Cy5 NHS modification is a post synthesis conjugation to a primary amino group thus an additional modification with an amino group is required. A C6 or C12 amino group can be placed at the 5' or for the 3' end a C3 or C7 amino and for internal positions an amino modified base is used, e.g Amino dT C6.
Cyanine 3 (Cy3) is a fluorescent dye that belongs to the Cyanine family of synthetic polymethine dyes. Cy3 is reactive, water-soluble, and has an absorbance maximum of 550 nm and an emission maximum of 570 nm. It is available as both a phosphoramidite and an NHS ester, and is used to fluorescently label oligonucleotides at either the 5’- or 3’-end, or internally. Cy3 plays a particularly important role in real-time PCR applications, being used as the reporter moiety in TaqMan probes (1), Scorpion primers (2) and Molecular Beacons (3). For such probes, Cy3 is most commonly paired with the dark quencher BHQ-2, as the two have excellent spectral overlap.
Cy3 can also be used to label DNA oligos for use as hybridization probes in other applications, such as Fluorescent In-Situ Hybridization (FISH). In 2010, Stoeckler and co-workers (4) reported that Cy3 double-labeling of FISH probes (at both ends) that were specific to ribsosomal RNA targets in microorganisms at least doubles FISH signal intensity without affecting specificity. This “Double Labeling of Oligonucleotide Probes for Fluorescence In Situ Hybridization (DOPE-FISH)” strategy may provide an effective solution to the problem of low signal intensity, which is commonly observed when using corresponding singly-labeled FISH probes for microbe identification. As an added benefit, Cy3-doubly labeled probes were shown to increase the in situ accessibility of rRNA targets sites in microbes, which allows for greater probe design flexibility.
Reaction scheme for primary amine labelled oligos with NHS ester is shown in the figure below.
1. Livak, K.J., Flood, S.J.A., Marmaro, J., Giusti, W., Deetz, K. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization.PCR Methods Appl. (1995), 4: 1-6.
2. Thelwell, N., Millington, S., Solinas, A., Booth, J., Brown, T. Mode of action and application of Scorpion primers to mutation detection. Nucleic Acids Res. (2000), 28: 3752-3761.
3. Tyagi, S., Kramer, F.R. Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. (1996), 14: 303-308.
4. Stoecker, K., Dorninger, C., Daims, H., Wagner, M. Double Labeling of Oligonucleotide Probes for Fluorescence In Situ Hybridization (DOPE-FISH) Improves Signal Intensity and Increases rRNA Accessibility. Appl. Environ. Microb.. (2010), 76: 922-926.