Cell‐Penetrating Peptides as Delivery Vehicles for a Protein‐Targeted Terbium Complex

Mohandessi, Shabnam; Rajendran, Megha; Magda, Darren; Miller, Leon K.

doi:10.1002/chem.201201805

Cited by 32 publications

(38 citation statements)

References 75 publications

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“…10 μM or greater concentrations of TMP-Lumi4-R 9 and other Lumi4-CPP analogs, even when incubation occurs at 4 °C. 24 By contrast, when the peptide conjugate concentration in culture medium is too low, a punctate staining pattern is observed that is suggestive of endocytosis (Figure 3a). Cytoplasmic delivery is desirable as it allows for targeting the widest variety of proteins.…”

Section: Resultsmentioning

confidence: 96%

“…19–23 Our lab recently showed that conjugation to nonaarginine or Tat-derived sequences allowed for cytoplasmic delivery of an otherwise membrane-impermeant Tb 3+ complex, Lumi4-Tb. 24 We further showed that CPP-coupled heterodimers of Lumi4-Tb and trimethoprim (TMP) rapidly (<30 min) entered the cytoplasm of various cell types, diffused freely throughout the cytoplasm and nucleus, and bound selectively to Escherichia coli dihydrofolate reductase (eDHFR) fusion proteins.…”

Section: Introductionmentioning

confidence: 97%

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Cytoplasmic Delivery and Selective, Multicomponent Labeling with Oligoarginine-Linked Protein Tags

et al. 2015

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Strategies that leverage bio-orthogonal interactions between small molecule ligands and genetically encoded amino acid sequences can be used to attach high-performance fluorophores to proteins in living cells. However, a major limitation of chemical protein labeling is that cells’ plasma membranes are impermeable to many useful probes and bio-labels. Here, we show that conjugation to nonaarginine, a cell penetrating peptide (CPP), enables passive cytoplasmic delivery of otherwise membrane-impermeant, small molecule protein labels. Heterodimers consisting of a luminescent Tb3+ complex, Lumi4®, linked to benzyl guanine, benzyl cytosine and trimethoprim were conjugated to the peptide CysArg9 with a reducible disulfide linker. When added to culture medium, the peptide conjugates rapidly (<30 min) enter the cytoplasm and diffuse freely throughout cells. The benzyl guanine, benzyl cytosine and trimethoprim derivatives bind selectively to fusion proteins tagged with SNAP-Tag®, CLIP-Tag® and E. coli dihydrofolate reductase (eDHFR), respectively. Furthermore, eDHFR and SNAP-Tag fusions can be labeled with Lumi4 analogs in the same cell, and this labeling can be detected using two-color, time-gated Förster Resonance Energy Transfer (FRET) microscopy. Finally, we present quantitative data showing that cytoplasmic uptake of nonaarginine-conjugated probes occurs in multiple cell types (MDCK, HeLa, NIH 3T3), most cells in a culture (>75%) are loaded with probe, and the cellular probe concentration can be controlled by varying incubation conditions. CPP-mediated delivery of Lumi4-linked protein labels will greatly increase the utility of lanthanide-based FRET microscopy. Moreover, our results strongly suggest that this approach can be adapted to deliver a wide variety of protein-targeted fluorophores or other functional probes that were previously unavailable for intracellular imaging studies.

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Section: Resultsmentioning

confidence: 96%

Section: Introductionmentioning

confidence: 97%

Cytoplasmic Delivery and Selective, Multicomponent Labeling with Oligoarginine-Linked Protein Tags

et al. 2015

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Section: Cellular Measurements Of Tb(iii) Luminescencementioning

confidence: 99%

“…2 A) (36). When added to culture medium, TMP-Lumi4-R 9 directly enters the cytoplasm of living cells and binds selectively to overexpressed E. coli dihydrofolate reductase (eDHFR) fusion proteins with high affinity (~1 nM K D ) (36)(37)(38). Time-gated images were acquired on an epifluorescence microscope equipped with an ICCD camera as the gated detector and an LED emitting at 365 nm as the pulsed light source (28).…”

Section: Cellular Measurements Of Tb(iii) Luminescencementioning

confidence: 99%

Evaluating the Performance of Time-Gated Live-Cell Microscopy with Lanthanide Probes

Rajendran

Miller

2015

Biophysical Journal

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Probes and biosensors that incorporate luminescent Tb(III) or Eu(III) complexes are promising for cellular imaging because time-gated microscopes can detect their long-lifetime (approximately milliseconds) emission without interference from short-lifetime (approximately nanoseconds) fluorescence background. Moreover, the discrete, narrow emission bands of Tb(III) complexes make them uniquely suited for multiplexed imaging applications because they can serve as Förster resonance energy transfer (FRET) donors to two or more differently colored acceptors. However, lanthanide complexes have low photon emission rates that can limit the image signal/noise ratio, which has a square-root dependence on photon counts. This work describes the performance of a wide-field, time-gated microscope with respect to its ability to image Tb(III) luminescence and Tb(III)-mediated FRET in cultured mammalian cells. The system employed a UV-emitting LED for low-power, pulsed excitation and an intensified CCD camera for gated detection. Exposure times of ∼1 s were needed to collect 5-25 photons per pixel from cells that contained micromolar concentrations of a Tb(III) complex. The observed photon counts matched those predicted by a theoretical model that incorporated the photophysical properties of the Tb(III) probe and the instrument's light-collection characteristics. Despite low photon counts, images of Tb(III)/green fluorescent protein FRET with a signal/noise ratio ≥ 7 were acquired, and a 90% change in the ratiometric FRET signal was measured. This study shows that the sensitivity and precision of lanthanide-based cellular microscopy can approach that of conventional FRET microscopy with fluorescent proteins. The results should encourage further development of lanthanide biosensors that can measure analyte concentration, enzyme activation, and protein-protein interactions in live cells.

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“…1-6 These features enable highly sensitive diagnostic assays, high throughput screening methods or optical imaging approaches that employ time-gated detection strategies. Time-gating delivers high signal-to-background ratio by exciting a sample with pulsed light and collecting long-lived emission signals after a brief delay that eliminates ns-scale fluorescence background.…”

Section: Introductionmentioning

confidence: 99%

Brightly Luminescent and Kinetically Inert Lanthanide Bioprobes Based on Linear and Preorganized Chelators

Mohamadi

Miller

2016

Bioconjugate Chem.

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The synthesis, photophysical properties and kinetic stability of a series of water-soluble, highly emissive Tb(III) and Eu(III) complexes featuring triethylenetetraamine hexaacetic acid (TTHA) and cyclohexyl triethylenetetraamine hexaacetic acid (cyTTHA) chelator scaffolds and carbostyril sensitizers are reported. The unique and modular design of the chelators gives rise to striking quantum yields of emission in aqueous solutions (up to 54%) as well as the characteristic lanthanides’ photophysical properties (long excited state lifetimes, large effective Stokes shifts, and narrow emission peaks). Furthermore, the pre-organized chelators (L3, L4 and L6) bind metal within minutes at ambient temperature yet exhibit substantial resistance to transchelation in the presence of a challenge solution (EDTA, 1 mM). Moreover, the Eu(III) complex of L4 remains stably luminescent in HeLa cells over hours, demonstrating the suitability of these compounds for live cell imaging applications. Representative chelators suitable for derivatization and protein bioconjugation were also prepared that were functionalized with clickable azide and alkyne moieties, biotin and trimethoprim (TMP). With exceptional long-wavelength brightness, enhanced kinetic inertness, and an adaptable synthetic route, the reported lanthanide complexes are promising probes and labels for time-gated bioanalysis, biosensing and optical microscopy.

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Cell‐Penetrating Peptides as Delivery Vehicles for a Protein‐Targeted Terbium Complex

Cited by 32 publications

References 75 publications

Cytoplasmic Delivery and Selective, Multicomponent Labeling with Oligoarginine-Linked Protein Tags

Cytoplasmic Delivery and Selective, Multicomponent Labeling with Oligoarginine-Linked Protein Tags

Evaluating the Performance of Time-Gated Live-Cell Microscopy with Lanthanide Probes

Brightly Luminescent and Kinetically Inert Lanthanide Bioprobes Based on Linear and Preorganized Chelators

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