Fluorescein Labeling of Oligonucleotides

What is Fluorescein?

Fluorescein is a synthetic fluorescent pigment-like compound that emits a bright green fluorescence when exposed to ultraviolet light. The molecule fluoresces very strongly, with peak excitation occurring at 495 nm and peak emission at 520 nm. The chemical structure of fluorescein consists of an aromatic ring, an oxygen heteroatom, and three carboxyl groups that make it water-soluble, and it exists in a deprotonated form in solution. Because fluorescein fluorescence is pH sensitive, changes in pH in the external environment may affect its fluorescent properties, making it useful in certain pH-dependent applications. Because of its bright green color, fluorescein is widely used as a fluorescent tracer in many biological and chemical applications.

Figure 1. A method of synthesizing fluorescein using a fusion of phthalic anhydride and resorcinol.

Common and widely used fluorescein derivatives:

• Fluorescein Isothiocyanate 1 (FITC)
• Succinimidyl Ester Modified Fluorescein(NHS-Fluorescein)
• Carboxyfluorescein(FAM), Carboxyfluorescein Succinimidyl Ester(CFSE), Pentafluorophenyl Esters (PFP), Tetrafluorophenyl Esters (TFP)

What is Fluorescein Labeling Oligonucleotide?

Fluorescein labeling of oligonucleotides is achieved by attaching a fluorescein molecule to the oligonucleotide sequence, which is mainly shown to be a probe with the advantages of high sensitivity, high specificity, low toxicity and easy monitoring.

The application of fluorescently labeled oligonucleotides is based on the principle of nucleic acid hybridization. The target DNA or RNA is analyzed by various forms of hybridization reactions with the target nucleic acid molecules, followed by relevant fluorescent probe detection techniques. Among the diagnostic tests relevant to the application of modified oligonucleotides, fluorescence-based detection is an extremely important class of technology. For example, for some specific applications of DNA Sanger sequencing and in situ hybridization (e.g., FISH ), oligonucleotides require a single label, whereas probes used for real-time fluorescence quantification of DNA/RNA (fluorescent motif-quencher probes) and probes used for allele identification (e.g., molecular beacon s), are dual-labeled. In the use of fluorescent motif-quencher pairs, dynamic quenching occurs via FRET (fluorescence resonance energy transfer) or collisional quenching. The quenching mechanism is related to the probe type. In general, either the probe opens, increasing the distance between the fluorophore and the quencher so that quenching stops (e.g., molecular beacons), or the fluorophore or quencher cleaves from the probe (e.g., Taqman probes).

Figure 2. Schematic representation of the structure and schematic representation of fluorescently labeled oligonucleotides.

Applications of Fluorescein Labeling Oligonucleotides

• Fluorescence in situ hybridization (FISH), used to look at specific nucleic acid sequences within a cell or tissue.
• Real-Time Rapid PCR, for real-time PCR monitoring and giving results in a short period of time.
• Fluorescence-based biosensor development, aiding in the establishment of various nucleic acid tests and quantitative assays.

BOC Sciences Offers Fluorescein Labeling Oligonucleotides

BOC Sciences is dedicated to providing you with labeled oligonucleotides of fluorescein and its derivatives and custom synthesis services to assist you with your nucleic acid-based detection and analysis projects in visual biology. We offer a one-stop shop for the design and synthesis of fluorescein-labeled oligonucleotides and get you the products you need in the shortest possible time.

Service Workflow

• Sequence Design and Modification Site Selection

We have a team of experienced experts to provide consultation and technical support, including a range of services starting from sequence design. Of course, you can also provide the target sequence and we will provide synthesis services starting from fluorescein modification.

• Chemical Synthesis and Coupling

Based on our expertise in oligonucleotide synthesis, we use an optimized preparation process to incorporate modified nucleotides containing active groups coupled to fluorescein into the oligonucleotide sequence. Upon completion of the synthesis, the oligonucleotide will undergo a coupling reaction with the fluorescein derivative.

• Purification and Characterization

In the last but not least step, we will purify the labeled oligonucleotides using standard purification and characterization methods to remove any unreacted fluorescein molecules or by-products to ensure purity.

Types of Fluorescein Available

Based on industry-leading oligonucleotide synthesis technology platforms and state-of-the-art equipment, more tailor-made services not mentioned are available at BOC Sciences. Please relax and contact us, we always look forward to partnering with you.

Reference

1. D, Egloff.; et al. Sequence-Specific Post-Synthetic Oligonucleotide Labeling for Single-Molecule Fluorescence Applications. ACS Chem. Biol. 2016, 11(9): 2558–2567.