Evolution of a designed protein assembly encapsulating its own RNA genome

Abstract

The challenges of evolution in a complex biochemical environment—coupling genotype to phenotype and protecting the genetic material—are solved elegantly in biological systems by nucleic acid encapsulation. In the simplest examples, viruses use capsids to surround their genomes. While these naturally occurring systems have been modified to change their tropism1 and to display proteins or peptides2–4, billions of years of evolution have favored efficiency at the expense of modularity, making viral capsids difficult to engineer. Synthetic systems composed of non-viral proteins could provide a “blank slate” to evolve desired properties for drug delivery and other biomedical applications, while avoiding the safety risks and engineering challenges associated with viruses. Here we create synthetic nucleocapsids—computationally designed icosahedral protein assemblies5, 6 with positively charged inner surfaces capable of packaging their own full-length mRNA genomes—and explore their ability to evolve virus-like properties by generating diversified populations using Escherichia coli as an expression host. Several generations of evolution resulted in drastically improved genome packaging (>133-fold), stability in whole murine blood (from less than 3.7% to 71% of packaged RNA protected after 6 hours of treatment), and in vivo circulation time (from less than 5 minutes to 4.5 hours). The resulting synthetic nucleocapsids package one full-length RNA genome for every 11 icosahedral assemblies, similar to the best recombinant adeno-associated virus (AAV) vectors7, 8. Our results show that there are simple evolutionary paths through which protein assemblies can acquire virus-like genome packaging and protection. Considerable effort has been directed at “top-down” modification of viruses to be safe and effective for drug delivery and vaccine applications1, 9, 10; the ability to computationally design synthetic nanomaterials and to optimize them through evolution now enables a complementary “bottom-up” approach with considerable advantages in programmability and control.

Supercharging enables organized assembly of synthetic biomolecules

“…While previous studies have shown that it is possible to design synthetic protein oligomers either by modifying extant structures [13][14][15][16] or de novo, [8][9][10][11][12] we now show that it may be possible to induce the formation of novel quaternary structures through the simple expedient of supercharging. In so doing, we demonstrate that precise molecular configurations can be obtained by focusing on the driving forces for assembly (charge:charge interactions) rather than the details of the interface.…”

“…Control SuPrA protomers containing entirely Ceru+32, entirely GFP-17, or an octamer of Ceru+32 juxtaposed with an octamer of GFP-17 were unstable at all NaCl concentrations, consistent with the experimental observations ( Supplementary Fig. 12, 13) Finer-grained structural modeling (i.e., of hydrophobic effects and counterion release) must await specific structural and physical details that should become manifest with further imaging (i.e., cryo-EM) experiments that better determine the precise symmetric arrangement of monomers.While previous studies have shown that it is possible to design synthetic protein oligomers either by modifying extant structures [13][14][15][16] or de novo, [8][9][10][11][12] we now show that it may be possible to induce the formation of novel quaternary structures through the simple expedient of supercharging. In so doing, we demonstrate that precise molecular configurations can be obtained by focusing on the driving forces for assembly (charge:charge interactions) rather than the details of the interface.…”

“…To the extent that these hypotheses can be realized, the ability to readily engineer synthetic, scalable molecular assemblies into almost any protein via supercharging should prove useful for technologies ranging from pharmaceutical targeting to artifical energy harvesting to "smart" sensing and building materials. 1,16,17,39 All rights reserved. No reuse allowed without permission.…”

“…[1][2][3][4][5][6][7] Repeated, symmetric interactions between molecular building blocks enable the formation of complex, defined, assemblies from a small total number of components as well as dynamic propagation of conformational change. [3][4][5][6][7][8] However, while de novo engineering of artificial symmetrical biomolecular complexes has begun to be explored, [9][10][11][12][13][14][15] these efforts generally rely upon precise design of molecular interfaces. 16,17 In contrast, studies of simple synthetic colloids suggests that higher order structures can be derived based solely on packing and energetic and considerations.…”

“…[3][4][5][6][7][8] However, while de novo engineering of artificial symmetrical biomolecular complexes has begun to be explored, [9][10][11][12][13][14][15] these efforts generally rely upon precise design of molecular interfaces. 16,17 In contrast, studies of simple synthetic colloids suggests that higher order structures can be derived based solely on packing and energetic and considerations. Computational and experimental studies of polyhedral or spherical colloids indicate that complementary particle shapes 18-24 and simple attractive "patches" [24][25][26][27][28][29] alone can enable formation of complex symmetrical assemblies.…”