Fluorophores cyanines

The tunability on emission wavelength of cyanine derivatives is based on the understanding of structure-photophysical property relationships, which allows the development of near-IR fluorophores [80, 84—87]. Enhancement of the rigidity in... 

DNA modified with a diamine compound to contain terminal primary amines may be coupled with amine-reactive fluorescent labels. The most common fluorophores used for oligonucleotide labeling are the cyanine dyes and derivatives of fluorescein and rhodamine (Chapter 9). However, any of the amine-reactive labels discussed throughout Chapter 9 are valid candidates for DNA applications. 

Figure 16 Fluorophores with efficient chemical excitation in the PO-CL reaction. TMP, 2,4,6,8-tetrathiomorpholinopyrimido 5,4-rf pyrimidine DTDCI, 3,3 -diethylthiadicarbo-cyanine iodide.

The optical properties of organic dyes (Fig. ld-f, Table 1) are controlled by the nature of the electronic transition(s) involved [4], The emission occurs either from an electronic state delocalized over the whole chromophore (the corresponding fluorophores are termed here as resonant or mesomeric dyes) or from a charge transfer (CT) state formed via intramolecular charge transfer (ICT) from the initially excited electronic state (the corresponding fluorophores are referred to as CT dyes) [4], Bioanalytically relevant fluorophores like fluoresceins, rhodamines, most 4,4 -difluoro-4-bora-3a,4a-diaza-s-indacenes (BODIPY dyes), and cyanines (symmetric... 

Malicka J, Gryczynski I, Gryczynski Z, Lakowicz JR (2003) Effects of fluorophore-to-silver distance on the emission of cyanine-dye-labeled oligonucleotides. Anal Biochem 315 57-66... 

Cyanines have been widely used as laser dyes, and as saturable absorbers in modelocked and Q-switched laser systems. 8, 50) The propensity of most cyanines to photooxidize which makes them useful in photographic film and as saturable absorbers makes them less than desirable as fluorophores in other applications. The use of... 

Wessendorf, M. W. and Brelje, T. C. (1992) Which fluorophore is brightest-A comparison of the staining obtained using fluorescein, tetramethylrhodamine, lissamine rhodamine, Texas Red, and cyanine 3.18. Histochem. 98, 81-85. 

Brightness. Brighmess of a fluorophore is proportional to the product of the molar absorption coefficient at the excitation wavelength times its quantum yield. This is the theoretical value, but in practice it can be much reduced by fluorescent quenching on interaction with other labels on the protein or DNA surfaces. Sulfonic acid groups on the aromatic rings of cyanines reduce this interaction, giving very much improved protein fluorescence. 

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]. 

Much attention has been focussed lately on the family of asymmetric cyanine dyes for use in fluorescence detection of nucleic acids. These dyes show a significant enhancement in fluorescence intensity (100- to 1000-fold) upon binding to double-stranded DNA as compared to that from the fluorophore in solution. Use of cyanine fluorophores may be advantageous for use in assay design and sensor applications with respect to some of the more commonplace dyes, such as ethidium bromide and Hoechst 33342, as these latter dyes exhibit significant fluorescence intensity as background when in solution and have significantly lower enhancement in emission intensity [42]. 

Cyanines are by far the most popular and extensively researched chromo-phore for use in biological and medical imaging applications [70,72], They are used almost exclusively in fluorescence-related protocols. Their popularity may be attributable to factors such as their excellent synthetic flexibility and fluorescence properties, the latter being very high in some instances. Cyanines are the only synthetic near-infrared fluorophores which are commercially available (for... 

Squarylium dyes such as (83) [75] have probably received less attention than cyanine dyes due to the fact that the majority of syntheses furnish symmetrical species which are difficult to monofunctionalize in reactions such as the formation of peptide conjugates. The unsymmetrical types have been reported but seem to suffer from about 50% decrease in extinction coefficient. Squaiyliums are also more difficult to handle due to their low solubility. Very few water-soluble systems have been reported. These compounds are also used exclusively as fluorophores, but quantum yields are highly dependent on substituents and environment. 

By contrast, the cyanine fluorophores interact strongly with DNA and RNA. We have shown that both Cy3 (Norman et al., 2000) and Cy5 (Iqbal... 

Figure 8.2 Orientation of transition moments of cyanine fluorophores terminally attached to double-stranded DNA. (A) The orientation parameter K2. The transition dipole vectors for the coupled donor and acceptor fluorophore are indicated by the arrows, labeled D and A. Vector A is generated by the in-plane translation of vector A to share its origin with vector D. The definition of K2, given in Eq. (8.5), is based upon the angles shown. (B) If the fluorophores he in parallel planes, the orientation parameter simplifies to K2 — cos2 T and varies between 0 and 1. The schematic shows the limiting cases, where the transition moments are parallel (k2 = 1) and crossed (K2 — 0). If the transition moments are colinear, K2 = 4.

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