Synonymous Modification Enables High Fidelity Expression of Biosensors and Probes with Repetitive Protein and Nucleotide Sequences

Abstract

Repetitive nucleotide or amino acid sequences are often engineered into biosensors and probes to achieve functional readouts and robust signal amplification. However, these repeated sequences are notoriously difficult to construct and maintain for stable expression. They are easily deleted or truncated, which occurs randomly and is not a priori predictable, resulting in aberrant expression and impacting the ability to correctly detect and to interpret biological functions. Here, we introduce a simple and generally applicable approach to solve this often unappreciated problem by modifying the nucleotide sequences of the target mRNA to make them non-repetitive but still functional (“synonymous”). We first demonstrate the procedure by designing a cassette of synonymous MS2 RNA aptamers, and tandem coat proteins for RNA imaging and show a dramatic improvement in signal and reproducibility in single RNA detection in live cells. The same approach is extended to enhance the stability of engineered fluorescent biosensors containing FRET-pair of fluorescent proteins, upon which a great majority of systems thus far in the field are based. Using the synonymous modified FRET biosensors, we achieve correct expression of full-length sensors, eliminating the aberrant truncation products that were often assumed to be due to non-specific proteolytic cleavages. Importantly, the biological interpretations of the sensor are significantly different when a correct, full-length biosensor is expressed. Thus the method we describe here should be routinely employed in generation of probes and sensors containing multiple, repetitive motifs to achieve correct expression these state-of-the-art tools.