Team:SYSU-CHINA/Human Practices
Overview
Timeline:
- Project selection
February 2,2020
- Online meeting with Professor Zhang Rui & 1st Brainstorm
April 8,2020
- Visit Professor Zhang Rui
September 14,2020
- Communicate with Yang Guorong, a salesman from Ribobio Biological Technology Co., LTD
September 12,2020
- Communicate with Zhang Tong, a salesman from Guangzhou Zhuan yan Biotechnology Co., LTD
September 22,2020
- Visit Professor Lee TienMing
September 25,2020
- Visit Professor Yang Jianhua
September 29,2020
- 2 nd brainstorm
September 29,2020
- Communicate with Huangtujin, project leader of Tsingke Biological Technology Co., LTD
October 7,2020
- 3 rd brainstorm
October 13,2020
Integrated Human Practices：
1．What are we aiming to achieve
- Develope an algorithm to predict the sequence features of dsRNA sequences with high affinity to specific proteins
- To explore experimental models suitable for dsRNA directed evolution
- Expand the understanding of editing mechanism and function of ADAR1
- To provide assistance for the screening of cancer therapy drugs targeting ADAR1
- Understand the application status and prospect of relevant technologies
2．From whom did we get help?
Professors:
- In order to explore experimental models suitable for dsRNA directed evolution, improve our experimental design, and better develop algorithms to predict sequence characteristics of dsRNA with high affinity to specific proteins, we visited Professor Zhang Rui (click to learn more) from Sun Yat-sen University.
We got the following advice:
① Our original plan was to recruit substrates with high affinity for ADAR. Professor Zhang Rui suggested that we modify them into GUIDE RNA and transfer them into cells to recruit endogenous ADAR for RNA editing.This made the project more feasible and had a larger clinical significance page.
② Professor Zhang Rui suggested that we select mammalian cells instead of yeast in the experiment: The editing effect of ADAR1 transferred from external source is poor. The existing dsRNA cyclization technology relies on endogenous nucleic acid ligase in mammalian cells, so the selection of mammalian cells is of greater clinical significance.
③ Professor Zhang believed that the stem ring of Focus ADAR1 could be reconstructed in yeast cells, which was a rapid molecular cloning experiment.
④ In the aspect of detecting the influence of ADAR2 on stem and ring editing, Mr. Zhang Rui believed that we did not need transcriptomic sequencing technology, but could direct Sanger sequencing, using ADAR2 to knockout cells and co-rotating vector +ADAR2 to see whether the editing level increased significantly.
⑤ For the screening of resistance genes, Professor Zhang Rui suggested that the toxic genes should be pretreated with induction promoter, and expressed separately with dsRNA and the toxic genes with a vector.DsRNA was expressed first and the resistance genes were screened
⑥ Mr. Zhang Rui thinks that we can do experiments and computer learning at the same time.On the one hand, directional evolution is carried out directly from the existing substrates with relatively high binding; on the other hand, machine learning is carried out simultaneously.There are few applications related to deep learning, and data may be insufficient, so it is not recommended. Machine learning can be done, which is similar to feature extraction. For captured substrates, direct input and features can be extracted.
⑦ Through communication with our Professor PI Zhang Rui, we plan to use a fixed dsRNA sequence mutation rate in the experiment to make it a constant;The concentrations of IFN and DOX were determined as independent variables in the experimental data, and the cell mortality was determined as dependent variables in the experimental data.
- In order to understand the application status and prospect of relevant technologies, better communicate with biological companies and get better feedback from the society on the project. We spoke to Professor Lee TienMing (click to learn more).
We got the following advice:
① As a basic research project, our project is still far from becoming a mature technology, so it is not suitable to carry out investigations directly into immunotherapy related aspects at present. Look to a biotech or pharmaceutical company for practical direction.
② The difficulties in the promotion of a new technology lie in: whether it can be accepted, whether the current difficulties can be solved or some bottlenecks can be broken, the risks this technology has and whether it can be avoided. These issues need to be consulted with the pharmaceutical industry.
③ Foreign companies are more advanced in directional evolution, but domestic companies have a large market. By comparing and combining with the current situation in China, it is confirmed that there is indeed a lack of products and RESEARCH and development in this aspect in China. Compared with the status quo of various foreign products, domestic research and development and the lack of products, our technology may provide a platform or accelerator to catch up.
④ Several suggestions when asking the company: Don't ask about the technical part, which involves the company's privacy; you can get useful Suggestions from the sales part; Look for large companies with advanced research that can lead the technological trend; Look for companies that are more willing to embrace new technology and find points that are relevant to the company's interests, so that the company is more willing to accept and promote the new technology.
- Developed algorithms to predict the sequence characteristics of dsRNA with high affinity to specific proteins, and expanded the understanding of the editing mechanism and function of ADAR1，we consulted Professor Yang Jianhua (click to learn more).
We got the following advice:
① In terms of algorithms, primary structure-based algorithms are motif-based, which may not be very instructive. Can be based on secondary structure consideration, machine learning/stem ring structure such as statistical analysis.
② RNA binding proteins are highly correlated with neurological diseases, such as Parkinson's disease and als. The current mainstream treatment is using technologies such as CRISPR-Cas9, and inhibitors of RNA-binding proteins are not yet mature.
③ ADAR is a hairpin RNA, and no one has reported that ADAR has a preference for binding. We can find the structural characteristics of ADAR by studying the secondary structure of ADAR.
④ RNA binding proteins have no affinity data to determine whether the binding or not, and divide the positive and negative sequence levels artificially. Professor Yang Jianhua suggested that we randomly generate a negative control set, randomly arrange the combination sequence and disrupt the base arrangement as a negative control.
⑤ To find out whether binding proteins with high affinity have similar secondary structures, so as to find the binding characteristics of ADAR proteins
⑥ Professor Yang Jianhua suggested that we should do statistical analysis instead of machine learning.
Biotechnology Company:
- We contacted Ribobio Biotechnology Co., LTD., and consulted Mr. Yang Guorong, the salesman of the company, for several questions. In addition, through a series of exchanges over several days, the other side has gained a certain level of understanding of iGEM and expressed a certain interest. Here are our questions and the answers provided by the company:
① How does your company promote these dsRNA products, or what is the marketing strategy? Last year, our company made a self-service order in our official website mall, the link is www.ribobio.com
Other customer LABS that need to customize the synthesis of double-stranded RNA can place orders directly on the website. Customized double-stranded RNA, conventional siRNA and miRNA can be promoted more effectively.
In addition, the official website can increase customers' attention to other products of our company. Currently, some animals use siRNA or miRNA injected directly, and many high marks have been published for such products, so in vivo experiments can be carried out without the traditional way of packaging viruses.
In addition, details of each product can be found on our official website, as well as the corresponding high-score literature that has been published with this product, which can be given to customers for reference in operation and usage.
② How does the market or various laboratories respond to these dsRNA products of your company?
Our company RIBOBIO Reibo biological double-stranded RNA products have been published in many high score literature, this kind of products is similar to primer, theoretically can give positive results, but all need experimental verification.The cell transfection efficiency has a great impact on the results. Most of the customer feedback products have no problems, but a small part of the feedback results are not very ideal. Such results are inevitable before the experimental verification and need to be screened.
③ Does your company plan to develop nucleic acid drugs?
The nucleic acid drug R&d Department is under development. For details, please consult the front desk at 02032290221.
- Then we got in touch with Guangzhou Zhuan Yan Biotechnology Co., LTD., and communicated with Mr. Zhang Tong, a salesman. After nearly three days of communication, we successfully let the other party know about iGEM and obtained a lot of valuable information. Here are our questions and their responses:
① What kinds of dsRNA nucleic acid products or drugs do you have at present?What research or experiments are they used in?
dsRNA nucleic acid products mainly include primer design and synthesis, CAS9 gene knockout, siRNA/shRNA design and synthesis, virus packaging, and sequencing should also be considered. We mainly provide scientific research services for some experiments.Applied to design verification upstream of experiment.
② [If there is a product/drug] how does your company promote these drugs or products?
Our company mainly promotes through online WeChat public account, offline visit customers, hold academic lectures and other ways.
③ [if there are products/drugs] what is your company's marketing strategy for these drugs/products?
The marketing strategy is mainly to let customers hand to mouth through high-quality services, to establish a good reputation.
④ [if there are drugs] Does your company have relevant strategies to make the general public understand and accept nucleic acid drugs?
The general public's awareness of nucleic acid drugs is not high, but still needs to be popularized by hospital doctors. WeChat public account can be more widely used to let the general public know.
⑤ [if so] What is the market's feedback on your nucleic acid drugs?
The market to my company's nucleic acid products or more recognized.Cooperative units have continued orders and good cooperation!
⑥ Do you have any products (such as antibodies, proteases, nucleic acid products, etc.) that have been developed with the method of directed evolution to improve the work efficiency or affinity of the products?
Product development USES directed evolution to improve product efficiency, mainly through upstream genetic design adjustments to improve product performance.
- We got in touch with Huang Tujin, the project leader of Beijing Tsingke Biotechnology Co., LTD., but unfortunately, no valuable information was finally obtained through the communication of Tsingke's lack of relevant dsRNA project and the application of directional evolutionary technology.But we also succeeded in letting each other know about iGEM and our project;
3．How do we practice the advice and information?
- According to the suggestion of Professor Zhang Rui, we abandoned the construction of plasmids in yeast cells and selected Hela cells in mammalian cells, and the toxic gene also selected apoptotic genes targeting Hela cells.
- After visiting Professor Li TieMing, we thought it was unreasonable to design questionnaires directly for tumor patients.We discussed and believed that our project, as a basic research, was far away from clinical trials and was not suitable to be taken as the most important direction in HP.
- By visiting Professor Li Tianming, we will invest the future application direction of the project in the field of biological companies.At the recommendation of Professor Zhang Rui, we contacted the relevant personnel of Ribobio company and Tsingke Company.
- We decided to refer to a set of existing evaluation criteria (binding Affinity parameters obtained from experiments) in the computer learning part.
- In the part of the algorithm, we standardized the affinity. The part with large value was taken as the positive training set with high affinity, the part with small value was taken as the negative training set with low affinity, and part of the data with similar affinity values was eliminated.
- The experimental modeling part, as a result of the experiment for the construction of the three fine demand is not high, the mathematical model between through brainstorming, we intend to adopt surface fitting method: for each specific IFN concentration and the concentration of DOX treatment leading to cell death, using a modeling tool matlab the curvefitting application data fitting to find their relationship.
- After considering the marketing and publicity Suggestions given by Professor Li Tianming, Ribobio Biotechnology Co., Ltd. and Guangzhou Zhuanyan Biotechnology Co., LTD., we finally decided to jointly hold two online publicity lectures for high schools with Shanghai Jiao Tong University, respectively introducing iGEM and our projects.
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No.135,Xingang Xi Road,
Guangzhou,510275,P.R.China
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