The Ultimate Guide to GFP mRNA

GFP is a widely used fluorescent reporter gene that enables you to visualize protein expression and transfection efficiency with standard filter sets. It is also an excellent control for testing different transfection reagents.

eGFP mRNA is expressed primarily by epidermal keratinocytes in appendageal structures (skin surface to 120-150 mm depth) and vascular cells and, to a lesser degree, in dermal fibroblasts.

Excitation and Emission Wavelengths

When irradiated with low-intensity UV, wild-type GFP reveals a neutral and anionic peak (F1 and F2, respectively) in its emission spectrum. This is due to a phenomenon known as excited-state proton transfer, in which a fraction of the chromophore's phenolic oxygen becomes more acidic after irradiation, thus changing from its ground state into an active, excited species.

Site-directed and random mutagenesis studies have shown that fluorescence depends very heavily on the three-dimensional structure of the amino acid residues surrounding the chromophore. Mutations close to the chromophore, as expected, have the most significant impact on fluorescence properties; however, mutations far removed from the chromophore can also significantly alter fluorescent characteristics.

To evaluate how these spectral changes affect the brightness of a fluorescent protein, a 3D fluorescence spectra is conducted using an eGFP standard obtained from a quantification kit. 

Molecular Weight

Green Fluorescent Protein (GFP) is a widely used direct detection reporter in mammalian cell culture. It produces bright green fluorescent light with an emission peak at 509 nm when exposed to blue ultraviolet light. GFP has a structure that is a cylinder with 11 beta-sheets outside and a single alpha helix in the center, which binds to aequorin, a blue molecule that gives rise to the chemiluminescence observed in GFP.

A single point mutation in the GFP gene created the eGFP mRNA protein with improved spectral characteristics. eGFP is 18-fold brighter than native GFP and has a central excitation peak at 395 nm with a minor one at 488 nm.

Relative Brightness

The relative brightness of a fluorescent protein is the ratio of its extinction coefficient (e) to its fluorescence quantum yield. The e of the wild-type GFP (wtGFP) is about 55,000 M-1cm-1, which allows it to be detected by standard filters for fluorescein and related synthetic dyes. However, wtGFP has a relatively low quantum yield, which makes it challenging to image in cells. A mutation in the chromophore of wtGFP, replacing Tyr66 with histidine (Y66H), improved the quantum yield and shifted the emission spectrum towards blue light. This variant, enhanced GFP or eGFP, is among the brightest of the Aequorea-based fluorescent proteins.

Further mutations in the chromophore of GFP have led to other color variants, including blue and cyan fluorescent proteins. These offer distinct advantages for applications like multicolor labeling but are still relatively weaker than eGFP. Biochemists and biologists continue to engineer fluorescent proteins to improve their brightness, photostability, and other properties. As a result, it's essential to consider multiple options when choosing a fluorescent protein for your experiments. 

Quantum Yield

Typically measured as a percentage, fluorescence quantum yield (Ph) represents the ability of a molecule to convert an absorbed photon into a fluorescence emission. Higher Ph values are associated with brighter fluorescence and a value of 1 means that every absorbed photon is converted to a fluorescence emission.

The concept of quantum yield is well-established in the field of photochemistry. It is used to quantify the amount of reaction that occurs per absorbed photon, as opposed to the total amount of energy lost through radiative transitions (internal conversion and vibrational relaxation), which deactivate the excited state and return it to its ground state. In other words, a chemical reaction with a high quantum yield is more efficient than one with a low quantum yield.

For example, eGFP has a Ph of 1.0, meaning that each absorbed photon produces a fluorescence emission. Compared to its competitors, such as the standard fluorescent proteins Fluo-3 and Fluo-4, eGFP has a much higher quantum yield, which translates into a stronger fluorescence signal. This is why eGFP is used as a direct detection reporter in cell biology experiments.

Relative Stability

The stability of mRNA is a critical factor for envisaged applications in which the expression of a gene of interest must be maintained over time. The mRNA must withstand cellular degradation and translation to produce the desired protein, which may require several hours. The half-life of GFP (wild type) has been reported to be 25-54 h.

Using a lipid nanoparticle (LNP) delivery system, mRNA and pDNA encoding for the same sequence of eGFP can be delivered to cells. LNPs are formulated with Ionizable lipid, DSPC, cholesterol, and DMG-PEG2000 at an optimal molar concentration to ensure high encapsulation rates and a fast uptake into the cell.

Results showed that mRNA transfection yielded fluorescence intensity time courses with an early onset and steady state of eGFP expression comparable to pDNA. In contrast, pDNA transfection yields sigmoidal intensity time courses with a distribution of onset times of varying durations.

In addition, mRNA transfection resulted in a more uniform expression level of d2EGFP than pDNA transfection. This is because mRNA molecules can be translated directly from the cytoplasm after endosomal escape, while pDNA needs to enter the nucleus before translation begins.