Tight Binding of a Dimeric Derivative of Vancomycin with Dimeric l-Lys-d-Ala-d-Ala

The ligand/receptor pair consisting of a synthetic dimeric derivative of vancomycin (V), linked at the C terminus by p-xylylenediamine (V-CONHCH2C6H4CH2NHCO-V), and a dimeric derivative of l-Lys-d-Ala-d-Ala, [CH2CONεH(Nα-Ac)-l-Lys-d-Ala-d-Ala-CO2-]2, provides a new system with which to study the influence of divalency on the strength of binding. A competitive assay using affinity capillary electrophoresis (ACE) has been developed and used to estimate the dissociation constant of the divalent complex (≈ 1.1 nM) and the enhancement in binding (∼103) relative to the corresponding monomeric interaction between unmodified monomeric vancomycin and diacetyl-l-Lys-d-Ala-d-Ala

Single-Cell Detection of Trans-Splicing Ribozyme In Vivo Activity

The Tetrahymena trans-splicing ribozyme can edit RNA in a sequence-specific manner, but its efficiency needs to be improved for any functional rescues. This communication describes a simple method that uses a bacterial enzyme β-lactamase to report trans-splicing activity of Tetrahymena ribozyme in single living mammalian cells by fluorescence microscopy and flow cytometry. This enzyme-based single-cell detection method is highly sensitive and compatible with living cell flow cytometry, and should allow a cell-based systematic screening of a vast library of ribozymes for better trans-spliced ribozyme variants

Cell-Permeable Near-Infrared Fluorogenic Substrates for Imaging β-Lactamase Activity

This communication describes a design of cell-permeable near-infrared fluorogenic substrates for imaging β-lactamase expression in living mammalian cells. This design is based on fluorescence energy transfer resonance and utilizes a peracetylated d-glucosamine to facilitate the transport of the near-infrared probe across cell membranes. This new type of fluorogenic probe may also be applied to image gene expression in living animals

In Vivo Bioluminescence Imaging of Furin Activity in Breast Cancer Cells Using Bioluminogenic Substrates

Furin, a proprotein convertases family endoprotease, processes numerous physiological substrates and is overexpressed in cancer and inflammatory conditions. Noninvasive imaging of furin activity will offer a valuable tool to probe furin function over the course of tumor growth and migration in the same animals in real time and directly assess the inhibition efficacy of drugs in vivo. Here, we report successful bioluminescence imaging of furin activity in xenografted MBA-MB-468 breast cancer tumors in mice with bioluminogenic probes. The probes are conjugates of furin substrate, a consensus amino acid motif R-X-K/R-R (X, any amino acid), with the firefly luciferase substrate d-aminoluciferin. In the presence of the luciferase reporter, the probes are unable to produce bioluminescent emission without furin activation. Blocking experiments with a furin inhibitor and control experiments with a scrambled probe showed that the bioluminescence emission in the presence of firefly luciferase is furin-dependent and specific. After furin activation, a 30-fold increase in the bioluminescent emission was observed in vitro, and on average, a 7−8-fold contrast between the probe and control was seen in the same tumor xenografts in mice. Direct imaging of furin activity may facilitate the study of furin function in tumorigenicity and the discovery of new drugs for furin-targeted cancer therapy

Protease-Modulated Cellular Uptake of Quantum Dots

Quantum dots (QDs) are often cell-impermeable and require transporters to facilitate crossing over cell membranes. Here we present a simple and versatile method that utilizes enzymes, matrix metalloprotease 2 (MMP-2) and MMP-7, to modulate the cellular uptake of QDs. QD-peptide conjugates could be efficiently taken up into cells after the MMP treatment. This enzyme-modulated cellular uptake of QDs may be applied to other nanoparticles for biological imaging and selective drug delivery into tumor cells

Near-Infrared Light Emitting Luciferase via Biomineralization

Near-Infrared Light Emitting Luciferase via Biomineralizatio

Novel Fluorogenic Substrates for Imaging β-Lactamase Gene Expression

A new class of small nonfluorescent fluorogenic substrates becomes brightly fluorescent after β-lactamase hydrolysis with up to 153-fold enhancement in the fluorescence intensity. Less than 500 fM of β-lactamase in cell lysates can be readily detected, and β-lactamase expression in living cells can be imaged with a red fluorescence derivative. These new fluorogenic substrates should find uses in clinical diagnostics and facilitate the applications of β-lactamase as a biosensor

Design, Synthesis, and Characterization of a High-Affinity Trivalent System Derived from Vancomycin and l-Lys-d-Ala-d-Ala

A trivalent derivative of vancomycin, tris(vancomycin carboxamide), [C6H3-1,3,5-(CONHC6H4-4-CH2NHCOV)3 (RtV3; V = vancomycin)], binds an analogous trivalent derivative of d-Ala-d-Ala, R‘tL‘3, (C6H3-1,3,5-[CONεH(Nα-Ac)-l-Lys-d-Ala-d-Ala]3) in water with a dissociation constant that is approximately 4 × 10-17 M, as estimated by HPLC using a competitive assay against Nα,ε-diacetyl-l-Lys-d-Ala-d-Ala (L). This binding is one of the tightest known for low molecular weight organic species. The dissociation of RtV3·R‘tL‘3 in the presence of an excess of L could be followed by HPLC. The kinetics of dissociation are quite different from those of monovalent tight-binding systems such as avidin and biotin. In particular, the rate of dissociation of the aggregate RtV3·R‘tL‘3 is rapid in the presence of monovalent L at concentrations greater than the value of the dissociation constant for the complex of L with V; by contrast, the rate of dissociation of biotin·avidin is independent of the concentration of biotin. Two mechanisms by which the dissociation may occur are postulated and discussed. Calorimetric measurements for the trivalent system indicate that the enthalpy of association is ∼−40 kcal/mol, about three times that of V + L, and thus the entropy of association is ∼−18 kcal/mol, approximately 4.5 times that of V + L

A Magneto-Optical Nanoplatform for Multimodality Imaging of Tumors in Mice

Multimodality imaging involves the use of more imaging modes to image the same living subjects and is now generally preferred in clinics for cancer imaging. Here we present multimodalityMagnetic Particle Imaging (MPI), Magnetic Resonance Imaging (MRI), Photoacoustic, Fluorescentnanoparticles (termed MMPF NPs) for imaging tumor xenografts in living mice. MMPF NPs provide long-term (more than 2 months), dynamic, and accurate quantification, in vivo, of NPs and in real time by MPI. Moreover, MMPF NPs offer ultrasensitive MPI imaging of tumors (the tumor ROI increased by 30.6 times over that of preinjection). Moreover, the nanoparticle possessed a long-term blood circulation time (half-life at 49 h) and high tumor uptake (18% ID/g). MMPF NPs have been demonstrated for imaging breast and brain tumor xenografts in both subcutaneous and orthotopic models in mice via simultaneous MPI, MRI, fluorescence, and photoacoustic imaging with excellent tumor contrast to normal tissues