2018 Fiscal Year Research-status Report
Verification of the functional role of coacervate droplets in in vitro RNA evolution
| Project/Area Number |
18K14354
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| Research Institution | Tokyo Institute of Technology |
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Principal Investigator |
ジャー トニー 東京工業大学, 地球生命研究所, 研究員 (10800328)
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| Project Period (FY) |
2018-04-01 – 2021-03-31
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| Keywords | Compartments / Self-Assembly / Chemical Evolution / Prebiotic Chemistry / Protocells / Polyesters |
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| Outline of Annual Research Achievements |
We were able to generate polydisperse polyesters from drying of α-hydroxy acid (αHA) monomers. These polyesters then formed membraneless microdroplets, similar to coacervate droplets, when subjected to aqueous conditions. The polyester microdroplets could be generated from various chemistries, providing diverse segregation and compartmentalization functionalities. Specifically, using confocal microscopy, we found that the droplets could segregate the hammerhead ribozyme, while providing differential segregation and compartmentalization for the fluorescent dyes and fluorescently-tagged RNA. These studies suggested that the membraneless microdroplets could compartmentalize nucleic acids for future evolution and functional assays, as a system similar to coacervate droplets.
We next performed pilot studies regarding biopolymer function, and found that both proteins (superfold GFO) and RNA (Hammerhead Ribozyme) preserve their function in the presence of the droplets. The protein is functional within the droplet, while the RNA is functional in the presence of the droplet. This suggests the potential relevance of the droplets to various origins of life models including peptide first and RNA-first, but also the GARD/composome model. Facile polymerization of αHAs provides a novel pathway for the assembly of combinatorially diverse primitive compartments that could specifically host a variety of possible prebiotic metabolic and replication processes that could be subjected to further chemical evolution, including RNA evolution.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
In between application and receipt of the grant, a study was published by Dora Tang et al (https://www.nature.com/articles/s41467-018-06072-w) which reported the catalysis of an RNA hammerhead ribozyme within a peptide-nucleotide coacervate droplet, similar to what we proposed. Instead, we have now changed our system to a different membraneless microdroplet system (similar to the original coacervate), except now with prebiotic polyesters generated from drying of alpha hydroxy acids. We have characterized these droplets with respect to their compartmentalization of small molecules and biopolymers. We have also performed seminal functional assays of biopolymers within the droplets. We have one paper under review at PNAS, which is about characterization of the membraneless microdroplets, and their ability to segregate and compartmentalize nucleic acids. We are also planning to file a patent application related to this system (compartmentalization of nucleic acids and proteins in membraneless polyester microdroplets).
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| Strategy for Future Research Activity |
We have only performed pilot studies regarding RNA and protein function, and now we will perform more detailed functional studies with the hammerhead ribozyme. Specifically, we will test more microdroplet systems for functional compatibility with the ribozyme. We will also explore more of the polyester sequence space, combinatorially, to find other polyester chemistries that could be amenable to compartmentalization of RNA within the microdroplets. Specifically, we are exploring cationic polyester microdroplets, due to the preferential electrostatic interaction between cationic polymers and the anionic backbone of RNA. These will be accomplished through a combination of microscopy and gel-shift assays.
We have also performed pilot studies regarding lipid assembly around the polyester microdroplets. Combining lipids into our system could allow even further the microdroplet systems to stably encapsulate and compartmentalize RNA molecules, as we would test this through confocal microscopy and fluorescence-recovery after photobleaching studies. Finally, we then hope to study the function and exchange of RNA polymers within a combined microdroplet/lipid layer system.
We hope to then subsequently start to testing enzyme activity within the droplets (we have only tested autofluorescent protein activity), starting with those that would be used within an in vitro evolution system. Finally, combining all of these aspects will allow us to complete a nucleic acid evolution system within a primitive membraneless droplet.
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| Causes of Carryover |
This amount remained for two reasons. I received a small external grant which was used for some of the consumables used in this project. We also had planned a portion to remain for miscellaneous costs such as publication fees. We had originally planned to publish a paper in FY 2018, but because the review is slow, it will be published in 2019. As such, this amount left over will be used as publication fees for FY2019.
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