Cyanine Dyes for DNA Labeling

Cyanine dyes are a class of organic dyes with extremely high fluorescence sensitivity and diverse bonding capabilities, and are widely used for DNA labeling in biological and medical research. Since cyanine dyes have special optical and chemical characteristics that make them a valuable tool for molecular biology and genetics research, they are used in the DNA labeling process. For example, cyanine dyes offer a high temporal and spatial resolution method for examining the dynamics of DNA molecules in single-molecule FRET (Förster resonance energy transfer) investigations.

Deoxyribonucleic Acid

Deoxyribonucleic acid (DNA) is a double helix molecule composed of nucleotides, which is composed of four bases: adenine (A), guanine (G), cytosine (C) and thymine (T), together with phosphate and deoxyribose. In cells, genetic information is carried by DNA. It regulates the growth, development, function, and reproduction of all living things and is found in their cells. Base pairing (A-T, G-C) joins two antiparallel DNA strands to form the double helix structure, while hydrogen bonding and base stacking keep the helix structure stable. The replication, transcription and translation processes of DNA jointly ensure the transmission and expression of genetic information.

Fig. 1. Cyanine for DNA labeling (RSC Advances. 2022, 12(43): 28063-28078).

In the field of medicine and genetic engineering, therapies related to DNA are becoming increasingly diverse and progressive. Gene therapy aims to treat genetic diseases or alleviate their symptoms by introducing, removing or modifying genes in patients. Common gene therapy methods include gene replacement, gene insertion and gene silencing. CRISPR-Cas9 technology is currently the most popular and widely used gene editing tool. Using this technology, the target DNA sequence can be cut and edited efficiently and accurately. In addition, antibody drugs, nucleic acid vaccines and DNA-encoded drugs have also been successfully used in the treatment of cancer, infectious diseases and immune system-related diseases. In the process of DNA research and diagnosis, DNA labeling technology plays a vital role. Cyanine is a commonly used fluorescent dye that is widely used in DNA labeling. The exceptional photostability, high fluorescence yield, and broad absorption spectrum range of cyanine dyes are well known. Its molecular makeup typically consists of two polar groups and a polyene chain, which allow it to readily embed into the double helix structure and bind firmly with DNA molecules. By combining with cyanine dyes, DNA molecules can be observed and detected with high sensitivity and high resolution under a fluorescence microscope or fluorescence measurement equipment.

Cyanine Dyes

Cyanine dyes are a class of fluorescent dyes widely used in bioimaging and bioanalysis. These are chemically synthesized organic compounds having a benzothiophene structure that glow in various wavelength ranges. The characteristics of cyanine dyes include:

• Fluorescence properties: Cyanine dyes have a broad wavelength range that spans the ultraviolet to near-infrared spectrum. They can also produce strong fluorescence signals when activated. They are therefore extensively employed in the biological sector.
• Photostability: It is crucial for investigations that cyanine dyes be able to sustain high fluorescence intensity under continuous, long-term illumination.
• Solubility and biocompatibility: Cyanine dyes have specific solubility and biocompatibility due to the long alkyl chains or ionic groups in their molecular structure. This allows them to interact with biological components and exist steadily in organisms or biological samples.

Cyanine dyes are extensively utilized in the biological area for tasks like bioanalysis, protein labeling, immunostaining, cell and tissue imaging, and nucleic acid probes. By attaching themselves to particular target molecules, they may visualize molecules in cells or organisms with high sensitivity and resolution. It is important to remember that in order to get better experimental results when using cyanine dyes, it is crucial to choose the right dye structure, wavelength, affinity, chemical modification, and other aspects based on particular experimental requirements and research goals.

Cyanine Structure

The basic structure of cyanine includes a polyene chain connecting two heterocyclic nitrogen rings or carbon rings. The core structure is usually represented as [N-(CH=CH)_n-N], in which two nitrogen atoms are connected by a conjugated chain. The nitrogen atom can be joined to the carbon chain directly or via a variety of heterocyclic rings (pyridine, imidazole, etc.). Because of its stable conjugated form, cyanine exhibits strong light absorption and luminescence properties. A heteroaromatic ring containing nitrogen, which could be a pair of homologous nitrogen atoms, is typically the end group as well. Cyanine can display emission and absorption properties in various visible light spectrum regions by varying the end group's or the bridge chain's length. A greater wavelength of light is absorbed when the conjugated system is longer, which is determined by the bridge chain length (n value). Based on the structure and properties, cyanine can be roughly classified into the following types: monoselene type (Cy3 type), diselene type (Cy5 type), triselene type and longer chain type, polycyclic cyanine and polychromatic cyanine.

• Cyanine 3

Cyanine 3 (Cy3) is an orange-yellow fluorescent anthocyanin dye. The excitation peak and emission peak of Cy3 dye are around 550 nm and 570 nm respectively. Its fluorescence is very bright when observed with the naked eye and is insensitive to pH. It can be excited with a laser beam of 532 nm (shoulder peak) or 556 nm (peak) in a confocal microscope, and can be observed with a TRITC (tetramethylrhodamine) filter in an ordinary fluorescence microscope, so it can be used on most fluorescence instruments.

• Cyanine 5

Cyanine 5 (often referred to as Cy5) has strong fluorescence properties in the near-infrared region of the visible spectrum (approximately 650-670 nanometers). This property makes Cy5 particularly suitable for various fluorescence imaging and labeling experiments, such as fluorescence confocal microscopy, flow cytometry, and DNA sequencing technologies. One of the main advantages of Cy5 is its high quantum yield and photostability, which can maintain a stable fluorescence signal even under long-term illumination. In addition, since its excitation and emission spectra do not overlap with the natural fluorescence spectra of many biological molecules, Cy5 can significantly reduce background noise and improve the signal-to-noise ratio. This is particularly important for accurate detection and quantitative analysis. In general, Cy5 can be covalently coupled to various biomolecules, such as antibodies, oligonucleotides, proteins, etc., to form fluorescent markers. These Cy5 markers can then be used to identify and quantify specific target molecules in biological samples. In multiplex fluorescence experiments, Cy5 is often used in combination with other fluorescent dyes (such as FITC, Cy3) to achieve simultaneous detection of multiple molecular targets.

DNA Molecule Labeled

Fluorescent dye-labeled DNA is an important technique for studying genome, detecting gene expression, analyzing DNA sequence, and monitoring the dynamics of biomolecules. It enables DNA to emit fluorescence under the irradiation of a specific light source by specifically binding the fluorescent dye molecule to the DNA sequence, thereby realizing the visualization and quantitative analysis of DNA. Common dyes include fluorescein (FITC), rhodamine, cyanine, methylene blue, etc. These dye molecules have specific excitation and emission spectra, and multi-color labeling and multi-parameter analysis can be achieved by selecting different fluorescent dyes. Dye-labeled DNA has a wide range of applications, including but not limited to microscopic fluorescence imaging, fluorescence in situ hybridization (FISH), quantitative polymerase chain reaction (qPCR), DNA sequencing, and single-molecule detection analysis.

Cyanine for DNA Labeling

In DNA labeling, cyanine dyes usually bind to DNA molecules through covalent or non-covalent interactions. One of the most popular techniques is covalent labeling, which uses a chemical reaction to link a cyanine dye to an amino or hydroxyl group in a DNA base. Labeling usually involves the use of chemically modified cyanine dyes, such as maleimide compounds or active esters. This technique enables researchers to create DNA probes with high fluorescence intensity and stability for real-time qPCR, chromatin immunoprecipitation (ChIP), and FISH studies.

In addition to covalent labeling, scientists also use non-covalent interactions to label DNA with cyanine dyes. For example, some cyanine dyes, due to their hydrophilic and hydrophobic components, can be attached to the DNA double helix through hydrophobic or electrostatic interactions. Under certain circumstances, this dye can be inserted between DNA base pairs to achieve DNA labeling. This non-covalent labeling technique is particularly suitable for large-scale high-throughput screening. It is usually used for fluorescent dye dyeing, which has the advantages of simple operation and high labeling efficiency. Anthocyanin dyes are used in DNA labeling for a wide range of applications, from basic science to clinical testing. Its special uses in various fields include :

• Fluorescence in situ hybridization (FISH): Probes tagged with cyanine dye are used to identify particular DNA sequences on chromosomes. Genome positioning and structural variation analysis can be carried out by looking at the position and intensity of the fluorescent signal under a microscope. This has major implications for cancer research and genetic disease detection.
• Real-time quantitative PCR (qPCR): The qPCR method uses variations in fluorescence signals to measure the quantity of DNA or RNA. Cyanine dye-labeled probes can sensitively identify the expression level or copy number variation of specific genes, providing a powerful tool for pathogen identification and gene expression analysis.
• Chromatin immunoprecipitation (ChIP): Utilizing ChIP technology, researchers may examine how proteins and DNA interact. Certain DNA segments in chromatin can be precisely identified using cyanine dye-labeled antibodies and probes, illuminating the function of transcription factors in the regulation of gene expression.
• DNA sequencing: Nucleotides tagged with cyanine dye are employed as fluorescent markers in next-generation sequencing technology, and the DNA sequence may be precisely read by measuring the light signal emitted by each fluorophore. This significantly increases the accuracy and speed of genome sequencing.
• Flow cytometry: Flow cytometry employs DNA probes tagged with cyanine dye for precise analysis and sorting of particular cell types. This method has many uses in drug screening, cancer research, and immunology research.

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Reference:

1. Biaggne, A. et al. Molecular dynamics simulations of cyanine dimers attached to DNA Holliday junctions. RSC Advances. 2022, 12(43): 28063-28078.

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