Light-mediated spatial control via photolabile fluorescently quenched peptide cassettes

Abstract

Light-regulatable compounds are finding increasing utility as spatial and temporal probes of biological behavior. An independent measure of successful light-induced structural change is possible when alteration (e.g., activation, deactivation, etc.) of the bioprobe can be directly linked to a fluorescent readout. We have identified a series of photolabile fluorescently quenched cassettes that display extraordinarily large fluorescence enhancements upon photolysis. A pair of cassettes has been inserted into mitochondrial localization sequences to assess an organelle-targeted light-mediated release strategy for controlling biological activity. The peptide constructs are readily absorbed by mitochondria and subsequently can be cleaved in a light-dependent fashion as assessed by the predicted changes in absorbance and fluorescence.

Figure 1

Mitochondrial-targeted light-mediated release strategy. Photolysis releases the quencher (and any associated biochemically active agent “X”) from the mitochondrial surface into the cytoplasm.

Figure 2

HeLa cells exposed to CPMLS-4-5-7 and mitotracker FarRed. Row A (unphotolyzed) and Row B (photolyzed), Column 1 (Cy 3 window, green, TAMRA-labeled peptide), Column 2 (Cy 5.5 window, red, mitotracker FarRed), Columns 3 (merged Columns 1 and 2, orange signifies regions of overlap, Pearson coefficient: 0.88 ± 0.03), and green circle (laser focus).

Chart 1

Quenched fluorescent cassette library (1 and 2) derived from fluorophores 3 and 4, photolabile linkers 5 and 6, and quenchers 7 – 10.