Abstract：
:During cellular uptake of fluorescently labeled cell-penetrating peptides (CPPs), intense fluorescent signals are commonly observed in the nucleus of the cell, suggesting intracellular CPP relocation and potential binding to the genome of the host. We therefore investigated the interaction of the CPP HIV-1 Tat(47-57) with double-stranded DNA, and we also tested whether the fluorescence intensity of the labeled CPP allows for linear predictions of its intracellular concentration. Using isothermal titration calorimetry, we observe that the CPP has a high affinity for salmon sperm DNA as characterized by a microscopic dissociation constant of 126 nM. The binding is exothermic, with a reaction enthalpy of -4.63 kcal/mol CPP (28 degrees C). The dissociation constant and reaction enthalpy decrease further at higher temperatures. The affinity of the CPP for DNA is thus 1-2 magnitudes higher than for extracellular heparan sulfate, the likely mediator of the CPP uptake. Accordingly, the high affinity for DNA confers stability to extracellular transport complexes of CPP and DNA but potentially affects the regulation and molecular organization of the host's genome after nuclear uptake. Moreover, the CPP leads to the condensation of DNA as evidenced by the pronounced increase in light-scattering intensity. The fluorescence quantum yield of the FITC-labeled CPP decreases considerably at concentrations > 5 micromol/L, at pH < 7, and upon binding to DNA and glycosaminoglycans. This change in fluorescence quantum yield impedes the microscopic identification of uptake routes and the comparison of uptake efficiency of different CPPs, especially if the accumulation in subcellular compartments (self-quenching and pH difference) and transitory binding partners (quenching and condensation) is unknown.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Ziegler A,Seelig Jdoi
10.1021/bi700416hsubject
Has Abstractpub_date
2007-07-10 00:00:00pages
8138-45issue
27eissn
0006-2960issn
1520-4995journal_volume
46pub_type
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