Design

Meeting the users’ demands is always the key of a successful product. Bearing this in mind, with our initial idea in developing miRNA inhibitors, we talked with the potential users of our product to gather their specific needs. Some interesting points that never came into our mind was raised in these interviews. For example, during the talk with Dr. Song Zhuo, the technical director of Genetalks, he talked about a few recently-published papers about cross-talks among different miRNAs. Building on these papers, he highlighted the potential needs of multi-targeting miRNA inhibitors and encouraged us to try crating one. For another example, during our interview with Professor Ping Li, she spoke highly of our dsDNA library based assembly system, and suggested us that a software assisting users to select correct nucleotides from the library might be necessary.

With these constructive suggestions, we adjusted our project design in the following aspects. Firstly, we tried assembling multiple-targeting miRNA inhibitors in our wet-lab experiments. Two miRNA Lockers, targeting two and four different miRNA molecules simultaneously were designed, assembled, and generated in our lab work and passed sequencing verification. Though cellular experiments on those Lockers have not been finished yet due to the time limits of iGEM, our current work have shown a great

Market demand:

We evaluated the cost of our product. It contains mainly three steps:

1. Major Instruments and Reagent

(1)Reagents

Oligo, EcoR I, Spe I, T4 Polynucleotide Kinase, T4 DNA ligase, DH5α, 2×Tap Master Mix, Tiangen Plasmid Microkit Kit.

(2)Instrument

Since the instruments needed for synthetic locker are the most common in the lab, they are not included in the evaluation.

2. Experimental Procedure

Click here to see the protocol

3.Cost Evaluation

Name of reagents	Unit price	Use level	Equivalent	Price for per-million cells
Oligo	$0.12 / base	0.00254 bases	$0.000302	$0.00114156
EcoR I	$19.55/4000U(15U/µL)	0.1µL	$0.0073	$0.027594
Spe I	$19.55/150U(10U/µL）	0.1µL	$0.13	$0.4914
T4 Polynucleotide Kinase	$25.87/200U(10U/µL)	0.2µL	$0.26	$0.9828
T4 DNA ligase	$25.87/28000U(350U/µL)	1µL	$0.32	$1.2096
2×Tap Master Mix	$102.27 / 5mL(3 pieces/mL)	10µL	$0.05	$0.189
Tiangen Plasmid Microkit Kit	$102.27 / 200 times	One time	$0.51	$1.9278
The cost of our product per-million cells				$4.83

As a comparison to our product, we investigated similar products available on the market, we managed to contact with some companies to inquire price of their miRNA inhibitors. According to the instruction given by those companies, we calculated the cost of each products.

Company	Product	Product's name	Price		Price for per-million cells
Sigma Aldrich	TuD RNA	mission Lenti microRNA inhibitor	$1053.27/		$52.68
Guangzhou RiboBio Co.	micrOFF™ miRNA Inhibitors are synthetic antisense oligonucleotides, which is complement to mature miRNAs sequence , for loss-of-function studies of miRNAs.	micrOFF miRNA inhibitor	$75.65/nmol		$37.83
Shanghai Integrated Biotech Solutions Co.,Ltd.	A special designed RNA molecule can bind with specific miRNA.	miRNA inhibitors	$76.65/nmol		$38.33
GenePharma Co.	An 21-23nt RNA molecule, and was  2'-O-Methoxyethyl-Modified ,can serves as an efficiently microRNA inhibitor	GMR-miRTM microRNA inhibitors	$105.91/nmol		$52.96
Biomics Biotechnologies.	This is a miRNA inhibitor, which is especially chemical modified, so that it can serves as an efficiently and specifically inhibitor.	miRNA inhibitor	$98.35/10nmol		$ 1.02
BIONEER Trade(Shanghai) Co.,Ltd.	This specially designed RNA molecule, which has been specially modified, can bind with specific miRNA.	AccuTarget™ miRNA Inhibitor	5 n mole	$55.68	$1.16
			10 n mole	$84.31	$0.88
			20 n mole	$108.40	$0.57

During our market research, we found that most miRNA inhibitors on market, besides the Sigma Aldrich’s mission Lenti microRNA inhibitor, are antisense oligonucleotides, which can only target one specific miRNA, and aren’t stable enough to serve as a long-term inhibitor. Compared with those miRNA inhibitors, our product is not only low cost, but also more stable and can bind with different miRNAs.

Application prospect

Nowadays, miRNA inhibitors plays an important role in medical treatment. To know more information of miRNA antagonist therapy, we interviewed Professor Li LingProfessor and master’s supervisor of biochemistry and molecular biology department, school of Basic Medical Sciences, Southern Medical University, his main research direction is molecular mechanism of bone metastasis in tumor.

Professor Li Ling mentioned that the FDA program approved gene therapy technology and the first gene therapy was expected to be approved in January 2018. In addition, an RNAi (RNA interference) drug has entered the main clinical endpoint of phase three clinical trials. MicroRNA antagonist therapy is a promising new technique for treating diseases at gene level. With the wide application of this technology in clinic, many clinical drugs and therapies harmful to human body will disappear. However, new therapies will have their own advantages and shortcomings, old things cannot fade away immediately, and new things will also face many challenges to retain. Hospitals and clinicians should keep up with the breakthrough of medical technology, enrich the knowledge reserve of gene therapy technology and its treatment, and meet the new challenges at any time to serve the human health.

Safety

For foundational research

When we interviewed Dr. Song Zhuo, the technical director of Genetalks, he put forward two questions:

Will the sponge affect the normal function of cells?

Will MicroRNA inhibitor work in positive correlation with level gradient?

These provided us new ideas of safety inspection of product beyond the mainline; to fully solve these questions, we added cytotoxicity test and concentration gradient experiment to our future work.

For medical field

Systematic drug-given method of the current microRNA antagonist therapy may induce adverse consequences, such as complement activation AND hepatotoxicity, which will cause potential security issues. We interviewed Professor Li LingProfessor and master’s supervisor of biochemistry and molecular biology department, school of Basic Medical Sciences, Southern Medical University, his main research direction is molecular mechanism of bone metastasis in tumor. and Tu ShuoTeaching assistant of biochemistry and molecular biology department of Nanchang University, her main research direction is tumor cell apoptosis.teaching assistant in different medical universities. Here are their answers:

Li Ling: Obviously, microRNA antagonists can treat diseases from the source by inhibiting the expression of target genes at the gene level, and have shown good efficacy in a large number of clinical studies and clinical trials. Side effects associated with new therapies also exist. There may be individual differences, and there may be some deficiencies need to be improved. When microRNA antagonist therapy enters clinical trials and FDA (sFDA) approval process, the treatment effect and its risk will be evaluated, and then mature therapy will be approved. For the new treatment, the patient's acceptance may be different, most of them need to be informed before the medication, and it is necessary to sign the informed consent.

Tu Shuo: Well, liver toxicity depends on whether the drug itself is toxic or only after metabolized in vivo, or it is only toxic to the individual populations. If the drug itself is toxic or metabolized to produce toxicity, then it is necessary to control the dosage strictly and conduct periodic blood biochemical examination during the drug use, as for complement activation, make clear whether it is due to individual specificity or universal situation. It can be taken simultaneously with drugs that inhibit complement activation or optimize its structure.so once something unexpected happens, we can stop using the drug or replace therapeutic plan. There are many drugs have liver toxicity, so the drug can still be used in clinic even it has liver toxicity.

Idea Exchange

In the end of August, we attended FAFU_CCICFujian Agricultural and Forestry University Conference of China iGEAMer Community, and spent three days there.

The meetup is held for teams to share ideas and help each other. When communicating with other teams, we heard team BGIC-Union need plasmids which we designed a year before, then we decided to offer the plasmids, click here for details in collaboration page. In a few words, we had a fulfilling time in CCIC!

Exactly at this meeting, we found the project of team Lanzhou has some relevance to our project; their project is A novel method in controlling weeds and pests by tandem RNA Interference. Then we made communication in specific experiments at the venue.

After CCIC, we made deeper online interactionS with team Lanzhou with more team member participated.

we talked about each teams’ project mechanism, new situations encountered in experiments and progress of modeling and human practice.

Policy

Attentions on policy-making

MiRNA antagonist therapy is a brand new area that there is no policy to regulate it; however, we decided to grasp the trend of policy. As the product developer, we also shoulder the responsibility of advising the government when needed. Thus, we consulted Professor Li Ling and teaching assistant Tu Shuo on wht should be paid attention to when making a policy for miRNA antagonist therapy.

Popularization

Publizing with new media

This year our track is foundational advance，and the product---miRNA inhibitors is not familiar to the public. Therefore, if we want to have the public engaged in our work, the very first thing is to let them make friends with miRNA and our project!

In order to achieve a greater influence and outreach, we decided to have our popular science article pushed by WeChat subscription (The WeChat ID of the subscription account is zaxue8, it aims to spread knowledge to the masses). In addition, we used many internet memes in this article for a more vivid expression, and it worked just as we expected!

By now, 2666 people have read this article, and the number is still increasing. Since it is on the internet, people can open it without limitation of time and space. And we can acquire the effectiveness of the publizing conveniently by reading rate and top comments.

You can read the article below as well as scaning the QR code by WeChat to read our articles on your phone:

Since we estimate miRNA is not popular among masses, we attached a small survey at the end of the article, and the result is:

This chart shows that the miRNA popularization still has a long way to go, and it might take a long time before taking the second step.

Do you want a picture book?

Many people, like me, will probably feel bored to read something contain nothing but words, especially when the words is about learning. I realized it after I entered college to find there are too few illustrations in textbooks. We do not want others to feel the same when they try to read our pamphlets, so we made it a beautiful picture book, printed and separated.

Education

Lecture in high school

High school students are young, vibrant, and always open to new things.

Last year, our team had Lab open day for some high school students. One named Zhang Herui, showed great interest in iGEM competition and synthetic biology and was recruited into our team. This year, considering there are few references of miRNA mentioned in textbooks, we walked into her school to fill the blanks and introduce our project.

Moving our glow sticks for synthetic biology

In June, our team was invited to give a lecture on iTED Sharing held by the Squirrel Club of NUDT, the theme of the lecture is biotech 2.0-the synthetic biology and iGEM in my eyes. Through this lecture, we gave the present students an introduction of our research field. We aimed to share our ideas with undergraduates and get feedbacks form the interactive section. Also the popularization of synthetic biology is achieved.