FITC labeled siRNA

What is the FITC labeled siRNA?

FITC-labeled siRNA entails small interfering RNA (siRNA) molecules conjugated with fluorescein isothiocyanate (FITC), a fluorescent dye. This tagging enables the detection and tracking of siRNA molecules in biological research. FITC is a commonly utilized fluorescent dye that generates green light upon excitation by certain wavelengths, such as 488 nm. When attached to biomolecules such as siRNA, FITC facilitates the tracking of tagged molecules using a fluorescence microscope or flow cytometry. FITC-labeled siRNA is essential for examining the absorption, localization, and distribution of siRNA within cells and tissues, particularly in the development of gene-silencing therapies.

FITC-siRNA hybrids with sense-strand modifiers. (Kozuch S D., et al., 2018)

How to prepare FITC-labeled siRNA?

Following synthesis, a 5′-DMS(O)-MT-Amino C6 modifier was coupled to the 5′ terminal hydroxyl group using an extended 15 min coupling time on the DNA/RNA synthesizer. The amino-functionalized RNA templates were then subjected to a solid-phase bioconjugation reaction for the attachment of the fluorescent probe, fluorescein isothiocyanate (FITC). The attachment of FITC was achieved by reacting FITC (75 equiv, 30 mg) dissolved in 20% aqueous N,N-dimethylformamide (DMF) in 0.1 M carbonate–bicarbonate buffer (pH 8.5) to the amino-modified RNA templates. The reaction was agitated at room temperature (RT) for 48 h on a benchtop shaker. Following the reaction, the CPG-bound RNA was washed with DMF and acetonitrile (MeCN) until no visible orange color remained in the supernatant. The sample was then cleaved and deprotected using 3:1 v/v NH4OH/EtOH at 55 ℃ for 12-14 h. The 2′-TBDMS protecting groups were desilylated using 1:1.5 v/v trimethylamine-trihydrofluoride TEA-3HF/DMSO at 65 °C for 2 h. The crude FL-RNA templates were precipitated in 3 M NaOAc (25 μL) and n-BuOH (1 mL) and dissolved in diethyl pyrocarbonate-treated Millipore water, RNase-free, for analysis and purification.

Solid-Phase FITC Bioconjugation of Linear, V-, and Y-Shape siRNA. (Kozuch S D., et al., 2018)

Self-Assembly of (i) Linear, (ii) V-, and (iii) Y-Shape multi-FITC-Labeled siRNA constructs. (Kozuch S D., et al., 2018)

RNA conjugation technology at BOC Sciences

Applications of FITC labeled siRNA

In Vivo imaging: The FITC label facilitates real-time observation of siRNA dispersion throughout cells or tissues. This assists in ascertaining if the siRNA attains the designated target location (e.g., cytoplasm or nucleus) and if it aggregates in certain cellular compartments. TRITC-Fol-PαC (G3, DSF 4) can accumulate in neoplasms. One study assessed the biodistribution of FITC-siRNA and TRITC-Fol-PαC (G3, DSF 4) following intravenous administration to tumor-bearing mice by measuring their fluorescence in different organs and tumors using a Maestro EX imaging system. The fluorescence from FITC-siRNA was mostly noticed in the kidneys 10 minutes post-intravenous injection and was significantly identified in tumors 30 minutes after delivery. Conversely, the fluorescence originating from TRITC-Fol-PαC (G3, DSF 4) was mostly observed in the liver and kidney during the first hour, then escalating in tumor tissues. The findings indicate that FITC-siRNA and TRITC-Fol-PαC (G3, DSF 4) accumulate in tumor tissues following intravenous delivery of the complex to tumor-bearing mice. The tumor tissue concentration of FITC-siRNA following intravenous administration of the combination was markedly elevated compared to that of FITC-siRNA administered alone. The concentrations of FITC-siRNA and TRITC-Fol-PαC (G3, DSF 4) in tumor tissue rose until one-hour post-administration, followed by a slow decline. After 5 hours of intravenous injection, the tumor tissue concentrations of FITC-siRNA and TRITC-Fol-PαC (G3, DSF 4) were approximately 6 μg/tissue (10 ng/mg of tissue) and 0.4 μg/tissue (0.75 ng/mg of tissue), respectively. The results indicate that although the FITC-siRNA combination with TRITC-Fol-PαC (G3, DSF 4) may partially dissolve following intravenous injection into tumor-bearing mice, both FITC-siRNA and TRITC-Fol-PαC (G3, DSF 4) can accumulate in tumors.

Intracellular distribution of FITC-siRNA complexes. (Arima H., et al., 2012)

In vivo imaging after intravenous injection of TRITC-Fol-PαC (G3, DSF 4)/FITC-siRNA complex. (Arima H., et al., 2012)

For cellular uptake studies: FITC-labeled siRNA is frequently employed to investigate the cellular uptake of siRNA molecules. Evaluate the efficacy of different delivery mechanisms, such liposomes, nanoparticles, or viral vectors, by monitoring fluorescence. A research shown that KB and A549 cells were treated with FITC-siRNA–loaded polyplex nanoparticles (PNPs) for four hours, followed by staining with α-FR antibody. The flow cytometry results indicated that the absorption of FITC-siRNA supplied by H/G(PC8kF6) was very effective in FR+ KB cells, characterized by elevated FR expression, but exhibited reduced efficiency in FR− A549 cells, which had low FR expression. No selectivity was noted in the delivery of FITC-siRNA by the nontargeted carrier H alone, further confirming that the cellular absorption of H/G(PC8kF6)-based PNPs was facilitated by FR-mediated endocytosis.

Cellular uptake of PNPs formed by FITC-siRNA. (Wen Y., et al., 2020)

Advantages of FITC labeled siRNA

Visualization and tracking in real-time: A principal benefit of FITC-labeled siRNA is its capacity for real-time imaging of siRNA molecules. The FITC (fluorescein isothiocyanate) fluorophore emits a vivid green fluorescence upon excitation, allowing researchers to observe the cellular uptake, intracellular localization, and distribution of siRNA within live or fixed cells through methods such as fluorescence microscopy or confocal microscopy. This real-time picture is essential for comprehending the dynamics of siRNA-based delivery systems.

Efficient monitoring of transfection: Labeling siRNA with FITC enables effective monitoring of siRNA transfection efficacy into target cells. This labeling facilitates the evaluation of siRNA uptake by cells and the efficacy of siRNA delivery systems, enabling researchers to refine transfection techniques and implement alterations to enhance delivery efficiency. Furthermore, the efficacy of transfection may be measured using flow cytometry or other fluorescence-based methods.

Non-invasive detection: FITC-labeled siRNA offers a non-invasive method for monitoring siRNA molecules. FITC labeling facilitates non-invasive detection in biological systems, obviating the need for more sophisticated and time-consuming approaches like radioactive labeling. This facilitates safer usage in laboratory environments while providing precise data on the siRNA's activity and localization within the cell.

Can be used for quantification: FITC-labeled siRNA is applicable in flow cytometry for the quantitative assessment of cell populations according to siRNA absorption levels. This facilitates high-throughput screening of cells, allowing for accurate assessments of transfection effectiveness across various cell types or circumstances. Researchers can quantify the percentage of cells that have effectively internalized siRNA, facilitating swift evaluations of siRNA delivery mechanisms.

Limitations of FITC labeled siRNA

Photobleaching: A significant disadvantage of employing FITC as a fluorescent marker is its propensity for photobleaching, characterized by the permanent loss of fluorescence during extended light exposure. This might restrict the duration of investigations necessitating real-time monitoring of siRNA, particularly in live-cell imaging, as the signal diminishes with prolonged observation, resulting in reduced sensitivity in long-term studies.

Quenching of fluorescence: The fluorescence of FITC can be diminished in the presence of certain biological components or environmental conditions, such as variations in pH within distinct cellular compartments (e.g., acidic environments like lysosomes). The pH sensitivity may compromise the precision of monitoring the tagged siRNA inside intricate biological systems, since the signal might not correctly represent the true location and distribution of siRNA within the cell.