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.

基于 RNA 的大分子机器，例如核糖体，具有依赖于跨越序列距离区域的结构相互作用的功能部分。这些特征限制了突变体库的进化探索，并混淆了三维结构引导设计。为了应对这些挑战，我们描述了 Evolink（进化和连接），一种能够在大型大分子机器中实现序列远距离区域高通量进化的方法，以及由计算 RNA 建模指导的文库设计，以探索结构稳定的设计。使用 Evolink，我们进化出了一种栓系核糖体，与之前开发的栓系核糖体相比，SQ171 细胞的正交蛋白质翻译活性提高了 58%，倍增时间提高了 97%，并在具有先前探索的生物学功能的一对核糖体螺旋中揭示新的允许序列。Evolink 方法可以增强大分子机器的工程设计，以实现合成生物学的新功能和改进功能。