Cyanine is a non-systematic name of a synthetic dye family belonging to polymethine group. The word cyanin is from the English word “cyan”, which conventionally means a shade of blue-green (close to “aqua”) and is derived from the Greek “kyanos” which means a somewhat different color: “dark blue”.
Cyanines were and are still used in industry, and more recently in biotechnology (labeling, analysis). Cyanines have many uses as fluorescent dyes, particularly in biomedical imaging. Depending on the structure, they cover the spectrum from IR to UV. There are a large number reported in the literature [1].
Cyanines were first synthesized over a century ago. They were originally used, and still are, to increase the sensitivity range of photographic emulsions, i.e., to increase the range of wavelengths which will form an image on the film, making the film panchromatic. Cyanines are also used in CD-R and DVD-R media. The ones used are mostly green or light blue in color, and are chemically unstable. This makes unstabilized cyanine discs unsuitable for archival CD and DVD use, as they can fade and become unreadable in a few years, however, recent cyanine discs contain stabilizers that slow down the deterioration significantly. These discs are often rated with an archival life of 75 years or more. The other dyes used in CD-Rs are phthalocyanine and azo.
Cy 3 and Cy5 are the most popular cyanine dyes, used typically combined for 2 color detection. Cy3 dyes fluoresce orange (~550 nm excitation, ~570 nm emission), while Cy5 is fluorescent in the red region (~650/670 nm) but absorbs in the orange region (~649 nm) [2].
They are usually synthesized with reactive groups on either one or both of the nitrogen side chains so that they can be chemically linked to either nucleic acids or protein molecules. Labeling is done for visualization and quantification purposes. They are used in a wide variety of biological applications including comparative genomic hybridization and in gene chips, which are used in transcriptomics. They are also used to label proteins and nucleic acid for various studies including proteomics and RNA localization.
In microarray experiments DNA or RNA is labeled with either Cy3 or Cy5 that has been synthesized to carry an N-hydroxysuccinimidyl ester (NHS-ester) reactive group. Since NHS-esters react readily only with aliphatic amine groups, which nucleic acids lack, nucleotides have to be modified with aminoallyl groups. This is done through incorporating aminoallyl-modified nucleotides during synthesis reactions. A good ratio is a label every 60 bases such that the labels are not too close to each other, which would result in quenching effects.
Many analogs of standard Cy 2 / Cy 3 / Cy 3.5 / Cy 5 / Cy 5.5 / Cy 7 / Cy 7.5 dyes were developed, using modification with moieties such as carboxyl, acetylmethoxy, sulfo,…: Alexa Fluor dyes, Dylight, FluoProbes dyes, Sulfo Cy dyes, Seta dyes and others can be used interchangeably with Cy dyes in most biochemical applications, with claimed improvements in solubility, fluorescence, or photostability.
For protein labeling, Cy3 and Cy5 dyes sometimes bear maleimide reactive groups instead. The maleimide functionality allows conjugation of the fluorescent dye to the sulfhydryl group of cysteine residues. Cysteines can be added and removed from the protein domain of interest via PCR mutagenesis.
Cy5 is sensitive to the electronic environment it resides in. Changes in the conformation of the protein it is attached to will produce either enhancement or quenching of the emission. The rate of this change can be measured to determine enzyme kinetic parameters. The dyes can be used for similar purposes in FRET experiments.
Cy3 and Cy5 are used in proteomics experiments so that samples from two sources can be mixed and run together through the separation process. This eliminates variations due to differing experimental conditions that are inevitable if the samples were run separately. These variations make it extremely difficult, if not impossible; to use computers to automate the acquisition of the data after the separation is complete. Using these dyes makes the automation trivial [3].
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Reference
[1] Fare TL, Coffey EM, Hongyue D, et al. Effects of Atmospheric Ozone on Microarray Data Quality. Analytical Chemistry. 2003;75:4672-4675. [2] K. Umezawa,A. Matsui, Y. Nakamura, D. Citterio, K. Suzuke (2009). Chem. Eur. J. 15: 1096. [3] Ilya A. Osterman, Alexey V. Ustinov, et al. Dontsova (January 2013). “A nascent proteome study combining click chemistry with 2DE”. PROTEOMICS 13 (1): 17–21.
