Proposed Implementation

TrigGate's Proposed Implementation

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mRNA has the potential to change the world as we know it today. The last two years has revealed to us the glimpse of what this technology is capable of: mRNA-based vaccines were in the front line of the battlefield against SARS-CoV-2 and completely changed the face of it. Vaccination is not, of course, the only field upon which mRNA-based technologies could be implemented: they might revolutionize the ways by which cancer is treated, to enable editing of various genomes and to break the glass ceilings of many current treatments. However, one major stumbling block standing in the way of mRNA-based technologies to take the medicine and biological spheres to the next level is the ability to create a successful cell-specific expression system. As a part of our project, we desire to implement a system that will allow a cell-specific expression of mRNA molecules, supposed to be translated to desired proteins, for any application of synthetic biology. This will hopefully allow the mRNA-based therapies to fulfill their full potential as the leading edge of personalized medicine.

The current approaches for achieving selective translation of mRNA molecules in certain tissues focus mostly on the delivery vehicle of the mRNA, not in the sequence of the mRNA or the structure of the mRNA imposed by its sequence. For example, a much frequent method for achieving specificity is generating combinatorial lipid libraries: the anionic mRNA is regularly encapsulated within a lipid cationic particle. The latter keeps the mRNA safe from degradation until reaching the target cell/tissue, to where the mRNA is injected while the lipid particle is dissolved. Speaking so, the way of achieving specificity is to generate different lipid particles that will encapsulate the mRNA, by that reaching different tissues/cell surfaces. This method, however, requires manual design of the libraries, consuming time and resources, in addition to the fact that this design method cannot ensure the particles will reach their target solely.

We offer something else - instead of taking into account the delivery vehicle in a manual manner, why not utilizing the properties of the mRNA itself? Specifically, our solution is a sequence-based one. Its novelty is by taking advantage of a toehold switch, a secondary structure located in the 5’-UTR and acting as a form of a riboswitch - in the presence of a trigger molecule, presumably an RNA one, it will enable translation, while without the trigger translation will be blocked: even though there were some works in the past that have utilized toeholds switches, including one pioneering work in prokaryotes that has founded that sort of implementation (for more details, please refer to our “Project Description” page), most of the attempts to implement it in eukaryotic systems were not quite successive. We try to make something different: not only that we aspire to implement toehold structures in eukaryotic cells for their various possible applications, we also plan to generate a computational tool that will generate automatically optimized toehold switches for various applications of specific translation. Even though a primary aspect of our project is to enable a cell-specific translation for eukaryotic applications, it might be of importance to note that our tool is widely directed to be fitted to any type of a model organism, from bacteria through yeast up to mammalian cells. For example, an exciting possibility to utilize our tool for a prokaryotic implementation is that of selective translation in the population level. In case we utilize a certain community of bacteria, we might want to enable a differential expression of mRNA molecules among which: certain bacteria would express one protein, while other bacteria would express another one. Utilizing our tool in that fashion, we might be able to induce desired evolutionary processes upon the population: while certain bacteria may express a protein which functions as antibiotic-resistance, the other bacteria might express another protein which functions as an antibiotic. Such processes might be efficient in terms of required applications, for example performing a selection for specific bacteria in the microbiome.

In our vision, the user only has to enter as an input a certain feature/some features according to which its desired Toehold construct will be designed, for example a tissue in which it will be expressed, then the tool will generate the construct's sequence and will return it as an output. It is important to note that even though we strive for simplicity in the level of the user interface, behind the scenes the tool itself would perform complex actions, utilizing the sophisticated algorithms we are developing in order to verify the indeed optimized Toehold switch will be provided as an output.

We strive to implement our tool in the real world in a way by which it will not only be essential for every application utilizing Toehold switches, but it also will have an effect of directing the world of research and therapeutics towards the realm of mRNA-based technologies. Since many current technologies utilize the many advantages mRNA-based therapies have to offer, we hope that our tool, aimed at a vulnerable point of those promising developments, the one of specific translation, will assist them to make the next steps towards constituting a reliable and sustainable primary form of treatment. We believe that by enabling specificity, more research entities will internalize the advantages of mRNA-based treatments, therefore will start to develop and manufacture more of them. We believe that once the frequency of mRNA-based treatments will rise (a process which has already started: the vaccines against SARS-CoV-2 are the primary example for that), there will also rise a need for an automatic tool that will enable the design of such treatments to be much more productive and efficient. In that case, our tool would serve as an integral step in the process of designing an mRNA-based therapy/development: in order for the development to avoid the limitations imposed by a systematic injection of the mRNA (detailed throughout different sections of our Wiki), it must be ensured that it will be selectively expressed in a target tissue, this is when our tool gets into action. However, safety issues must be taken into account while considering an implementation of the tool in the real world: while utilizing the tool, the sequences it generates must be safe for later uses, since it is safe to assume that once a sequence will be generated by the tool, the user would like to generate it and utilize it for research/therapeutic causes, so the sequence will be synthetized and will be tested throughout laboratory experiments. Therefore, we believe that when our tool will be implemented in the real world, it is rather important it will contain certain features, which will inform the user in any case the sequence that will be generated as an output will constitute some sort of a danger or might be utilized in an unsafe manner. That way, the user will be able to make necessary preparations for the examination of the generated construct throughout lab work, and will also be able to make an aware choice regarding the use of the construct as a possible future form of treatment/tool of research.