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An improved cell-penetrating, caspase-activatable, near-infrared fluorescent peptide for apoptosis imaging. Bioconjug Chem 2009 Apr;20(4):702-9



Pubmed ID


Pubmed Central ID




Scopus ID

2-s2.0-65249177729 (requires institutional sign-in at Scopus site)   104 Citations


Apoptosis is required for normal cellular homeostasis, and deregulation of the apoptotic process is implicated in various diseases. Previously, we developed a cell-penetrating near-infrared fluorescence (NIRF) probe based on an activatable strategy to detect apoptosis-associated caspase activity in vivo. This probe consisted of a cell-penetrating Tat peptide conjugated to an effector recognition sequence (DEVD) that was flanked by a fluorophore-quencher pair (Alexa Fluor 647 and QSY 21). Once exposed to effector caspases, the recognition sequence was cleaved, resulting in separation of the fluorophore-quencher pair and signal generation. Herein, we present biochemical analysis of a second generation probe, KcapQ, with a modified cell-penetrating peptide sequence (KKKRKV). This modification resulted in a probe that was more sensitive to effector caspase enzymes, displayed an unexpectedly higher quenching efficiency between the fluorophore-quencher pair, and was potentially less toxic to cells. Assays using recombinant caspase enzymes revealed that the probe was specific for effector caspases (caspase 3 > 7 > 6). Analysis of apoptosis in HeLa cells treated with doxorubicin showed probe activation specific to apoptotic cells. In a rat model of retinal neuronal excitotoxicity, intravitreal injection of N-methyl-d-aspartate (NMDA) induced apoptosis of retinal ganglion cells (RGCs). Eyecup and retinal flat-mount images of NMDA-pretreated animals injected intravitreally with KcapQ using a clinically applicable protocol showed specific and widely distributed cell-associated fluorescence signals compared to untreated control animals. Fluorescence microscopy images of vertical retinal sections from NMDA-pretreated animals confirmed that activated probe was predominantly localized to RGCs and colocalized with TUNEL labeling. Thus, KcapQ represents an improved effector caspase-activatable NIRF probe for enhanced noninvasive analysis of apoptosis in whole cells and live animals.

Author List

Maxwell D, Chang Q, Zhang X, Barnett EM, Piwnica-Worms D


Edward M. Barnett MD, PhD Professor in the Ophthalmology and Visual Sciences department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Amino Acid Sequence
Caspase Inhibitors
Cell Survival
Enzyme Activation
Enzyme Inhibitors
Fluorescent Dyes
HeLa Cells
Infrared Rays
Retinal Ganglion Cells
Spectrophotometry, Ultraviolet