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Here we developed an orange light-absorbing chromoprotein named ShadowR as a novel acceptor for performing fluorescence lifetime imaging microscopy-based Förster resonance energy transfer (FLIM-FRET) measurement in living cells. ShadowR was generated by replacing hydrophobic amino acids located at the surface of the chromoprotein Ultramarine with hydrophilic amino acids in order to reduce non-specific interactions with cytosolic proteins. Similar to Ultramarine, ShadowR shows high absorption capacity and no fluorescence. However, it exhibits reduced non-specific binding to cytosolic proteins and is highly expressed in HeLa cells. Using tandem constructs and a LOVTRAP system, we showed that ShadowR can be used as a FRET acceptor in combination with donor mRuby2 or mScarlet in HeLa cells. Thus, ShadowR is a useful, novel FLIM-FRET acceptor.

ShadowR: a novel chromoprotein with reduced non-specific binding and improved expression in living cells