Fluorescence Labeling of Nucleic Acids

Since RNA/DNA does not contain naturally occurring fluorescent groups, and the introduction of fluorescent groups is necessary for fluorescence determination. BOC Sciences has extensive experience in fluorescence chemistry and has developed a wide range of widely used and trusted solutions for fluorescently labeling a wide variety of substances. Fluorescently labeled nucleic acids offer many advantages for gene characterization, quantification, integration and expression studies in molecular biology research, diagnostics and imaging studies.

Fig.1 Fluorescent labeling of nucleic acids. (Rombouts, K., 2016)

What is fluorescence labeling of nucleic acids?

Fluorescence labeling of nucleic acids is the process of attaching a fluorescent molecule or fluorophore to a nucleic acid molecule to introduce a fluorescent signal. The fluorescent labeling process involves covalent or non-covalent attachment of a fluorescent dye or fluorophore to a nucleic acid. Covalent labeling typically involves attaching a reactive group on the fluorescent molecule to a specific functional group on the nucleic acid, such as an amino or thiol group. Non-covalent labeling methods utilize specific interactions between certain probes and the nucleic acid sequence of interest.

Methods of fluorescence labeling of nucleic acids

BOC Sciences employs advanced methods for fluorescence labeling of nucleic acids, ensuring minimal impact on their structure and function:

Chemical labeling: We utilize reactive dyes that covalently attach to specific functional groups (e.g., amino or thiol groups) on nucleic acids, allowing stable incorporation of fluorescent tags.

Enzymatic labeling: Enzymes such as DNA polymerases or ligases can incorporate fluorescently labeled nucleotides during nucleic acid synthesis or modification processes.

Click chemistry: Click chemistry reactions, such as CuAAC (copper-catalyzed azide-alkyne cycloaddition), provide a bioorthogonal approach to selectively attach fluorescent probes to nucleic acids.

Interstrand crosslinking: Fluorescent intercalating agents or crosslinkers can be used to label double-stranded nucleic acids, offering insights into DNA/RNA secondary structure and dynamics.

Advantages of fluorescence-labeled nucleic acids

Sensitive detection: Fluorescent labels provide high sensitivity, allowing for the detection of small amounts of nucleic acids. This sensitivity is crucial in applications like PCR, sequencing, and gene expression analysis.

Multiplexing capability: Different fluorescent dyes can be used simultaneously to label multiple targets within the same sample, enabling multiplexed analyses. This is particularly useful in microarray analysis and multiplex PCR.

Real-time monitoring: Fluorescent labeling enables real-time monitoring of nucleic acid processes such as PCR amplification, hybridization kinetics, and RNA localization. Changes in fluorescence intensity can be measured continuously, providing dynamic information about biological processes.

Visualization and imaging: Fluorescent labels allow for visualization and imaging of nucleic acids within cells or tissues. This is important for studying gene expression patterns, RNA localization, and chromosomal abnormalities.

High specificity: Fluorescent probes can be designed to specifically bind to target sequences, providing high specificity in applications like FISH and RNA imaging. This reduces background noise and increases assay accuracy.

Versatility: Fluorescent labeling can be applied to various types of nucleic acids, including DNA, RNA, and oligonucleotides. This versatility allows for the development of diverse molecular biology techniques and assays.

Quantitative analysis: Fluorescent signals can be quantified using imaging systems or spectroscopy, allowing for quantitative analysis of nucleic acid concentrations, gene expression levels, and sequence variations.

Compatibility with high-throughput techniques: Fluorescent labeling is compatible with high-throughput techniques such as microarrays and next-generation sequencing (NGS), enabling rapid and efficient analysis of large numbers of samples.

Non-destructive labeling: Many fluorescent labels are non-destructive and do not interfere significantly with the structure or function of nucleic acids. This makes them suitable for live-cell imaging and dynamic studies of nucleic acid behavior.

Ease of detection: Fluorescent signals are easily detectable using fluorescence microscopy, plate readers, or flow cytometry systems. This simplifies data collection and analysis in molecular biology experiments.

Applications of fluorescence-labeled nucleic acids

Real-time PCR: Fluorescent probes enable quantitative and specific detection of target DNA/RNA sequences.

Fluorescence in situ hybridization (FISH): Labeled probes visualize nucleic acids within cells and tissues, aiding in genetic and cytogenetic analyses.

DNA sequencing: Fluorescently labeled nucleotides are used in DNA sequencing methods like Sanger sequencing and next-generation sequencing (NGS). Each nucleotide is labeled with a unique fluorescent dye, allowing for high-throughput sequencing and accurate base identification.

RNA/DNA imaging: Fluorescence microscopy allows visualization of nucleic acid dynamics in live cells, elucidating cellular processes and interactions.

Cell cycle analysis: Fluorescent nucleic acid dyes like propidium iodide (PI) or DAPI (4',6-diamidino-2-phenylindole) are used to stain DNA in cells for flow cytometry analysis. By measuring the fluorescence intensity, the DNA content of cells can be determined, allowing for cell cycle phase identification and analysis of DNA ploidy.

Single-molecule imaging: Fluorescently labeled nucleic acids can be used for single-molecule imaging techniques to study DNA-protein interactions, transcription dynamics, and DNA repair processes at the single-molecule level

Drug delivery: Functionalized nucleic acids with fluorescent tags can be used in targeted drug delivery systems, guiding therapeutic agents to specific tissues or cells.

Biosensing: Fluorescently labeled nucleic acids serve as probes in biosensors for detecting pathogens, mutations, or biomarkers with high sensitivity.

Services for fluorescence-labeled nucleic acids

BOC Sciences offers fluorescent labeling services for nucleic acids. In addition to the fundamental DNA and RNA fluorescent labeling services, BOC Sciences provides a range of specialized services for fluorescence-labeled nucleic acids to support various research applications.

Fluorescence labeling of DNA

DNA fluorescent labeling involves binding a fluorescent dye to a DNA molecule in order to track and detect the DNA sequence in an experiment. Our researchers use end-labeling method for labeling, the following are the labeling steps of end-labeling method:

(1) Label the end of the DNA molecule with a fluorescent dye, commonly used markers include fluorescein dyes and so on.

(2) In the 3' end-labeling method, DNA terminal deoxynucleotidyl transferase (TdT) and fluorescent dye-modified reactants (e.g., fluorescein-dUTP) are used for labeling. TdT is able to incorporate fluorescent dye-modified nucleotides on the OH group at the end of the DNA to form fluorescently labeled DNA.

(3) In the 5' end labeling method, several methods can be used, such as the PCR or the Klenow fragmentation reaction. These reactions introduce fluorescent markers at the 5' end of the DNA, such as PCR amplification using fluorescein phosphate and 5' phosphorylated primers.

(4) The labeled DNA can be detected and analyzed by equipment such as gel electrophoresis or fluorescence microscopy.

Fluorescence labeling of RNA

BOC Sciences provides reverse transcription labeling, RNA-conjugated dye labeling, fluorescent protein labeling and other methods to combine fluorescent substances with RNA for better application in genetic disease diagnosis, viral infection analysis, prenatal diagnosis, tumor genetics and genomic research.

Fluorescence labeling of oligonucleotide

BOC Sciences offers custom oligonucleotide labeling services, where specific sequences of DNA or RNA can be conjugated with fluorescent dyes. This facilitates applications such as probe development for in situ hybridization, fluorescence in situ hybridization (FISH), and detection of specific nucleic acid targets in biological samples.

Fluorescence labeling of nucleotide

Fluorescence-labeled nucleotides can be enzymatically incorporated into DNA or RNA probes for FISH (fluorescence in situ hybridization), DNA arrays/microarrays and other hybridization techniques. Our diverse fluorescent labels provide the ideal tools for multicolor techniques such as spectral karyotyping, multilocus FISH analysis, chromosome painting and comparative genome hybridization.

Fluorescence labeling of dNTPs (deoxynucleoside triphosphates) is a technique used to incorporate fluorescent tags into DNA molecules during processes like PCR (Polymerase Chain Reaction) or DNA synthesis. This labeling method allows for real-time monitoring and detection of DNA amplification or synthesis.

Fluorescence labeling of ddNTPs is a key aspect of Sanger sequencing, enabling the accurate determination of DNA sequences and facilitating various applications in molecular biology and genetic research. The technique relies on the specific properties of ddNTPs to terminate DNA synthesis and generate fluorescently labeled DNA fragments that can be analyzed for sequence information.

Fluorescence labeling of nucleoside

Alkyne-modified nucleoside EU (5-ethynyl uridine) is supplied to cells and incorporated into nascent RNA. The small size of the alkyne tag enables efficient incorporation by RNA polymerases without any apparent changes to the RNA levels of several housekeeping genes. Detection of incorporated EU is accomplished by copper (I)–catalyzed click coupling to an azide-derivatized fluorophore. The multiplexing capability of the assays makes them ideal for toxicological profiling or interrogation of disease models using high-content imaging platforms.

Cells can naturally incorporate the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) into their DNA during cell division, making this nucleoside analog an excellent marker of both cell cycle and cell proliferation. Analysis of incorporated BrdU can be either by detection with an antibody to BrdU-modified DNA or by modification of the fluorescence of a nucleic acid stain. For instance, the fluorescence of TO-PRO-3 and LDS 751 is considerably enhanced by the presence of BrdU in DNA,ref whereas that of the Hoechst dyes is specifically quenched. 5-Bromo-2'-deoxyuridine 5'-triphosphate (BrdUTP) is commonly used in TUNEL-based methods to detect proliferating or apoptotic cells.

Fluorescence labeling of primers

Fluorescently labeled primers are specialized DNA or RNA primers that have been chemically modified to include a fluorescent dye molecule. These primers are used in various molecular biology techniques, particularly in methods like PCR (Polymerase Chain Reaction) and DNA sequencing, to enable the detection and visualization of amplified DNA fragments.

Our Advantages

• Fluorescence reagent and probe are economical and safe
• Deep knowledge and rich experience in biomaterial modification and conjugation
• The probe is stable and can be used within two years after one labeling
• The experimental period is short, the results can be obtained quickly, the specificity is good, and the location is accurate
• FISH can be located in the DNA sequence of 1kb, and its sensitivity is similar to that of radioactive probe
• Polychromatic FISH can detect multiple sequences at the same time by displaying different colors in the same core
• All samples are carefully monitored for stability and characterized to ensure batch to batch consistency

Case Study

Case Study 1

Chemical selective modification reactions are widely used in the labeling of biomolecules, including copper-catalyzed or tension-promoted azide-alkyne cycloaddition (CuAAC/SPAAC) and Diels-Alder (IEDDA) reaction with anti-electron demand. Although these reactions can be effectively labeled in vitro, the labeling of nucleic acids in vivo is still limited to single-stranded RNA or requires membrane-permeable or phototoxic methods to efficiently label chromatin-encapsulated double-stranded DNA (dsDNA). In this study, the author designed a new imaging probe (PINK) for nucleoside olefin groups. PINK consists of fluorescent embedded reagent and tetrazine group. Tetrazine group is a bioorthogonal group and a fluorescence quenching group, and the fluorescence embedded reagent partially enables it to reversibly interact with double-stranded DNA until it is connected with olefin-containing nucleotides. The combination of the two can label double-stranded DNA very quickly and fluorescently.

Fig. 2 Schematic diagram of PINK probe. (Loehr, M. O., 2022)

Case Study 2

Marcus Wilhelmsson et al. synthesized a tricyclic cytosine analog with fluorescence properties, called tC^O. The excitation wavelength was 369nm, and the strongest fluorescence wavelength was 457nm. It can be doped into the target RNA molecule by terminal deoxynucleotidyl transferase (TdT) or RNA polymerase to realize internal fluorescence labeling. Different from other fluorescent markers, tC^OTP is more similar to natural CTP, which can be correctly recognized by RNA polymerase or deoxynucleotidyl transferase and the translation machine in the cell, with little interference to the translation process, and can be correctly folded and located in the right position in the cell. More importantly, for the first time, they realized the global living cell imaging of mRNA molecules labeled with fluorescent base analogues from cell uptake to protein expression, which will bring great help to the mechanism of cell uptake and intracellular escape. It is expected that this convenient, universal and minimally interfering fluorescent labeling method can be applied to the development of nucleic acid delivery systems and promote the upgrading of RNA drugs.

Fig. 3 Translation of tC^O-labeled mRNA molecules in human cells by chemical transfection. (Baladi, T., 2021)

FAQ

1. Why is fluorescence labeling useful for nucleic acids?

Fluorescence labeling enables researchers to track and study nucleic acids in vitro and in vivo. It is valuable for applications such as in situ hybridization, fluorescence in situ hybridization (FISH), DNA sequencing, real-time PCR, and various cellular imaging techniques.

2. How are nucleic acids typically labeled with fluorescence?

Nucleic acids can be labeled by directly incorporating fluorescently modified nucleotides during synthesis (e.g., in PCR or sequencing), or by post-synthetic labeling methods where a fluorescent dye or probe is attached to the nucleic acid molecule using chemical conjugation techniques.

3. What are the commonly used fluorescent dyes for labeling nucleic acids?

Some popular fluorescent dyes used for nucleic acid labeling include fluorescein (FITC), Cy3, Cy5, Texas Red, SYBR Green, and various Alexa Fluor dyes. These dyes emit light at specific wavelengths when excited by a light source.

4. How does fluorescence labeling affect nucleic acid properties?

Fluorescence labeling generally does not significantly alter the properties or functions of nucleic acids if performed correctly. However, bulky or inappropriate labeling may interfere with hybridization or enzymatic reactions.

5. How can labeled nucleic acids be visualized or detected?

Labeled nucleic acids can be visualized using fluorescence microscopy, fluorescence plate readers, or imaging systems that detect specific emission wavelengths corresponding to the fluorescent dye used.

6. What factors should be considered when choosing a labeling method?

• Compatibility with downstream applications
• Sensitivity and specificity of the labeling method
• Impact on nucleic acid function
• Stability and photostability of the fluorescent label
• Cost and ease of use

7. What are the limitations of fluorescence labeling of nucleic acids?

Limitations include potential interference with nucleic acid function, background fluorescence, photobleaching (loss of fluorescence over time), and the need for optimization based on specific experimental requirements.