Fluorescent Protein Variants Generated by Reassembly between Skeleton and Chromophore

[Image: see text] Fluorescent proteins (FPs) can be used as intrinsic molecular tags to track the dynamic activity in live cells. To obtain variants in an available and massive manner is always a challenge. Here, we adopted a computer-based microarray synthesis method to realize the reassembly between the chromophore and the skeleton. DNAWorks was used to segment the input FP templates into a set of overlapping oligonucleotides (20–43 mer) with a balanced annealing temperature, G + C content, and codon frequency. The constitution of the chromophore was kept in the same section by switching the divided sites during segmentation and the codon was optimized to further keep the balanced parameters. The designed oligonucleotides were synthesized on photo-programmable microfluidic arrays. Sequence analysis and the subsequent conditional induced expression of FPs revealed that oligonucleotides were highly reassembled. Spectra, photostability, and molecular size detection of randomly selected variants showed that they were distinct monomeric proteins that preserved photoactivity. Our study provides an effective means of obtaining FP variants based on a computer-designed parallel synthesis.