High-affinity tuning of single fluorescent protein-type indicators by flexible linker length optimization in topology mutant.

Genetically encoded Ca 2+ indicators (GECIs) are versatile for live imaging of cellular activities. Besides the brightness and dynamic range of signal change of GECIs, Ca 2+ affinity is another critical parameter for successful Ca 2+ imaging, as the concentration range of Ca 2+ dynamics differs from low nanomolar to sub-millimolar depending on the celltype and organism. However, ultrahigh-affinity GECIs, particularly the single fluorescent protein (1FP)-type, are lacking. Here, we report a simple strategy that increases Ca 2+ affinity through the linker length optimization in topology mutants of existing 1FP-type GECIs. The resulting ultrahigh-affinity GECIs, CaMPARI-nano, BGECO-nano, and RCaMP-nano (K d  = 17-25 nM), enable unique biological applications, including the detection of low nanomolar Ca 2+ dynamics, highlighting active signaling cells, and multi-functional imaging with other second messengers. The linker length optimization in topology mutants could be applied to other 1FP-type indicators of glutamate and potassium, rendering it a widely applicable technique for modulating indicator affinity.