RNA-based macromolecular machines, such as the ribosome, have functional parts reliant on structural interactions spanning sequence-distant regions. These features limit evolutionary exploration of mutant libraries and confound three-dimensional structure-guided design. To address these challenges, we describe Evolink (evolution and linkage), a method that enables high-throughput evolution of sequence-distant regions in large macromolecular machines, and library design guided by computational RNA modeling to enable exploration of structurally stable designs. Using Evolink, we evolved a tethered ribosome with a 58% increased activity in orthogonal protein translation and a 97% improvement in doubling times in SQ171 cells compared to a previously developed tethered ribosome, and reveal new permissible sequences in a pair of ribosomal helices with previously explored biological function. The Evolink approach may enable enhanced engineering of macromolecular machines for new and improved functions for synthetic biology.
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The authors declare that all experimental data supporting the findings of this study are available within the paper and its supplementary files. Publicly available data, such as the 4YBB (PDB) ribosome structure are mentioned explicitly when used. All data related to models are available upon request from the authors. The map and fitted model for the cryo-EM data are reported as Electron Microscopy Data Bank entry no. EMD-26666 and PDB structure 7UPH. Source data are provided with this paper.
All inputs and command files used in setting up computational modeling are available at https://github.com/everyday847/ribotv3_simulations.
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This work was supported by the National Science Foundation (grant no. MCB-1716766), the Human Frontiers Science Program (grant no. RGP0015/2017), the Army Research Office (grant no. W911NF-16-1-0372), all to M.C.J. R.D. thanks the NIGMS MIRA R35 award for funding. We thank J. Lucks and M. Evans at Northwestern and R. Kretsch at Stanford for discussions. Some of this work was performed at the Stanford-SLAC Cryo-EM Center (S2C2), which is supported by the National Institutes of Health Common Fund Transformative High-Resolution Cryo-Electron Microscopy program (grant no. U24 GM129541). The US Government is authorized to reproduce and distribute reprints for Governmental purposes notwithstanding any copyright notation thereon. The views and conclusions contained herein are those of the authors and should not be interpreted as necessarily representing the official policies or endorsements, either expressed or implied, of the US Government or the National Institutes of Health.
M.C.J. and D.S.K. are coinventors on the US provisional patent application that incorporates discoveries described in this manuscript. M.C.J. has a financial interest in Pearl Bio, and his interests are reviewed and managed by Northwestern University in accordance with their competing interest policies. All other authors declare no competing interests.
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Extended Data Fig. 1 Optimization of molecular biology steps involved in library preparation workflow of Evolink.
a, A clonal sample of the tethered ribosome (Ribo-T v2) is linearized using different oligos compatible with multiple ligation protocols. b, From the different ligation products, generation of final amplicon for next-generation sequencing can happen with a wide range of ligation methods and starting template amounts in the PCR. Gel data representative of two independent experiments.
a–c, Positively enriched genotypes (purple) and negative enriched genotypes (dark gray) can be tracked throughout multiple time points throughout selection. Genotypes that drop out during selection can also be identified (light gray). Corresponding heat maps that reveal trends in selected tether lengths also helped inform designs. Generally, across the three libraries tested in this work, (a) the Broad Sampling Library, (b) the Designed Junction Library, and (c) the Designed Junction + Length Refined Library, log2-fold enrichment values between -6 to 6 are observed. Enrichment and heatmap data representative of three independent experiments.
Extended Data Fig. 3 Score vs. Root-Mean-Standard-Deviation analysis of FARFAR2 simulations of enriched tether sequences.
a–d, For the Broad Sampling Library, we observe striking differences between simulations that constrained (blue) or did not constrain (orange) 3D structures of the Tether-H101 junction. Of the four modeled genotypes, two sequence (c,d) exhibit particularly substantial differences, hinting at structural instability in the Tether-H101 junction. e–h, When similar simulations are performed with enriched tether sequences from the Designed Junction Library (designed sequences at the Tether-H101 junction), the results of FARFAR2 simulations reach similarly low scores in constrained vs. unconstrained modeling runs.
Extended Data Fig. 4 Representative constrained and unconstrained 3D models of Designed Junction Library winner.
The winning genotype from Fig. 4h was modeled using Rosetta, and representative outputs are shown. In both the (a) unconstrained and (b) constrained model, the Designed Junction residues are predicted to base pair, reinforcing structural stability to this region.
Targeted structure probing was performed on the tethers of both RiboTv2 and RiboTv3 polysomes via DMS-MaPseq. The per-nucleotide chemical reactivities of the tethers and their adjacent rRNA stems can be seen in the figure for both RiboTv2 (a) and RiboTv3 (b). Gray shaded nucleotides represent U and G residues that are not modified by DMS.
Single-particle Cryo-EM was carried out on RiboTv3 polysomes. a, A representative raw micrograph shows that RiboTv3 polysomes look like characteristic ‘beads on a string’ as expected for actively translating ribosomes. b, A tethered ribosome that has dissociated from an mRNA looks like an open clamshell, as would be expected for a tethered ribosome. The large and small subunits are indicated by white arrows.
Raw count data of genotypes from the Broad Sampling Library experiment.
Raw count data of genotypes from Ribo-T junction library experiment.
Raw count data of genotypes from Designed Junction Library experiment.
Raw count data of genotypes from targeted Designed Junction Library experiment. Raw growth time series data. Raw GFP expression data. Raw mass spectrometry data.
Raw image of Evolink molecular biology validation gel.
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Kim, D.S., Watkins, A., Bidstrup, E. et al. Three-dimensional structure-guided evolution of a ribosome with tethered subunits. Nat Chem Biol 18, 990–998 (2022). https://doi.org/10.1038/s41589-022-01064-w
Nature Chemical Biology (2022)