This project aims at synthesizing new luminescent probes for the biological imaging of Zn2+. These probes are based on model peptides of zinc finger for Zn2+ binding and lanthanide complexes for near-infrared emission.
The main objective of the LUMZIF project is to develop Zn2+ selective probes for biological imaging of zinc. We aim at synthesizing probes that (i) selectively detect Zn2+ over other physiological cations, (ii) emit in the near-infrared window where (a) where light scattering is also reduced and (b) absorption and autofluorescence of the biological systems are reduced compared to the UV and visible regions that results in the minimization of damages to the biological system and in the maximization of detection sensitivity by improving signal to noise ratio, (iii) penetrate in cells, (iv) allow ratiometric quantification of Zn2+ and (v) display a wide range of affinities for Zn2+ in order to adapt the probe to the concentration of zinc that has to be imaged and that can range over several orders of magnitudes. <br />These probes will be based on zinc finger peptides because they have the ability to bind Zn2+ selectively and on lanthanide cations emitting in the near-infrared for optical signaling. The combination of these two elements has never been envisaged so far and appears highly promising for the design of valuable Zn2+ sensors meeting all the criteria for successful in vitro and in vivo imaging of zinc. <br />
Emission of a large amount of light by lanthanides requires an “antenna effect”, i.e. the sensitization of the lanthanide by a neighboring organic chromophore which, once excited, transfers its energy to the Ln3+ cation which can then emit photons. Therefore, we will graft on a zinc finger peptide a lanthanide complex and a suitable organic chromophoric sensitizer. We will take advantage of the Zn2+-induced folding properties of zinc finger peptides to bring the antenna and the lanthanide sufficiently close to each other to trigger the lanthanide emission in the presence of Zn2+. Ratiometric probes will be obtained based on a similar strategy. The flexibility in peptide synthesis and the facile synthesis of such probes by playing with non-natural amino acids and orthogonal protecting groups allows such design.
The elaboration of successful probes will rely on the optimization of the positions at which the antennae and the lanthanides will be grafted on the zinc finger peptides and on the choice of antennae suited to sensitize near-infrared lanthanides. Finally, in order to have the peptides internalized into cell and targeted to specific cellular location, we will use a cell penetrating peptide sequence that we will add to our zinc finger peptide.
The first results concerning terbium-based probes have validated the design and shown that the properties of the probe can be tuned by varying the positions of the lanthanide and its antenna. Regarding ratiometric probes, the first trials are rather encouraging and validate the chosen principle though work is still to be done to optimize the synthesis. We are currently working on the synthesis of non-natural amino acids incorporating chromophores able to sensitize near-infrared emitting lanthanides.
Zinc is involved in many cellular processes including several signaling processes. Imbalance of zinc homeostasis can be associated to severe disorders and diseases. The LUMZIF project aims at elaborating new probes emitting in the near-infrared for the biological imaging of zinc. We will take advantage of the luminescence properties of lanthanides emitting in near-infrared to overcome the drawbacks of classical Zn2+ probes based on organic dyes. We are confident that our approach will give rise to valuable Zn2+ sensors that could be used both in vitro and in vivo. Especially, probes for in vivo Zn2+ imaging will be helpful to biologist working in the field of zinc homeostasis and zinc related diseases.
Sensors are in high demand today and the possibilities of the intellectual protection of lanthanide luminescent probes for their bioanalytical and imaging applications are extremely broad. Therefore, this project should lead to several patents and publications in high impact scientific journals.
The aim of this project is to develop new sensors for selective biological imaging of Zn2+ based on zinc finger peptides for the metal ion binding and on near-infrared emitting lanthanides for the optical signalling.
Metal ions are essential for life. They are involved in many biological processes and their concentrations are finely regulated at the cellular and whole organism levels by a complex machinery of biomolecules. An imbalance of metal homeostasis in living organisms can cause severe disorders at the cellular level and diseases at the organism level. Among physiological metal ions, Zn2+ plays a crucial and complex role in many biological processes. This metal ion is an essential structural and catalytic co-factor for many proteins but it is also involved in several signalling pathways. Its concentration varies depending on the cellular compartment and important zinc flux mediate biological information. Several diseases such as Alzheimer’s or Parkinson’s diseases or cancer were discovered to be associated with altered Zn2+ homeostasis. In order to get deeper insights into the physiological role of zinc and the relation between the imbalance in zinc homeostasis and physiological disorders, new tools are required to image zinc and its flux in living organisms. Hence, the development of molecular probes for the optical imaging of zinc at the cellular level has attracted a growing attention during the past decade. However, probes elaborated to date are based on organic dyes and suffer from several drawbacks related to their organic nature which most of the time prevent their use to image zinc in biological samples. The main drawback resides in the excitation and emission wavelengths, in the UV or visible range, where considerable absorption, autofluorescence and light scattering originate from the biological media. The ideal probe which fulfils all the criteria for successful biological imaging still remains to be designed.
In this project, we will design, synthesize and characterize new smart zinc probes operating on the basis of Ln3+ ions emitting in the near-infrared and of zinc finger peptides for the selective binding of Zn2+ to ensure specific and sensitive response of the probe. The probe will benefit from the Zn2+-induced folding properties of zinc fingers to modulate the energy transfer between the sensitizing antenna and the emitting lanthanide. We will take advantage of the versatility of peptide synthesis to optimize the design of the proposed peptidic sensors with near-infrared lanthanide cations and corresponding aromatic antennae for the sensitization of these lanthanide cations. Using a stepwise rational approach, we will be able to create efficient ratiometric sensors emitting in the near-infrared and able to penetrate cells.
The strength of our project resides in the ability of this peptidic and lanthanide-based system to be tuned and optimized. An important level of novelty in this project resides in the use of near-infrared emitting lanthanide as these cations offer promising advantages for the ultimate targeted biological imaging applications.
We are confident that this approach will give rise to valuable Zn2+ sensors that could be used both in vitro and in vivo. Especially, probes for in vivo Zn2+ imaging will be helpful to biologist working in the field of zinc homeostasis and zinc related diseases.
Monsieur Olivier Seneque (Laboratoire de Chimie et Biologie des Métaux UMR 5249/Equipe Physicochimie des Métaux en Biologie) – firstname.lastname@example.org
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
CNRS/LCBM/PMB Laboratoire de Chimie et Biologie des Métaux UMR 5249/Equipe Physicochimie des Métaux en Biologie
CNRS-CBM Centre National de la Recherche Scientifique-Centre de Biophysique Moléculaire UPR 4301
Help of the ANR 434,000 euros
Beginning and duration of the scientific project: December 2012 - 42 Months