Fluorophores FRET pair

The wide variety of absorption and emission wavelengths has allowed the use of different coumarin derivatives as FRET pairs [37], Contrary to most fluorophores, coumarin is uncharged which makes it intrinsically membrane-permeant. To induce water solubility polar groups are frequently introduced to the basic structure [35],... 

An important feature that influences the performance of the probe to a large extent is the selection of the FRET pair. Generally, fluorophores with high excitation wavelengths are preferred over blue fluorophores in single photon microscopy for applications in tissue since there is no autofluorescence in this region and the ratio... 

Proteases are one of the largest families of enzymes and are involved in a multitude of vital processes. Due to their biological relevance and diversity, multiple fluorescent reporters monitoring their activity have been designed and successfully applied in vitro and in vivo [112-114]. Standard small molecule FRET probes for proteases consist of an amino acid sequence flanked by a FRET pair, consisting of two fluorophores or one fluorophore and a quencher molecule. Upon cleavage of the peptide sequence, the emission of the donor fluorophore is dequenched and the intensity increases whereas the emission of the acceptor decreases and vanishes more or less completely in those cases where the acceptor is fluorescent (see Fig. 6.11). 

EPL, a FRET pair, tetramethylrhodamine and fluorescein, was incorporated in c-Crk-II. By judicious placement of the fluorophores within the c-Crk-II molecule, it was possible to monitor the phosphorylation state of the protein using FRET measurements (Fig. 10.1-6). In a subsequent study, an extremely sensitive dual labeled c-Crk-II analog was developed that enabled real-time monitoring of c-Abl kinase activity, and provided a nonradioactive assay for the screening of potential inhibitors of the kinase [69). 

This technique is used to sense the proximity of two fluorescently labeled molecules. The fluorophore couple is chosen to have a donor that in the excited state transfers its energy by dipole-dipole coupling to an acceptor, that is, the second fluorophore, which re-emits the light at a longer wavelength [117]. Among others, FRET pairs with appropriate spectral overlaps are BFP-GFP or CFP-YFP. Because the transfer efficiency correlates with the inverse sixth power of the distance between the fluorophores and depends on their spatial orientation, the experimental detection of FRET is possible only for pairs separated in space by 1-10 nm. If position and orientation of the fluorophore pair are favorable and ERET occurs, the fluorescent emission of the donor is quenched whereas the acceptor begins to fluoresce. The transfer efficiency can be derived by the ratio of the two emissions [118]. 

In principle, any couple of fluorophores can be used for FRET, provided that the emission spectrum of the donor overlaps with the absorption of the acceptor. For a review of FRET-couples (and RO values) of chemical dyes see [62]. Furthermore, donors with a high fluorescence quantum-yield and acceptors with a high molar absorbance will display increased FRET. For FLIM it will be important to tune the instrument-performance to ensure maximal sensitivity to small changes in lifetimes at the control donor lifetime. Usually this is easily achieved. Many FRET-pairs have been used for FRET-FLIM including chemical probes as Fluorescein-Rhodamine [54,93],calcein-sulforhodamine B [94], and Cy3-Cy5, [70]. Since 1996, the availability of genetic-encoded fluorophores such as CFP, GFP, YFP has boosted application of FRET-FLIM enormously [95]. Nowadays fluorescent-tagging of proteins no longer depends on laborious protein pu-... 

Mandecki, W. FRET-based single molecule fluorescence assay to sequence nucleic acids using ribosomal translation component labeled with fluorophore-quencher pairs. U.S. Pat. Appl. Publ. US 2005282173, 2005 Chem. Abstr. 2005, 144, 66376. 

Fluorescence or Forster resonance energy transfer (FRET) is widely accepted as being one of the most useful methods to observe biochemical events in vitro and in living cells. Generally, there are two forms of FRET sensors those based on a pair of genetically encoded fluorophores, usually employing fluorescent proteins from jellyfish or corals, or those based on small molecules that make use of small organic fluorophores. 

Many of the strategies for measuring FRET from spectral images that were mentioned above have been implemented to study FRET. We will now cover sRET [12], a specific implementation that uses the last approach where FRET is measured from a pair of spectral images collected at different excitation wavelengths. Recently, the sRET approach has been extended to explicitly consider paired and unpaired fluorophores, the impact of incomplete labeling (or for fluorescent proteins fractional maturation), and the... 

FRET is an extremely useful phenomenon when it comes to the analysis of molecular conformations and interactions. F or the analysis of interactions, in which two separate molecules are labeled with an appropriate pair of fluorophores, an interaction can be shown by observing FRET. Further, FRET can be used as a type of spectroscopic ruler to measure the closeness of interactions. Proteins, lipids, enzymes, DNA, and RNA can all be labeled and interactions documented. This general method can be applied not only to questions of cellular function like kinase dynamics [3] but also to disease pathways, for example, the APP-PS1 interaction that is important in Alzheimer s disease (AD) [4], Alternatively, two parts of a molecule of interest can be labeled with a donor and acceptor fluorophore. Using this technique, changes in protein conformation and differences between isoforms of proteins can be measured, as well as protein cleavage. 

The two most established methods of labeling a molecule or pair of molecules to make a FRET measurement are immunolabeling and fusion to genetically encoded FPs like GFP. Although these are well established techniques, they have certain drawbacks and so novel sensors like QDs and labeling techniques, like cysteine-reactive fluorophores continue to be developed and offer great promise for the future. 

FRET requires the presence of two fluorophores, one with a shorter emission wavelength (donor) and another with a longer emission wavelength (acceptor). The fluorophores must be chosen such that there is sufficient overlap of the donor emission spectrum and the acceptor excitation spectrum. When FRET occurs, which requires the proximity of the two fluorophores, excitation of the donor results in transfer of energy to the acceptor and, hence, emission at the wavelength characteristic for the acceptor. FRET can be seen with various kinds of fluorophores, but most recendy it has been used in particular with variants of GFPs because this permits FRET in intact cells. The most frequently used pairs of GFPs are the cyan fluorescent protein (CFP) and the yellow fluorescent protein (YFP) variants. The donor CFP is excited at its maximum... 

The duplex probe configuration provides for a close proximity of the donor/acceptor pair and may lead to two mechanisms of quenching FRET and direct transfer (contact-mediated) quenching [78]. Placement of the fluorophore and quencher toward the centre of the duplex probe sequence may further add to the quenching efficiency as the ends of duplexes are known to breathe and are not as tightly bound as internal base pairs. Duplex probes are relatively easy to synthesize as the fluorophore and quencher moieties do not have to be incorporated into the same strand. 

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