Bioconjugation in Fluorescence In Situ Hybridization (FISH)

What is fluorescence in situ hybridization (FISH)?

Fluorescence in situ hybridization (FISH) is a powerful and accurate molecular biology technique for detecting and localizing specific nucleic acid sequences in a cell or tissue slice. Since its development in the 1980s, FISH has become an important tool in genetics, cell biology and medical research.

Fluorescence in situ hybridization principle

The basic principle of FISH is to use the known fluorescency-labeled single strand nucleic acid as a probe, according to the principle of base complementarity, through denaturation, annealing and renaturation processes, specifically combined with the target single strand nucleic acid in the sample to form a hybrid double-stranded nucleic acid that can be detected. By using fluorescence microscopy or laser confocal observation and counting of fluorescence signals, it is possible to determine the morphology and distribution of cells or organelles that have been stained after hybridization with specific probes, or the location of DNA regions or RNA molecules in chromosomes or other organelles that have been combined with fluorescent probes.

Fluorescence in situ hybridization steps

Sample preparation: The cell or tissue sample to be tested is fixed and the cell membrane is permeated by an appropriate method so that the probe can enter the inside of the cell.

Probe preparation: Nucleic acid probes complementary to the target sequence were designed and synthesized, and fluorescently labeled. The design of the probe requires a high degree of specificity to ensure that it only hybridizes with the target sequence and does not produce non-specific binding to other sequences. A length is generally between 50-300 bases and should not exceed 400 bases. The probe is too long, it is not easy to enter the cell; Too short, the specificity is not high.

Hybridization reaction: The labeled probe is incubated with the fixed sample to bind the probe to the target nucleic acid sequence. The hybridization temperature is generally 37 or 42 ℃, and the reaction time is 16-20 hours. The time of hybridization reaction should be related to the length of the probe and cell permeability. After full permeability treatment, the oligonucleotide probe can complete hybridization in only 2-6 hours.

Washing and detection: Unbound probes are washed away, and probes hybridized with the target sequence are retained. Observe the sample using a fluorescence microscope to identify and locate the fluorescence signal. Different fluorescent dyes emit specific colors of fluorescence under different wavelengths of light, and these fluorescence signals can be selectively observed through the filter of fluorescence microscope.

In vitro and in vivo hybridization techniques

In vitro hybridization: In vitro hybridization is usually performed on an ex vivo section of cells or tissue. This method is easy to operate and highly controllable, and is suitable for various types of samples, such as blood chromosome analysis, paraffin-embedded tissue sections, etc. In vitro FISH has become a standard technique in genetic research, clinical diagnosis and drug development.

In vivo hybridization: In vivo hybridization is performed in living cells or tissues to study the dynamic changes and spatial distribution of nucleic acids under living conditions. This approach relies on efficient probe delivery systems and sensitive detection techniques that enable real-time monitoring of biological processes such as gene expression, chromosome behavior, and viral infection. However, due to the complexity of the internal environment, the technical difficulty and cost of FISH in vivo is relatively high, and it is mostly used for basic research and advanced clinical applications.

What is bioconjugation?

Bioconjugation is the process of chemically linking biomolecules (such as nucleic acids, proteins, or peptides) to other molecules, often for purposes such as functionalization, labeling, or targeted delivery. This technique allows for the modification of biomolecules, enhancing their properties and functionalities for various applications in molecular biology and diagnostics. Common methods of bioconjugation include covalent bonding, click chemistry, and enzymatic conjugation, each of which offers unique advantages in terms of efficiency and specificity.

Importance of bioconjugation in FISH

In the context of FISH, bioconjugation plays a pivotal role in enhancing the performance of hybridization probes. By improving the stability of probes during storage and hybridization, bioconjugation minimizes degradation and ensures reliable results. Moreover, the incorporation of fluorescent labels through bioconjugation significantly increases signal intensity, allowing for the detection of low-abundance targets.

Through bioconjugation technology, controlled binding and release between probe and target can also be achieved. By using specific chemical bonds, such as bonds that can be cut by specific environments or enzymes, the probe can achieve dissociation from the target under specific conditions, which is of great significance for dynamic study of the behavior and function of biomolecules. For example, when studying dynamic changes in gene expression, it is possible to track the distribution and changes of molecules in cells in real time by controlling the binding and release processes.

Bioconjugation can also enhance the spatial resolution of FISH technology. By combining nanotechnology and chemical modifications, fluorescent molecules can be incorporated into probes in a more refined manner, thereby improving the spatial resolution of FISH imaging. This is an important help for the study of genomic mapping of microstructures and gene expression in complex cell tissues.

Bioconjugation chemistry in FISH

The effectiveness of bioconjugation in FISH is significantly influenced by the selection of fluorescent probes and the methods used for their conjugation. The choice of probe determines not only the specificity and sensitivity of the assay but also its overall performance in detecting target nucleic acids.

Types of fluorescent probes used in FISH

Probes are strands of nucleic acid that can be composed of DNA, cDNA, or RNA. Regardless of the probe type, the sequence of the probe must be complementary to the target sequence to ensure proper hybridization. The probe may be modified with fluorophore directly attached to the probe for detection with fluorescence microscopy, or the fluorophore may be covalently linked to an antibody that binds to the antigen incorporated into the probe.

Schematic representation of the riboprobe and oligonucleotide in situ hybridization probe types. (A) Hapten-labeled RNA probes must be bound by an antibody labeled with a fluorophore to allow for visualization. (B) DNA oligomers directly labeled with a fluorophore can be directly visualized. (Young, A. P., 2020)

RNA probes: These probes target RNA sequences within cells for detecting and visualizing mRNA, lncRNA, and miRNA in tissues and cells. RNA FISH can help in elucidating the spatial and temporal patterns of gene expression within cells and tissues.

DNA probes: Typically these probes target DNA sequences on chromosomes and are used to identify and localize specific DNA regions or sequences. These probes are often used in genetic counseling and species identification.

Peptide nucleic acid (PNA) probes: PNAs are synthetic analogues of DNA that can provide greater binding stability and sensitivity than traditional DNA probes when used in FISH.

Quantum dots (QD) probes: Quantum dots are nanoscale inorganic fluorescens with light stability and narrow emission spectra that have been successfully used for FISH analysis of human metaphone chromosomes, human sperm cells, and bacterial cells.

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Chemical strategies for linking probes to nucleic acids

Several chemical strategies are employed for linking fluorescent probes to nucleic acids, each offering unique advantages. Covalent coupling is a prevalent method that utilizes reactive groups, such as NHS-esters, to form stable linkages between the probe and the nucleic acid. This approach ensures that the fluorescent label remains attached during hybridization, which is crucial for accurate detection. Another innovative strategy is click chemistry, a modular and efficient bioconjugation technique that facilitates high specificity and yield. Click chemistry reactions typically occur under mild conditions, minimizing potential side reactions and enhancing the purity of the final product.

Enzymatic conjugation methods

Enzymatic methods provide an alternative route for attaching fluorophores to nucleic acids through enzymatic incorporation. Techniques such as terminal deoxynucleotidyl transferase (TdT)-mediated labeling enable the addition of fluorescently labeled nucleotides to the ends of oligonucleotides. This method can offer higher specificity and reduced side reactions compared to traditional chemical methods, resulting in more reliable probe performance.

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Photostability and spectral properties of fluorophores

The selection of fluorophores is critical to the success of FISH applications. Photostability is essential, as it ensures that the fluorescent signal remains robust throughout the duration of the experiment, allowing for clear visualization of hybridized probes. Additionally, the spectral properties of the dyes must be compatible with the detection system being used. This includes considering the excitation and emission wavelengths to ensure optimal signal detection and minimal overlap between multiple probes in multiplex assays. By carefully selecting the appropriate fluorescent probes and conjugation methods, researchers can significantly enhance the sensitivity and specificity of FISH, making it an invaluable tool in molecular diagnostics and research.

Advantages of using bioconjugation in FISH

Increased specificity and sensitivity

By using chemically modified probes, researchers can achieve stronger binding affinities to their target nucleic acid sequences. This improvement reduces background noise and enhances the signal, allowing for the detection of low-abundance targets that might otherwise go unnoticed. As a result, bioconjugated FISH assays provide more accurate diagnostic information and facilitate the identification of subtle genetic variations.

Versatility and adaptability to various targets

Bioconjugation techniques are highly versatile, enabling the development of probes tailored to a wide range of targets, including DNA, RNA, and proteins. This adaptability is crucial for applications across different research fields, from cancer diagnostics to gene expression studies. Researchers can design specific probes that can bind to various sequences, thereby expanding the applicability of FISH in both clinical and research settings. The ability to customize probes according to specific research needs enhances the overall utility of the FISH technique.

Multiplexing capabilities

By utilizing different fluorophores that emit at distinct wavelengths, multiple targets can be detected simultaneously within a single sample. This capability allows researchers to conduct comprehensive analyses without the need for separate experiments for each target, saving time and resources. Multiplexed FISH assays enable more complex investigations into genetic interactions and expression patterns, providing a more holistic view of the biological system under study.

Predictable and reproducible results

Bioconjugation contributes to the predictability and reproducibility of FISH results. Standardized conjugation methods and optimized probe designs lead to consistent performance across experiments. This reliability is essential in clinical diagnostics, where accurate and reproducible results are critical for patient management. By ensuring that each assay produces comparable outcomes, bioconjugation facilitates the validation of results and enhances confidence in the findings.

Applications of bioconjugation in FISH

The integration of bioconjugation into Fluorescence In Situ Hybridization (FISH) has significantly broadened its applications across various domains, enhancing its utility in both clinical and research settings.

Clinical diagnostics

In clinical diagnostics, bioconjugated probes can specifically bind to chromosomal regions associated with various cancers, allowing for the precise identification of aneuploidies, translocations, and other chromosomal rearrangements. This capability is essential for guiding treatment decisions and monitoring disease progression. Additionally, FISH is employed in gene expression studies to analyze the expression patterns of specific genes within tissues. This application is crucial for understanding the molecular underpinnings of diseases and for developing targeted therapies.

Research applications

Bioconjugated FISH probes also play a vital role in various research applications. One key use is in chromosome mapping, where FISH is instrumental in determining the chromosomal locations of specific genes. This information is critical for genetic mapping studies and for understanding the relationships between gene location and function. Moreover, FISH provides insights into spatial gene expression within tissues, allowing researchers to visualize how gene expression varies across different cell types and conditions. Such studies are essential for elucidating developmental processes and the molecular mechanisms of diseases.

Environmental monitoring

Bioconjugated probes can be used to detect microbial communities in environmental samples, such as soil, water, and biofilms. This application contributes significantly to ecological studies, enabling scientists to assess biodiversity, ecosystem health, and the impacts of environmental changes. Additionally, FISH can aid in bioremediation efforts by identifying specific microorganisms involved in the degradation of pollutants, facilitating the development of strategies to enhance natural remediation processes.

Advanced techniques and innovations

The emergence of a new generation of fluorescent probes provides superior light stability and brightness. These enhanced fluorophores enable sharper and more consistent imaging, even under challenging experimental conditions. Coupled with click chemistry, researchers can now develop probes that maintain integrity and function throughout the experiment. This combination allows for more precise and multi-parameter analysis in clinical and research contexts.

Nanotechnology has facilitated the development of nanoprobes that can improve targeting and imaging capabilities and improve the sensitivity of FISH detection. At the same time, microfluidics offer the potential for high-throughput analysis to process multiple samples simultaneously, reducing the use of reagents and shortening turnaround times. These advances will revolutionize probe design and application, opening up new avenues for research and diagnostics. As the field continues to evolve, continued innovations in bioconjugation technologies will undoubtedly enhance FISH's capabilities and advance our understanding of complex biological systems.