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creating multilayered structure to understand and application

From planets to cells, even to atoms, they are all ordered structures with physico-chemically distinct multilayers (Figure 1.1). As the distinctive one of them, the natural biological materials are self-assembled into layered structures with near-atomic precision in living cells, formatting the basis of function and regulation, whereas our understanding about their detailed mechanisms is still very inadequate. A simple example of layered nanostructures is the biomolecular nanocage formed by viruses like MS2 or CPMV, with multiple protein components self-assembling in a highly symmetrical fashion to form a closed-shell structure out of its genomic RNA core (Figure 1.2). As a unique structure of eukaryotic cell, the nucleolus is featured by its multiple layers and the delicate coordination between these layers to fulfil its function in ribosome biogenesis, in which the biogenesis process starts from the inner layer and ends at the outer layer of the nucleolus [1].

Fig. 1.1(1) Multi-layered structures in nature

Fig. 1.1(2) Multi-layered structures in nature

Fig. 1.2 Molecular structures of CPMV coat proteins

In another way, although technologies such as DNA origami have made it possible to engineer biological molecules to form defined structures, 3D materials created through the use of synthetic DNA and/or protein generally lack the same precision and controllability as living cells [2]. Both proteins and DNA have been used as building blocks to create tunable nanoscale cages, but each molecular type has its own limitations [3]. Inspired by viral capsids, there has been extensive research about the self-assembled protein cages [4][5]. However, tuning the nanocage parameters (such as the size and volume) usually requires re-engineering the system with a different set of protein building blocks, which requires technical expertise and can be time-consuming. As an alternative approach, DNA nanotechnology, especially DNA origami technology, has been useful in the bottom-up fabrication of well-defined nanostructures [2]. Since DNA nanocages are usually negatively charged and may require supra-physiological concentrations of magnesium for stability, they are of low stability in organisms and must be further elaborated with receptor-binding peptides or proteins to imbue them with bioactivity [6]. In addition, although multilayered nanostructures are desired in numerous application scenarios such as nanofabrication, catalysis, drug delivery, and so on, there is currently no easy way to build them. The next-generation techniques for building multilayer nanocages will be in vivo synthesis and multiscale manufacturing through merging self-assembling protein building blocks with addressable DNA scaffolds, which could combine the bioactivity and chemical diversity of the former with the programmability of the latter.

Domestic Public Hospital Liposome Market

Liposome Market in the U.S

Publication trends at OMVe

Therefore, from an engineering perspective, the creation of directionally encoded multilayer functional nanostructures of sub-micrometres presents an exciting challenge. This not only provides a new opportunity for understanding our world, but also anticipates expanding the applications of synthetic nanostructures, through encoding assembly line logic into target materials [7].

From the application perspective, producing such structures meets the market demand in various aspects. Currently, the market is in need of a material that can act as a carrier, targeting specific positions and delivering its payloads to heal, kill pests, or provide protection. For years, materials like OMVs, liposomes and others have filled some of these roles. Here, our created structure, called XMU-egg, offers advantages to enhance targeting efficiency, payload delivery and multi-level regulation. Compared to existing materials like OMVs, which are used to envelope toxins to kill pathogens or represent antigens on their surface to trigger the immune system, our nanostructure exhibits more extensive functions. The protein on the outer-layer can served as ligands to recognize specific receptors, while inside the layer, in the space, various cargoes with different functions can be encapsulated. Moreover, the RNA scaffolds have the potential to be a RNA vaccine! The thing even better is that this process can be controlled using switches applied to its protein or RNA modules. It’s a picture of ligands recognizing receptors and sending toxins, medicines, enzymes, or anything else into the target to achieve a goal. The medical, scientific, technical, and engineering values are all there.


controllable formation of layered structures in vivo

Existing technical means

The remarkable breadth of applications not only highlights the power of nanostructures but also reveals roadblocks that need to be removed. The challenges of manufacturing multi-scale ordered materials in the complex cellular environment include:

Complex design process: Both protein-based and DNA-based nanostructures are currently created through engineering the complicate interaction among molecules, requiring professional expertise and even specialized software to assist their designs. Is there any other interaction that can be easily engineered and can be utilized to drive the formation of nanostructures?

Difficulty in controlling the formation of assemblies and tuning their sizes: It would be useful for further applications if one could control the assemble behaviors, layer components, and sizes of nanostructures.

Ordered assembly: For multilayer nanostructures, ordered assembly is essential. How to encode assembly line logic into target materials is a big challenge.

High cost and low stability in vivo: The in vitro assembly of nanostructures requires large amounts of high-purity DNA/protein and the in vitro conditions for nanostructure formation are usually beyond the physiological range. Is it possible to efficiently create nanostructures in vivo?

Our Solution

Here, through designing the phase separation behaviors of synthetic RNA and protein molecules, our project aims to construct ordered RNA-protein condensates called XMU egg. This provides a way to synthesize multi-scale ordered materials in vivo:

Liquid-liquid phase separation (LLPS) is a spontaneous assembly process both in vivo and in vitro that separates a homogeneous solution into dense and coexisting dilute phases [7]. The concept of phase-separation-mediated formation of biomolecular condensates provides a new and easier framework to drive the formation of nanostructures. By following the fundamental principles that drive LLPS, we use three protein domains with LLPS ability, including FUSN, RGG and IDP, to drive the formation of the three layers of XMU egg in vivo.

The formation of XMU egg is controlled by the inducible expression of its protein/RNA modules and by the exposure to blue light. Only when all modules are expressed and blue light is exposed, XMU egg can be efficiently formed. This is achieved through the use of inducible promoters and the fusion of Cry2, a photolyase homology domain that oligomerizes upon blue light stimulation.

The size of XMU egg and the thickness of each layer are defined by the RNA scaffolds. Three specific RNA-protein interaction pairs, including MS2-MBP, boxB-λN, and gRNA-Cas9, are used. The position, order, and interval of MS2, boxB and gRNA in the RNA scaffolds will encode the assembly line logic of XMU egg.

The mechanisms by which the XMU egg operates is detailed in depth in the Designsection


[1] Feric, M. et al. Coexisting liquid phases underlie nucleolar subcompartments. Cell 165, 1686-1697 (2016).

[2] Dey, S. et al. DNA origami. Nat Rev Methods Primers 1, 13(2021).

[3] Yang Xu. et al. Tunable Nanoscale Cages from Self-Assembling DNA and Protein Building Blocks. ACS Nano 13,3545-3554(2019)

[4] King, N. P. et al. Accurate Design of Co-Assembling Multi-Component Protein Nanomaterials. Nature 510, 103-104(2014)

[5] Bale, J. et al. Accurate Design of Megadalton-Scale Two-Component Icosahedral Protein Complexes. Science 353, 389-394(2016)

[6] Lee, H. et al. Molecularly Self-Assembled Nucleic Acid Nanoparticles for Targeted In Vivo SiRNA Delivery. Nat. Nanotechnol. 7, 389-393(2012)

[7] Dai, Y., You, L. & Chilkoti, A. Engineering synthetic biomolecular condensates. Nat Rev Bioeng 1, 466-480 (2023).