Cy3: A key fluorescent dye in molecular biology
Cyanine3 (Cy3) dye is a widely used orange-red fluorescence dye. Cy3 fluorescence dyes are commonly used to label nucleic acids, proteins or antibodies. The Cy3 dye family is valued for its bright fluorescence and stability and plays an important role in molecular biology applications, imaging and detection. However, it should be noted that fluorescein and rhodamine have better properties and have therefore largely replaced Cy3 dyes. E.g. 5- TAMRA (Alexa FluorTM 555 dye) is a Cy3 dye alternative that is generally more fluorescent and photostable than the Cy3 dye, making it well suited to applications such as imaging and flow cytometry.
Cy3: Definition, structure & function
Cy3 is a synthetic fluorescent dye belonging to the cyanine family. It contains a connecting chain of three methine groups, connecting two nitrogen-containing heterocyclic rings. The heterocycle allows efficient electron delocalization and strong fluorescence. Cy3 structure can vary and is available with different modifications such as methyl, ethyl or butyl substituents, carboxyl, acetylmethoxy, and sulfonic acid groups which alter their hydrophilicity. The Cy3 color is commonly used for fluorescence microscopy, flow cytometry, and nucleic acid labeling.
How does Cy3 work?
Cy3 contains a bridge chain of three methine groups, connecting two nitrogen-containing heterocyclic rings. The molecular structure of Cy3 enables its strong fluorescence through resonance energy transfer within its conjugated system. Cy3 can be excited using the 532 nm laser line and visualized with TRITC (tetramethylrhodamine) filter sets. When excited by a specific wavelength of light, Cy3 emits a bright orange-red fluorescence at approximately 570 nm. Due to its photostability and strong signal intensity, Cy3 fluorescence is an ideal marker for visualizing complex biomolecular assemblies. The Cy3 fluorophore can be attached differently e.g. by click chemistry, NHS ester reaction or Staudinger ligation to label proteins, nucleic acids and other biological molecules.
Applications of Cy3 in molecular research
Fluorescence microscopy
Nucleic acid labelling with Cy3 is frequently used in microscopy to visualize cellular structures and molecular interactions. In the last 10 years in particular, RNA labelling has become an interesting tool because it can be used to analyze biodistribution at the cellular level as well as in whole organisms. In experimental setups mRNAs labelled with e.g. Cy3 color and coding or fluorescent proteins are used to test biodistribution and protein translation. Click chemistry can be used to produce mRNA labelled with Cy3 fluorescence specifically at the 5′-end, 3′-end or within the ORF. In the figure below, HeLa cells were transfected with eGFP-encoding mRNA labelled with a Cy3 fluorochrome only at the poly(A) tail. The microscopy study shows intact mRNA in the cells, high translation efficiency of the labelled mRNA and possible biodistribution analysis.
Figure: Microscopy images of HeLa cells transfected with eGFP mRNA labeled with Cy3 fluorophore in the poly(A) tail. Left to right: bright field, green fluorescence, and Cy5 channel. Scale bar: 100 mm
Gene expression studies
Gene expression consists of several individual processes, including transcription, splicing, translation and post-translational modification as well as their regulatory mechanisms. Transcription is the first step in gene expression. It is carried out by RNA polymerases that produce a complementary RNA copy of a DNA strand, the primary transcript. To study gene expression patterns fluorescence in situ hybridization (FISH) is used. FISH is a molecular cytogenetic technique that enables the detection and localization of specific DNA or RNA sequences within cells/tissues. The principle is to hybridize e.g. small single stranded DNA molecules (probes) to complementary sequences on chromosome spreads or in nuclei of whole cells (target), which are immobilized on glass slides. The probes are labeled directly or indirectly using fluorescent e.g. Cy3 or radioactive nucleotides or through e.g Cy3 fluorescent antibodies. After Cy3 labeling, FISH probes and targets are thermally denatured and hybridized. Thus shows that Cy3-labeled probes are integral to gene expression analysis, especially in in situ hybridization and RNA detection techniques. These labeled oligonucleotides help track gene activity in tissues and organisms, providing insights into genetic regulation and disease mechanisms.
Flow cytometry & cell sorting
Flow cytometry is a widely used technique to analyze physical and chemical properties of cells or particles as they pass through a laser. It is commonly used to assess cell populations based on parameters like size, complexity, and fluorescence intensity.
But how does Cy3 labelling of cells work? For example, HeLa cells have been incubated with the nucleoside analog EdU (5-ethynyl-2’-deoxyuridine) for 2 hours, which incorporates into newly synthesized DNA during the S-phase of the cell cycle. A click chemistry reaction is then performed, labeling the EdU with the fluorescent dye Cy3 or alternative dyes like 5-TAMRA or Alexa FluorTM 555, allowing for detection via flow cytometry. With the right laser the measurement of the Cy3 labeled cells will be started. Using a TRITC laser, measurement of the Cy3-labelled cells will give a spectrum with two peaks. The first peak (low fluorescence) corresponds to non-proliferating cells, while the second peak (high fluorescence and labeling with Cy3) corresponds to proliferating cells.
Benefits of using Cy3
High sensitivity & specificity
High fluorescence intensity and sensitivity emphasize to use Cy3 for labeling biomolecules. Cy3 fluorescence enabling clear imaging and precise detection in diagnostic and research applications. Cy3 as well as its improved alternatives like 5-TAMRA or Alexa FluorTM 555 show low background interference and ensure high-quality data acquisition. 5- TAMRA (Alexa FluorTM 555 dye) is a Cy3 dye alternative that is generally more fluorescent and photostable than the Cy3 dye, making it well suited to applications such as imaging and flow cytometry.
Compatibility with other dyes
Multiplexed, real-time fluorescence detection is typically performed with 3–4 fluorochromes at a time due to signal-to-noise ratio, high spectral overlap and the need to maintain the chemical compatibility of dyes. Cy3 can be used alongside other fluorochromes, in multiplexing experiments, allowing simultaneous detection of multiple targets in a single assay. Suggested quenchers for common fluorochromes, used in multiplexed experiments are e.g. 6-FAM, 5-TAMRA (a Cy3 alternative), 5/6- Sulforhodamine and Eterneon-red 645. But be careful to choose dyes that are compatible with your instrument.
Future trends in fluorescent dyes & Cy3 research
Advancements in fluorescent dye technology continue to enhance the performance of Cy3 and related compounds. Researchers are developing brighter, more photostable dyes to improve imaging resolution and expand the applications of fluorescence-based assays. Emerging trends include the integration of Cy3 in advanced imaging platforms and novel labeling strategies for enhanced molecular tracking.