| Synonyms: | 6-carboxy-X-rhodamine, succinimidyl ester |
| CAS #: | 216699-36-4 |
| Molecular Formula: | C37H33N3O7 |
| Molecular Weight: | 631.7 |
| 6-ROX, SE (6-Carboxy-X-rhodamine, succinimidyl ester) is a long-wavelength, amine-reactive fluorescent dye widely used in molecular biology, biochemistry, and optical imaging. As a derivative of rhodamine, 6-ROX, SE is valued for its photostability, bright fluorescence, and compatibility with various applications, including real-time quantitative polymerase chain reaction (qPCR), fluorescence resonance energy transfer (FRET), DNA sequencing, and protein labeling. 1. Chemical and Structural Properties 6-ROX, SE is a single isomer of carboxy-X-rhodamine, specifically the 6-isomer, with a succinimidyl ester (SE) functional group that enables covalent attachment to amine groups on biomolecules such as proteins, peptides, and oligonucleotides. The chemical structure of 6-ROX includes a rhodamine core, which contributes to its fluorescence properties, and a linker arm with the SE group, facilitating conjugation without compromising the fluorophore’s spectral characteristics. • CAS Number: 216699-36-4 • Molecular Formula: C37H33N3O7 • Molecular Weight: 631.7 • Solubility: 6-ROX, SE is soluble in organic solvents like anhydrous dimethyl sulfoxide (DMSO) and dimethylformamide (DMF). It is less soluble in aqueous solutions, requiring careful preparation to avoid hydrolysis of the SE group. • Stability: The succinimidyl ester is susceptible to hydrolysis, particularly in aqueous environments, which can reduce labeling efficiency. For long-term storage, it is recommended to store 6-ROX, SE at -20°C, desiccated, and protected from light to maintain stability. 2. Spectral Properties 6-ROX, SE is a long-wavelength rhodamine dye with spectral properties that make it suitable for applications requiring minimal autofluorescence and deep tissue penetration. Its key spectral characteristics include: • Excitation Maximum (λex): ~575–580 nm • Emission Maximum (λem): ~602–610 nm • Molar Extinction Coefficient (ϵ): ≈ 82,000 M−1cm−1 (at λex) • Stoke’s Shift: ≈ 27 nm • Quantum Yield: Moderate to high, providing strong fluorescence intensity suitable for sensitive detection. • Comparison with 5-ROX: The 5-ROX and 6-ROX isomers have nearly identical spectral properties, with the primary difference being the position of the carboxyl group on the rhodamine core. This positional difference has minimal impact on absorption and emission spectra, making them functionally similar in most applications. The long-wavelength emission of 6-ROX, SE makes it ideal for multiplex qPCR, as it minimizes spectral overlap with other fluorophores like FAM or SYBR Green. 3. Applications 6-ROX, SE is a versatile fluorescent dye with applications across various fields of biological and biochemical research. Below are its primary uses: 3.1 Real-Time Quantitative PCR (qPCR) 6-ROX, SE is commonly used as a reference dye in qPCR to normalize fluorescence signals and correct for well-to-well variations caused by pipetting errors, instrument optics, or environmental factors like bubbles or condensation. Its fluorescence remains stable throughout the PCR process, making it an effective passive reference dye. Key features include: • Normalization: By dividing the reporter dye signal (e.g., FAM or SYBR Green) by the 6-ROX signal, normalized reporter values (Rn) are calculated, reducing variability and improving precision among technical replicates. • Instrument Compatibility: 6-ROX is compatible with qPCR instruments equipped with a ROX channel, such as Applied Biosystems platforms (e.g., ABI PRISM 7700). It is typically used at concentrations of 50 nM or 500 nM, depending on the instrument. • Troubleshooting: Monitoring 6-ROX fluorescence in a multicomponent plot helps diagnose issues like evaporation (increasing signal) or bubbles (spikes/drops in signal). 3.2 DNA Sequencing 6-ROX, SE is used for labeling oligonucleotides in automated DNA sequencing, particularly in four-color fluorescence-based sequencing methods. Its photostability and distinct emission spectrum make it suitable for high-throughput applications. 3.3 Fluorescence Resonance Energy Transfer (FRET) 6-ROX, SE serves as an acceptor or donor in FRET assays due to its spectral properties, enabling the study of molecular interactions, such as protein-protein binding or conformational changes. 3.4 Protein and Peptide Labeling The succinimidyl ester group allows 6-ROX, SE to covalently bind to amine groups on proteins and peptides, making it useful for fluorescence microscopy, flow cytometry, and other imaging applications. Its long-wavelength emission reduces background autofluorescence, enhancing signal clarity. 3.5 Optical Imaging and COX-2 Inhibition 6-ROX has been explored as a selective fluorescent probe for cyclooxygenase-2 (COX-2), binding to its active site and inhibiting the conversion of arachidonic acid to prostaglandins. This property makes it valuable for optical imaging of tumors and inflammation, where COX-2 is overexpressed. 3.6 Multiplex Assays The long emission wavelength of 6-ROX, SE makes it an excellent choice for multiplex assays, where multiple targets are detected simultaneously. Its minimal spectral overlap with shorter-wavelength dyes like FAM or SYBR Green ensures accurate signal detection. 4. Advantages • Photostability: Compared to fluorescein-based dyes, 6-ROX, SE is more resistant to photobleaching, allowing for prolonged imaging and reliable signal detection. • Long-Wavelength Emission: The emission at ~623 nm minimizes autofluorescence and enables deep tissue penetration, making it ideal for in vivo imaging and multiplex assays. • Versatility: The amine-reactive SE group allows conjugation to a wide range of biomolecules, expanding its utility across diverse applications. • Instrument Compatibility: 6-ROX, SE is optimized for use with qPCR instruments requiring a ROX channel, simplifying integration into existing workflows. • Improved Stability with Alternatives: Variants like 6-ROXtra SE offer enhanced stability and water solubility compared to standard 6-ROX, addressing some of its limitations. 5. Limitations • Stability Issues: 6-ROX, SE is less stable than other rhodamine dyes, particularly in aqueous solutions, due to the susceptibility of the succinimidyl ester to hydrolysis. This necessitates careful storage and handling. • Quenching in Certain Conditions: In digital PCR (dPCR), high concentrations of inhibitors like hyaluronic acid (HA) can quench 6-ROX fluorescence, leading to increased normalized fluorescence values and potential data misinterpretation. • Instrument Dependency: The use of 6-ROX, SE as a reference dye is limited to qPCR instruments with a ROX channel (e.g., Applied Biosystems). Instruments like the Rotor-Gene Q, which use a centrifugal rotary design, do not require a passive reference dye, limiting 6-ROX applicability. • Fluorescence Quenching in Protein Conjugates: When conjugated to proteins, 6-ROX fluorescence may be quenched, reducing signal intensity compared to newer alternatives like 6-ROXtra. • Solubility: 6-ROX, SE has poor water solubility, requiring organic solvents for stock solutions, which can complicate preparation for aqueous-based assays. 6. Practical Considerations To maximize the utility of 6-ROX, SE in research, consider the following guidelines: 6.1 Storage and Handling: • Store 6-ROX, SE at -20°C, desiccated, and protected from light to prevent hydrolysis and photodegradation. • Prepare stock solutions in anhydrous DMSO immediately before use to minimize hydrolysis of the SE group. • Aliquot solutions to avoid repeated freeze-thaw cycles, which can degrade the dye. 6.2 qPCR Applications: • Ensure the qPCR instrument’s ROX channel is enabled to record 6-ROX fluorescence. Failure to do so may result in incorrect normalization. • Use the recommended concentration (50 nM or 500 nM) based on the instrument manufacturer’s guidelines. • Monitor the multicomponent plot to detect anomalies like evaporation or bubbles, indicated by changes in 6-ROX fluorescence. 6.3 Labeling Protocols: • For protein or oligonucleotide labeling, use a buffer free of amine-containing compounds (e.g., Tris, glycine) to prevent unwanted reactions with the SE group. • Optimize conjugation conditions to achieve high labeling efficiency, as hydrolysis can reduce the yield of labeled biomolecules. 6.4 Troubleshooting: • If fluorescence signals are inconsistent, check for quenching by inhibitors like HA or heme in dPCR or qPCR samples. • Verify instrument calibration for the 6-ROX emission spectrum to minimize signal cross-talk in multiplex assays. 7. Comparison with Alternatives • 6-ROXtra SE: This variant offers improved stability and water solubility compared to 6-ROX, SE, with nearly identical spectral properties. It is less prone to quenching when conjugated to proteins and easier to purify when used in oligonucleotide labeling. • 5-ROX, SE: The 5-isomer of ROX has similar spectral properties but may differ slightly in stability and conjugation efficiency. Both isomers are often used interchangeably in qPCR applications. • CF Dyes: Biotium’s CF dyes, which use aliphatic carboxylic acid-derived SE groups, are less susceptible to hydrolysis and offer higher labeling efficiency compared to 6-ROX, SE. • Other Rhodamine Dyes: Compared to fluorescein-based dyes, 6-ROX, SE is more photostable and has a longer emission wavelength, but it may be less bright than dyes like Alexa Fluor® 594 in certain applications. 8. Future Directions The development of improved derivatives like 6-ROXtra suggests a trend toward addressing the stability and solubility limitations of 6-ROX, SE. Future research may focus on: • Enhancing the water solubility of rhodamine-based dyes to simplify preparation and conjugation protocols. • Developing hydrolysis-resistant reactive groups to improve labeling efficiency in aqueous environments. • Expanding the use of 6-ROX, SE in novel imaging techniques, such as super-resolution microscopy (e.g., STED), as demonstrated with hydroxylated derivatives. • Optimizing 6-ROX-based probes for in vivo imaging, leveraging its COX-2 inhibitory properties for targeted diagnostics. 6-ROX, SE is a robust and versatile fluorescent dye with significant applications in qPCR, DNA sequencing, FRET, protein labeling, and optical imaging. Its long-wavelength emission, photostability, and amine-reactive properties make it a valuable tool for sensitive and multiplexed assays. However, its susceptibility to hydrolysis, limited water solubility, and potential quenching in certain conditions necessitate careful handling and consideration of alternatives like 6-ROXtra SE for specific applications. By following proper storage, preparation, and instrument calibration protocols, researchers can maximize the performance of 6-ROX, SE in a wide range of experimental settings. References: 1. Dominko T, et al. (2000). Dynamic imaging of the metaphase II spindle and maternal chromosomesin bovine oocytes: implications for enucleation efficiency verification, avoidance of parthenogenesis, and successful embryogenesis. Biol Reprod 62, 150-4. 2. Yoshikawa, Y., et al., Differential display with carboxy-x-rhodamine-labeled primers and the selection of differentially amplified cdna fragments without cloning. Anal Biochem 1998, 256, 82-91. 3. Hung, S.C., et al., Cyanine dyes with high absorption cross section as donor chromophores in energy transfer primers. Anal Biochem 1996, 243, 15-27. 4. Li, Y. and A.N. Glazer, Design, synthesis, and spectroscopic properties of peptide-bridged fluorescence energy-transfer cassettes. Bioconjug Chem 1999, 10, 241-5. |
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