Fluorophores cyanine dyes

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. 

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... 

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]. 

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. 

Fluorescent dyes, especially the cyanine dyes Cy3 and Cy5, are the most popular choice for dual colour microarray analysis. The main benefit of using Cy dye fluorophors in particular is that the two dyes can be excited and detected from the same slide. Fluorescent dyes can be directly incorporated into nucleic acid by either enzymatic or chemical methods. Basically there are two fluorescent dye-labeling strategies ... 

When the absorption and / or the emission spectra of a fluorophore possess two or more bands, the Stokes shift will be equal to the difference that separates the two most intense bands of the two spectra (Fig. 2.5). Stokes shift is dependent on the structure of the fluorophore itself and on its environment (the solvent where it is dissolved and / or the molecule to which it is bound. For example, the cyanine dye, thiazole orange (TO) absorbs at 500 nm and emits at 535 nm when it is dissolved in solution at low concentrations. These spectral properties are characteristic of that of a monomer. Increasing the concentrations yields to the formation of aggregates that absorb at 473 nm and emit at 635 nm. Thus, aggregates how larger Stokes shift compared to the monomer. The same phenomenon is also observed when TO monomers interact with DNA at high concentration of TO compared to that of DNA. In this case, the Stokes shift is found equal to at least 100 nm (Ogul chansky et al. 2001). 

NIR fluorophores are generally considered as substances that emit fluorescence in the NIR region (650-900 nm). Particularly, the fluorescence quantum yield (QY) of NIR fluorophores is always lower than that of short wavelength emission ones. Over the past few decades, enormous progress has been made in the field of NIR fluorescent dyes. There are several major NIR organic fluorescent chromophores, such as bay-substituted perylene or naphthalene bisimides, cyanine dyes, BODIPYs, DPPs, and porphyrins. 

PNA FRET probes have been synthesised by attaching the fluorophores onto C5 of a uracil base, and PNA has also been used in molecular beacons " (see section 3.5). Cyanine dyes have been introduced as a PNA surrogate base to detect mismatches with complementary DNA. A DNA molecular beacon has been used as a probe to detect strand invasion of a DNA duplex by PNA. The molecular beacon is complementary to the single stranded region left by PNA strand invasion, and it is able to bind to that... 

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