Labeling with BODIPY fluorophores

The device has been used to capture DNA sequences in very complex mixtures [45]. For the experiment shown in Fig. 10, a model target sequence consisting of a 75-mer based on a sequence from group A Streptococcus pyogenes (GAS) was synthesized and labeled with the fluorophore Bodipy Texas Red. In addition, the unlabeled complement to the 75-mer was synthesized. Equimolar amounts of the two oligonucleotides in amounts varying from 0.1 to 10 nM were combined with 2 pg sheared and denatured human DNA with a final volume of... 

BODIPY fluorophores are a class of probes based on the fused, multi-ring structure, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (Figure 9.27) (Invitrogen) (U.S. patent 4,774,339). This fundamental molecule can be modified, particularly at its 1, 3, 5, 7, and 8 carbon positions, to produce new fluorophores with different characteristics. The modifications cause spectral shifts in its excitation and emission wavelengths, and can provide sites for chemical coupling to label biomolecules. 

BODIPY 530/550 C3 is insoluble in aqueous solution, but it may be dissolved in DMF or DMSO as a concentrated stock solution prior to addition of a small aliquot to a reaction. Coupling to amine-containing molecules may be done using the EDC/sulfo-NHS reaction as discussed in Chapter 3, Section 1.2 (Figure 9.29). However, modification of proteins with this fluorophore probably won t yield satisfactory results, since BODIPY fluorophores are easily quenched if substitutions on a molecule exceed a 1 1 stoichiometry. For labeling molecules which contain only one amine group, such as DNA probes modified at the 5 end to contain an amine (Chapter 27, Section 2.1), BODIPY 530/550 C3 will give intensely fluorescent derivatives. 

Since BODIPY fluorophores are easily quenched if substitutions on a molecule exceed a 1 1 stoichiometry, modification of proteins with this fluorophore probably will not yield satisfactory results. However, for labeling molecules that contain only one amine group, BODIPY FL C3-SE will give intensely fluorescent derivatives. 

Several BODIPY fluorophores with different excitation and emission maxima are available, and each can have alternative linkage chemistries. It is important to note that the linkage chemistry affects the fluorescence intensity. One cannot therefore quantitatively compare peptides labelled using different linkage chemistries. Once having selected a fluorophore/ linkage chemistry it is better to stick with it. 

Direct labeling of a biomolecule involves the introduction of a covalently linked fluorophore in the nucleic acid sequence or in the amino acid sequence of a protein or antibody. Fluorescein, rhodamine derivatives, the Alexa, and BODIPY dyes (Molecular Probes [92]) as well as the cyanine dyes (Amersham Biosciences [134]) are widely used labels. These probe families show different absorption and emission wavelengths and span the whole visible spectrum (e.g., Alexa Fluor dyes show UV excitation at 350 nm to far red excitation at 633 nm). Furthermore, for differential expression analysis, probe families with similar chemical structures but different spectroscopic properties are desirable, for example the cyanine dyes Cy3 and Cy5 (excitation at 548 and 646 nm, respectively). The design of fluorescent labels is still an active area of research, and various new dyes have been reported that differ in terms of decay times, wavelength, conjugatibility, and quantum yields before and after conjugation [135]. New ruthenium markers have been reported as well [136]. 

Consequently, we decided to use a lipidic substrate equipped with an appropriate fluorogenic moiety to measure lipase activity. Importantly, even huge moieties, such as BODIPY [23], rhodamine [24] or pyrene [25] coupled to typical lipase substrates usually do not interfere with their cleavage by lipolytic enzymes. The size of the fluorophore does not seem to be very critical. In general, it should be as small as possible, lipophilic, stable and insensitive to oxidation. For these reasons we preferred p-nitrobenzofurazan (NBD) as a fluorescent label. 

In addition to nonspecific lysine labeling. Lien et al. reported site-specific incorporation of a fluorescent tag into nascent proteins using a Cys-tRNA . After aminoacylation of E. coli Cys-tRNA , it was modified with a SH-reactive fluorophore (BODIPY-FL). This method may prove valuable when used in conjunction with site-directed mutagenesis to create protein containing single-cysteine residues by which one can prepare the nascent protein containing single fluorophore at a defined position. 

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