG will be edited to UGG(non-stop codon), both upstream and downstream genes could express normally. Then we could see one upstream signal and one downstream signal. Without target, we could only see upstream signal.

简而言之，这个系统可以检测特定的RNA，然后产生我们想要看到的信号。当我们想要在细胞中检测特定的RNA（目标RNA）时，我们会将带有设计好的传感器的质粒转染到细胞中。只有传感器上游的基因能正常表达。如果目标RNA存在，传感器的mRNA转录本将与目标配对。在传感器的中间，有一个终止密码子UAG与目标中的密码子ACC配对，这会形成一个A-C错配。当双链RNA形成时，ADAR会被招募，ADAR特异性地将错配的A-C编辑为I-C。终止密码子UAG将被编辑成UGG（非终止密码子），上游和下游的基因都能正常表达。然后我们就能看到一个上游信号和一个下游信号。如果没有目标RNA，我们只能看到上游信号。

Saccharomyces cerevisiae is an important model organisms in molecular bioloby, cellular biology and synthetic biology, widely used for basic scientific research and biomanufacturing. Reading the transcriptional information helps remoulding the cell factory for better performance. However, there still lacks an useful RNA sensor that can be easily applied in vivo.

酿酒酵母（Saccharomyces cerevisiae）是分子生物学、细胞生物学和合成生物学中的重要模式生物，广泛应用于基础科学研究和生物制造。读取转录信息有益于重塑细胞工厂，从而获得更好的性能。然而，目前仍然缺乏一种可以轻松应用于体内的有效RNA传感器。

The RADAR system has not yet been introduced into yeast so we decided to construct this new system in yeast.

该RADAR系统尚未引入到酵母中，因此我们决定在酵母中构建这一新系统。

Also, there were few research about the activity of adar in yeast. ADAR system has potential for broader applications. And no one has constructed this system in yeast. We decided to do this project for making contributions to the development of RNA detecting technology as well as helping our friends to do experiments more easily. Although, we admitted that it was really a great challenge for us.

目前，关于ADAR在酵母中的活性研究很少，而且还没有人在酵母中构建过这个系统。这说明ADAR系统具有更广泛研究价值和应用潜力。我们决定开展这个项目，以期为 RNA 检测技术的发展做出贡献，同时帮助研究者更轻松地进行实验。尽管如此，这对我们来说或是一个巨大的挑战。

Because various effector genes could be insert to the downstream of UAG stop codon, the system can perform multiple functions: if effector genes are reporter genes such as fluorescent proteins, the target RNA content in living cells can be quantitatively detected in real time based on fluorescence intensity; if effector genes are specific trans-regulatory elements, gene expression based on the expression status of a single gene can be controlled to establish feedback pathways; and when multiple RNA sensors are used as signal inputs, diverse outputs and controls can be achieved through logical gate operations and combinations of different downstream effector proteins. Compared to currently reported RNA sensing methods, this system offers lower detection limits and higher programmability, while the overall system is relatively small, only 2~3kb, allowing multiple loadings on a single plasmid.This system has potential for a widely use.

由于各种效应基因可以被插入到UAG终止密码子的下游，该系统可以执行多种功能：如果效应基因是报告基因，如荧光蛋白，则可以根据荧光强度实时定量检测活细胞中的目标RNA含量；如果效应基因是特定的反式调控元件，可以基于单个基因的表达状态来控制基因表达，从而建立反馈通路；当使用多个RNA传感器作为信号输入时，可以通过逻辑门操作和不同下游效应蛋白的组合来实现多样化的输出和控制。与目前报道的RNA感应方法相比，该系统具有更低的检测限和更高的可编程性，同时整个系统相对较小，仅2-3kb，允许在单个质粒上进行多重装载。这个系统具有广泛应用的潜力。

There are four applications we thought the most promising, and some of them we also tried to prove the concept in our project.

我们认为有三个应用最有前景，其中一些我们还在我们的项目中尝试进行了概念验证。

As shown in Figure 3, to achieve in situ detection of multigene expression, multiple mRNA signals need to be processed simultaneously while maintaining orthogonality. We propose handling multiple signals through methods such as signal cascading, signal oscillation, and protein logic gates. For instance, by constructing four exogenous controlled gene expression cassettes and integrating them into the yeast genome, we can design an ADAR-based sensing system to achieve signal cascading or oscillation through combinations of trans-regulatory elements, or by constructing logical pathways using protein logic gates, with fluorescence as the final output signal. The relationship between output signals and gene expression can be observed, mathematical models can be constructed, and process design parameters can be optimized.

如图3所示，为了实现多基因表达的原位检测，需要同时处理多个mRNA信号，同时保持正交性。我们提出通过信号级联、信号振荡和蛋白质逻辑门等方法来处理多重信号。例如，通过构建四个外源控制的基因表达盒并将其整合到酵母基因组中，我们可以设计一个基于ADAR的感应系统，通过反式调控元件的组合实现信号级联或振荡，或通过构建蛋白质逻辑门的逻辑通路，以荧光作为最终输出信号。可以观察输出信号与基因表达之间的关系，构建数学模型，并优化过程设计参数。

One possible application scenarios is to apply our system to the production of liquor. Liquor is fascilating and addictive, and its special flavor is widely loved by people all over the world. However, the production of liquor is very complicated and it's hard to uncover the secret of its flavor, thus making it hard to create liquor with new flavor. Esters are great flavour contributors for liquor, and Saccharomyces cerevisiae is responsible for a large amount of phenylethyl propionate and ethyl phenylacetate production$^8$. Using our system, we can count the key enzyme in real-time and make it easier and more intuitive to modify the production of flavor molecules.

一个可能的应用场景是将我们的系统应用于酒类生产。酒类令人着迷且具有成瘾性，其特殊风味受到全世界人们的广泛喜爱。然而，酒类的生产过程非常复杂，其风味的奥秘难以揭示，因此创造新风味的酒类变得困难。酯类是酒类风味的重要贡献者，而酿酒酵母（Saccharomyces cerevisiae）负责产生大量的苯乙基丙酸酯和苯乙酸乙酯$^8$。使用我们的系统，我们可以实时计数关键酶，从而使修改风味分子的生产变得更加容易和直观。

Engineering strains are the "chips" of bio-manufacturing, and protecting their genetic information is crucial. In China, since 2017, there has been many microbial invention patent infringement disputes, bring attentions to the field of strain security. In our project, we designed an RNA sensor-based security system for engineered strains. We innovatively utilize environmental stress as induction for cell survival (Fig.4). For yeast strain with our security system, it constitutively express a special RNA sensor with a suicide gene as basic gene and Cre as reporter gene. Two loxP sites are located in the two ends of sensor in the same direction. Once the sensor sense its target transcript, which is endogenous stress response gene, the sensor gene will be cut off by the downstream cre recombinase to save the cell.

工程菌株是生物制造的"芯片"，保护它们的基因信息至关重要。在中国，自2017年以来，出现了许多微生物发明专利侵权纠纷，引起了人们对菌株安全领域的关注。在我们的项目中，我们为工程菌株设计了一个基于RNA传感器的安全系统。我们创新性地利用环境压力作为细胞存活的诱导因素（图4）。对于带有我们安全系统的酵母菌株，它持续表达一种特殊的RNA传感器，其中包含一个自杀基因作为基本基因，以及Cre作为报告基因。两个loxP位点以相同方向位于传感器的两端。一旦传感器感应到其目标转录本（即内源性压力响应基因），下游的cre重组酶将切除传感器基因以拯救细胞。

The in vivo dynamic monitoring of splice variants is essential for elucidating the complexities of gene expression regulation, understanding disease mechanisms, and developing targeted therapies, but it faces significant challenges due to technical limitations, the complexity of splicing events, biological variability, and ethical considerations. Overcoming these obstacles requires advancements in detection techniques, quantitative analysis, and data integration, alongside careful experimental design and ethical considerations, to fully harness the potential of this approach in biomedical research. Our sequence-specific RNA sensor hold the potential to carry out in vivo dynamic monitoring of splice variant by designing our sensor to sense the unique end joining of exons for different variants. We will confirm this potential in our wet lab.

胞内剪接变体的动态监测对于阐明基因表达调控的复杂性、理解疾病机制和开发靶向治疗至关重要，但由于技术限制、剪接事件的复杂性、生物学变异性和伦理考虑，这一领域面临着重大挑战。克服这些障碍需要在检测技术、定量分析和数据整合方面取得进展，同时还需要仔细的实验设计和伦理考虑，以充分利用这种方法在生物医学研究中的潜力。我们的序列特异性RNA传感器有潜力通过设计传感器来感知不同变体独特的外显子连接，从而实现剪接变体的体内动态监测。我们将在湿实验室中验证这一潜力。

[1] Robertson, M. P. & Joyce, G. F. The origins of the RNA world. Cold Spring Harbor perspectives in biology 4, a003608 (2012).
[2] Kang, H. J. et al. Spatio-temporal transcriptome of the human brain. Nature 478, 483-489 (2011).
[3] Bustin, S. A. How to speed up the polymerase chain reaction. Biomolecular detection and quantification 12, 10-14 (2017).
[4] Shishkin, A. A. et al. Simultaneous generation of many RNA-seq libraries in a single reaction. Nature methods 12, 323-325 (2015).
[5] Wang, Y.-S. & Guo, J. Multiplexed single-cell in situ RNA profiling. Frontiers in Molecular Biosciences 8, 775410 (2021).
[6] Jiang, K. et al. Programmable eukaryotic protein synthesis with RNA sensors by harnessing ADAR. Nature Biotechnology 41, 698-707 (2023).
[7] Kaseniit, K. E. et al. Modular, programmable RNA sensing using ADAR editing in living cells. Nature biotechnology 41, 482-487 (2023).
[8] Wu, Q., Xu, Y. & Chen, L.-q. Diversity of yeast species during fermentative process contributing to Chinese Maotai‐flavour liquor making. Letters in applied microbiology 55, 301-307 (2012).

[1] Robertson, M. P. & Joyce, G. F. The origins of the RNA world. Cold Spring Harbor perspectives in biology 4, a003608 (2012).
[2] Kang, H. J. et al. Spatio-temporal transcriptome of the human brain. Nature 478, 483-489 (2011).
[3] Bustin, S. A. How to speed up the polymerase chain reaction. Biomolecular detection and quantification 12, 10-14 (2017).
[4] Shishkin, A. A. et al. Simultaneous generation of many RNA-seq libraries in a single reaction. Nature methods 12, 323-325 (2015).
[5] Wang, Y.-S. & Guo, J. Multiplexed single-cell in situ RNA profiling. Frontiers in Molecular Biosciences 8, 775410 (2021).
[6] Jiang, K. et al. Programmable eukaryotic protein synthesis with RNA sensors by harnessing ADAR. Nature Biotechnology 41, 698-707 (2023).
[7] Kaseniit, K. E. et al. Modular, programmable RNA sensing using ADAR editing in living cells. Nature biotechnology 41, 482-487 (2023).
[8] Wu, Q., Xu, Y. & Chen, L.-q. Diversity of yeast species during fermentative process contributing to Chinese Maotai‐flavour liquor making. Letters in applied microbiology 55, 301-307 (2012).