Fig. 2: Generation of diversified sensor sequences with the ABCD approach.

a Schematic representation of the combinatorial codon scrambling algorithm proposed by Tang et al. and the abundance-biasing modification that defines the ABCD algorithm. The efficient exploration of the very large sequence space and the estimation of the interaction energy between subsequence pairs enable a very high degree of diversification, while the codon biasing prevents the usage of low-abundance codons, diminishing the chances of impacting expression. b Scheme of the algorithm used in this work to diversify FP FRET pairs, using the original YFP sequence as an internal reference to produce a new YFP sequence that is maximally diverse from it. The diversified YFP sequence is then compared with the CFP sequence of the corresponding FRET donor. c Algorithm scheme for the simultaneous diversification of three FPs. After a first round of diversification, the diversified sequence is included in the reference sequence for a second round of diversification. d Obtained f values for the Citrine diversification runs (yellow) and after substitution of the reference Citrine sequence with the ECFP one (blue). The impact of both the FP switch and the different codon abundance bias is negligible, and all f values are below the approximative threshold for recombination observed by Tang et al. in their PCR study. e Distribution of identical subsequence length for different FRET pairs. While the original ECFP/Citrine pair contains very long identical stretches, all pairs containing a codon-diversified Citrine compare very favorably not only with the original pair, but also with the “naturally diversified” mTFP1/Venus pair. f Dot plots highlighting the presence of identical subsequences among FRET pairs. Codon diversification not only removes the very long stretches along the diagonal originating from the two FPs sharing a common ancestor, but also improves the diversity in the out-of-diagonal regions, corresponding to sequence comparisons involving a nucleotide shift between the donor and acceptor sequences.