Overcoming the design, build, test (DBT) bottleneck for synthesis of nonrepetitive protein-RNA binding cassettes for RNA applications

Abstract

AbstractThe design-build-test (DBT) cycle in synthetic biology is considered to be a major bottleneck for progress in the field. The emergence of high-throughput experimental techniques, such as oligo libraries (OLs), combined with machine learning (ML) algorithms, provide the ingredients for a potential “big-data” solution that can generate a sufficient predictive capability to overcome the DBT bottleneck. In this work, we apply the OL-ML approach to the design of RNA cassettes used in gene editing and RNA tracking systems. RNA cassettes are typically made of repetitive hairpins, therefore hindering their retention, synthesis, and functionality. Here, we carried out a high-throughput OL-based experiment to generate thousands of new binding sites for the phage coat proteins of bacteriophages MS2 (MCP), PP7 (PCP), and Qβ (QCP). We then applied a neural network to vastly expand this space of binding sites to millions of additional predicted sites, which allowed us to identify the structural and sequence features that are critical for the binding of each RBP. To verify our approach, we designed new non-repetitive binding site cassettes and tested their functionality in U2OS mammalian cells. We found that all our cassettes exhibited multiple trackable puncta. Additionally, we designed and verified two additional cassettes, the first containing sites that can bind both PCP and QCP, and the second with sites that can bind either MCP or QCP, allowing for an additional orthogonal channel. Consequently, we provide the scientific community with a novel resource for rapidly creating functional non-repetitive binding site cassettes using one or more of three phage coat proteins with a variety of binding affinities for any application spanning bacteria to mammalian cells.