Cyanine Dyes for Nucleotide Labeling

Cyanine dyes are a class of fluorescent dyes widely used in the fields of biomedicine and biotechnology. They can efficiently limit the interference of background fluorescence because of their high fluorescence quantum yield, wide spectrum range, and strong absorption and emission wavelengths in the visible and near-infrared areas. Because of these qualities, cyanine dyes are excellent choices for imaging and detecting applications. An important method in nucleotide labeling is the use of cyanine dyes to label DNA or RNA. To achieve high-sensitivity and high-specificity detection and imaging, cyanine dye-labeled nucleotide probes can be employed in methods including fluorescence microscopy, flow cytometry, and fluorescence in situ hybridization (FISH).

What is a Nucleotide?

Nucleotides are the basic units that make up nucleic acids (such as DNA and RNA). Each nucleotide consists of three parts: a five-carbon sugar (ribose or deoxyribose), a phosphate group, and a nitrogenous base (such as adenine, guanine, cytosine, thymine, or uracil). These bases are bound to each other through hydrogen bonds to form the double helix structure of DNA or RNA. In research and experiments, the quantification and tracking of nucleotides are important operations. For example, in PCR (polymerase chain reaction), a large number of precise nucleotides are required to amplify the target gene sequence. In order to facilitate the labeling and tracking of nucleotide sequences, researchers have developed a variety of dye labeling techniques, one of which is the use of cyanine dyes. Cyanine dye is a kind of water-soluble plant pigment, which widely exists in the plant kingdom. It has bright color and obvious fluorescence characteristics. These characteristics make it an ideal biomarker.

Nucleotide Structure

The structure and function of nucleotides, which are an essential carrier of genetic information in living organisms, are important aspects of biochemistry. A nitrogenous base, pentose sugar, and phosphate group are the three primary parts of a nucleotide. In addition to being the basic building blocks of nucleic acids, nucleotides are also involved in a variety of biological processes, including the transduction of cell signals.

Fig. 1. Structure of a nucleotide.

• Nitrogenous bases: There are two major categories of bases, purines and pyrimidines. Purines include adenine (adenine, A) and guanine (guanine, G); while pyrimidines include thymine (thymine, T, in DNA), uracil (uracil, U, in RNA) and cytosine (cytosine, C). These bases participate in hydrogen bonding between nucleotides to form base pairs in the double helix structure of nucleic acids.
• Pentose: Depending on the type of nucleic acid, the sugar contained is different. In DNA, the sugar is 2'-deoxy-D-ribose (deoxyribose); in RNA, the sugar is D-ribose (ribose). The difference between ribose and deoxyribose is that the latter lacks a hydroxyl (-OH) group at the 2' position. This slight difference leads to differences in the chemical properties of DNA and RNA. DNA is more stable and suitable for long-term storage of genetic information, while RNA is relatively easier to degrade and suitable for information transmission and regulation.
• Phosphate group: The phosphate group in the nucleotide is connected to the 5' carbon atom of the pentose through a phosphodiester bond. The phosphate group not only makes the nucleotide acidic, but also participates in the formation of the backbone structure connection of the nucleic acid molecule, and is the key to the polymer structure of the nucleic acid molecule.

Cyanine

Fluorescent dyes are key materials in the fields of bioimaging, photonics, and organic light-emitting diodes, and their luminescence efficiency plays a vital role in technological innovation. While designing new organic dyes with better performance, it is equally important to improve the luminescence efficiency of existing dyes. Cyanine dyes are an old and widely used dye with the advantages of high molar absorption, easy wavelength control, and easy derivatization. Structurally, cyanine dyes usually contain polycyclic aromatic hydrocarbons or heterocyclic compounds with different functional groups bridged at both ends. The bridging part is usually composed of conjugated chains, which give them excellent optical properties such as high absorbance, strong fluorescence, and good photostability. Cyanine dyes are generally classified as non-sulfonated cyanines and sulfonated cyanines. For most applications, they are interchangeable because their spectral properties are almost identical. Both sulfonated and non-sulfonated dyes can be used to label biomolecules such as DNA and proteins. The main difference between them is their solubility - sulfonated dyes are water soluble. They do not require the use of organic co-solvents for labeling in aqueous environments.

• Non-Sulfonated Cyanine Dyes

Available non-sulfonated dyes include Cy3, Cy3.5, Cy5, Cy5.5, Cy7, and Cy7.5. The structural changes lead to changes in the fluorescent properties of the dyes, and the cyanine series of fluorophores covers the most important parts of the visible and near-infrared spectra. Most non-sulfonated cyanine derivatives (except hydrazides and amine hydrochlorides) have low water solubility. When these molecules are used for biomolecule labeling, organic cosolvents (5-20% of DMF or DMSO) must be used for effective reaction. The cyanine dye is first dissolved in an organic solvent and then diluted in an appropriate polar buffer before use. In general, the fluorescence properties of non-sulfonated cyanines are less dependent on the solvent and the surrounding environment.

• Sulfonated Cyanine Dyes

Sulfonated cyanines include a sulfonic acid group, which effectively increases their solubility in water. The charged sulfonate group reduces aggregation between the dye molecule and the heavy label. Currently available sulfonated cyanines include Sulfo-Cy3, Sulfo-Cy5, and Sulfo-Cy7. Sulfonated cyanines are highly water-soluble and no organic solvent is required for labeling with these reagents. In terms of purification, sulfonated cyanines must be used to effectively remove unreacted dye when water or aqueous buffers are used for purification. When purified by gel filtration, chromatography (HPLC, FPLC, ion exchange chromatography), or electrophoresis, both sulfonated and non-sulfonated cyanines are reactive.

Nucleotide Labeled

Nucleotide labeling technology is of great significance in modern molecular biology and biotechnology, especially in genomics, proteomics and systems biology research. By chemically modifying and labeling nucleotides, scientists can track, locate and quantitatively analyze DNA and RNA molecules. At present, commonly used nucleotide labeling techniques include radioisotope labeling, fluorescent labeling, biotin labeling, enzyme labeling and rare nucleotide labeling. Among them, fluorescent labeling is one of the important methods, which is widely used due to its high sensitivity and visualization characteristics. By attaching fluorescent dyes (such as fluorescein FITC, rhodamine, cyanine dyes, etc.) to nucleotide molecules, these labeled nucleotides can emit fluorescence under light of a specific wavelength. Fluorescently labeled nucleotides can be detected by instruments such as fluorescence microscopes, flow cytometers and fluorometers. The advantages of fluorescent labeling are its high sensitivity, no radioactive risk and multiple labeling capabilities (using fluorescent dyes of different colors).

Cyanine for Nucleotide Labeling

In the process of cyanine dye labeling nucleotides, the nucleotide sequence is first linked to the cyanine dye by covalent bonding. This process usually involves the introduction of a reactive group such as an amine, carboxyl or azide group on the nucleotide molecule, which then chemically reacts with the active docking group of the cyanine dye. In this way, the dye and the nucleotide form a stable covalent bond, ensuring the stability of the fluorescent signal and the accuracy of the detection. The advantages of Cy dye labeling are its photostability, high sensitivity and multiple detection capabilities. Compared with other fluorescent dyes, cyanine dyes have higher brightness and lower background noise, which makes them more widely used in complex biological systems. In addition, the multiple chromatographic selectivity of cyanine dyes allows the simultaneous detection of multiple different targets in the same experiment, greatly improving the efficiency of the experiment and the credibility of the data. In practical applications, cyanine dye-labeled nucleotides are often used in techniques such as fluorescence in situ hybridization (FISH), microarray analysis and real-time fluorescence quantitative PCR (qPCR).

• Fluorescence in situ hybridization (FISH): Cyanine dye-labeled nucleotide probes can hybridize with target nucleic acid sequences, thereby accurately locating specific genes or genomic regions under a microscope. Common cyanine dyes such as Cy3 and Cy5 have different excitation and emission wavelengths, which can achieve multi-color labeling and simultaneous detection of multiple target sequences.
• Quantitative PCR (qPCR): qPCR uses primers or probes labeled with cyanine dyes to monitor the PCR amplification process in real time. By monitoring the changes in the fluorescence signal, the initial copy number of the target gene can be quantified, which has important applications in gene expression analysis, pathogen detection, etc.
• DNA sequencing technology: Cyanine dye-labeled nucleotides act as terminators or reversible terminators to undergo synthesis reactions with the sequence template to be tested. Since different nucleotides are labeled with cyanine dyes with different fluorescent properties, the system can determine the base sequence on the synthetic chain by detecting the fluorescent signal, thereby achieving high-throughput, fast and accurate genome sequencing.

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