Team:SYSU-CHINA/Description

Abstract

RNA binding proteins (RBPs) play an essential role in tumors and neurodegenerative diseases, while most of them lack effective inhibitors. Since directed evolution has shown its high efficiency in selecting new products, this year we provided a sample combining rational design and directed evolution to obtain dsRNA inhibitors of RBPs and took ADAR1, a dsRNA adenosine deaminase, as an example. In our project, an algorithm-guided model was trained from natural substrates of ADAR1 and used to established a candidate dsRNA library. In cells, dsRNA is forced to compete for binding ADAR1 with an editable stem-loop which has a toxic gene downstream of it. The cells will only survive when endogenous ADAR1 is inhibited by transferred dsRNAs. The intensity of the competition is regulated by IFN-α and Tet-on system. As the experiment progresses, efficient substrates are extracted and used to train the model above for the next round. Through the continuous cycles of this screening process, we can obtain high-efficient inhibitors of ADAR1 efficiently, while this model can also be extended to other RBPs.

Backgrounds

RNA-binding proteins and the inhibitors

RNA-binding proteins (RBPs) act as key players in post-transcriptional events, they have become hotspots since their close relation with lots of disease, which include cancer, genetic disease and neurodegeneration mechanisms.

Figure 1. Diseases associated with RBPs

Currently, the way to solve these problems is to find the inhibitors of the RBPs. Generally, the methods to obtain efficient and stable nucleic acid inhibitors are direct synthesis or chemical modification, but the cost is relatively high and the sequences may be unstable. In addition, through contact with biological companies, we learned that there are still many unknowns and uncertainties in the research and application of RNA inhibitors, which need to be further studied.

The most common nucleic acid inhibitors include small RNAs, RNA aptamers and so on. RNA aptamers are short RNAs that bind intracellular molecules or proteins and can thus modulate intracellular processes. They can bind specific domains and conformations of proteins so to either inhibit them or modulate their function. Though may have various potential applications, they have not been as widely used as other RNA-based technologies such as small interfering RNAs or guide RNAs. This is because RNA aptamers are not stable enough to accumulate in cells until the concentration is sufficient to function.

To solve the problem，Jacob L. Litke et al. developed an expression system for achieving rapid RNA circularization, resulting in RNA aptamers with high stability and expression levels but not affect the function. It worked by adding ligation sequences and ribozymes to both ends of the RNA of interest. When the transcripts are expressed, the ribozymes undergo spontaneous autocatalytic cleavage, leaving the remaining part with 5’ and 3’ ends that are then ligated by common endogenous RNA ligase RtcB. And it is proved that while RNA's stability improves, its function is not affected[1].

Figure 2. Schematic diagram of the autocatalytic circular RNA mammalian expression vector[1]

ADAR1 and its impact on fields of cancers

In our project, we chose ADAR1 as our instance, since it is a typical RNA binding protein that can act as a good representation, and it plays an important role in many fields.

ADAR (double-stranded RNA-specific adenosine deaminase) catalyzes the editing of nucleotide modification of adenosine to inosine in human body. It doesn’t exist in plants or prokaryotes, but in metazoan. There are three highly conserved members in the family: ADAR1, ADAR2 and ADAR3, among which ADAR1 and ADAR2 have been identified to have significant adenosine deamination activity.

Researches show that the high expression of ADAR1 promotes the growth and metastasis of many kinds of cancers such as liver cancer, breast cancer, multiple myeloma and so on. It has been proved that the loss of ADAR1 in tumor cells profoundly sensitizes tumors to immunotherapy and overcomes resistance to checkpoint blockade [2]. In the absence of ADAR1, A-to-I editing of interferon-inducible RNA species is reduced, and this leads to double-stranded RNA ligand sensing by PKR and MDA5 which respectively related to growth inhibition and tumor inflammation. In consequence, finding the inhibitor of ADAR is an effective way in assisting cancer immunotherapy[3].

Figure 3. Schema of the genetic dependencies of enhanced immune infiltration[3]

Figure 4. Loss of ADAR1 in tumours overcomes resistance to immune checkpoint blockade[3]

Directed evolution

Directed evolution is an emerging research technology. It refers to applying a specific selection pressure to a group that originally has mutant sequences, forcing the group to evolve in a way that accommodates this pressure.

The 2018 Nobel Prize in Chemistry was awarded to three scientists for their contribution of directed evolution of enzymes and the phage display of peptides and antibodies. In November 2019, Ron Milo of the Weizmann Institute of Science in Israel reported that they had successfully obtained E. coli that could produce all its biomass carbon from CO2 through directed evolution [4]. All these works show the advantage and great potential of directed evolution technology.

Synthesizing the literature we found and the instructions from Prof. Zhang Rui, compared to those we mentioned above, directed evolution should be a more effective way to obtain targeted RNA sequences. Additionally, the ability to rationally engineer directed mutation will be a paradigm shift in the capabilities afforded by synthetic biology, potentially resulting in organisms with vastly increased rates of evolution [5]. That’s why we came up with the idea of introducing algorithms into our project.

However, there is currently a lack of directed evolutionary strategies for dsRNA, so we think it is necessary to work on this area.

Our solutions

To find the dsRNA which can effectively inhibit ADAR1, we design a flexible platform with synthetic biology.

Figure 5. Flow chart of the project

We combine the ADAR1 editing event with a selectable trait. In the cell, the dsRNA we provide are forced to compete with an editable stem-loop which has a toxic gene downstream of it. When the dsRNA has a higher affinity to ADAR1, the stem-loop will not be edited and when the dsRNA has a higher affinity to ADAR1, the stem-loop will not be edited and block the expression of downstream toxic gene, so the cell survives. Conversely, if the stem-loop is edited, the cell dies, and the corresponding dsRNA is eliminated. In this way, we can screen out the effective dsRNA.

Figure 6. Schema of the directed evolution of dsRNA

Additionally, we combine directed evolution with algorithms. The dsRNA library is constructed under the guidance of computer program and by error-prone PCR, and the experimental results are fed back to the algorithm for further prediction.

That’s why our project is called Semi-rational evolution of ADAR1 inhibitor. It provides a reference for screening inhibitors of RBPs and gives a new paradigm to directed evolution of dsRNA. (To learn more, click here:https://2020.igem.org/Team:SYSU-CHINA/Design)

THE IMPACT OF EPIDEMIC

This year is a special year. Affected by the epidemic, we were not allowed to go back to our lab from February to September. Consequently, the time allowed us to carry out experiments is limited. Yet, during this period, we still continued to improve our projects through paper searching and brainstorming, as well as communication with different groups of people. We held weekly online meetings in our team and monthly ones with our PI to gradually improve the processing of the project.

Reference

[1] Litke JL, Jaffrey SR. Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts. Nat Biotechnol. 2019 Jun;37(6):667-675. doi: 10.1038/s41587-019-0090-6. Epub 2019 Apr 8. PMID: 30962542; PMCID: PMC6554452.
[2] Zaretsky JM, Garcia-Diaz A, et al. Mutations Associated with Acquired Resistance to PD-1 Blockade in Melanoma. N Engl J Med. 2016 Sep 1;375(9):819-29. doi: 10.1056/NEJMoa1604958. Epub 2016 Jul 13. PMID: 27433843; PMCID: PMC5007206.
[3] Ishizuka JJ, Manguso RT, Cheruiyot CK, et al. Loss of ADAR1 in tumours overcomes resistance to immune checkpoint blockade. Nature. 2019;565(7737):43-48. doi:10.1038/s41586-018-0768-9.
[4] Gleizer S, Ben-Nissan R, Bar-On YM, et al. Conversion of Escherichia coli to Generate All Biomass Carbon from CO2. Cell. 2019;179(6):1255-1263.e12. doi:10.1016/j.cell.2019.11.009
[5] Simon AJ, d'Oelsnitz S, Ellington AD. Synthetic evolution. Nat Biotechnol. 2019 Jul;37(7):730-743. doi: 10.1038/s41587-019-0157-4. Epub 2019 Jun 17. PMID: 31209374.