Experimental models and measurements to study cardiovascular physiology
Neil Herring, David J. Paterson in Levick's Introduction to Cardiovascular Physiology, 2018
Improved imaging technolog y and exploitation of genetically encoded fluorescence technolog y have also been combined to measure subcellular microdomains of second messengers. Transfected proteins contain two different fluorescent molecules that come into close proximity to each other on binding of a second messenger. This produces a transfer of fluorescence, or Forster resonance energy transfer (FRET) between donor and acceptor chromophores such that the emission ratio between their two wavelengths changes as demonstrated in Figure 19.3. For example, cyclic adenosine monophosphate (cAMP) FRET sensors are structurally based on exchange protein directly activated by cAMP (Epac), protein kinase A, or cyclic nucleotide-gated channels and often use a cyan fluorescent protein donor and a yellow fluorescent protein acceptor to tag the regulatory and catalytic subunits, respectively. The FRET signal can be pH-sensitive; important control experiments involve demonstrating that the FRET signal is within the dynamic range and not saturated, and also transfecting a dead sensor control that contains the chromophores but has lost the ability to bind the second messenger.
Application of Nonlinear Microscopy in Life Sciences
Lingyan Shi, Robert R. Alfano in Deep Imaging in Tissue and Biomedical Materials, 2017
Forster resonance energy transfer is a process of nonradiative energy transfer between an excited donor molecule and an acceptor molecule in ground state within close proximity (several nanometers). Figure 6.8 depicts one of requirements of FRET—appreciable spectral overlap between donor emission and acceptor absorption. FRET is often used to study protein-protein interactions on the nanometer scale. The donor quenching and the increased emission of the acceptor due to FRET [75] can be measured in cultured cells, but is unreliable in tissues due to high levels of autofluorescence and spectrally dependent and variable light absorption in tissue. The shortening of the fluorescence lifetime of the donor fluorophores, on the other hand, can be measured precisely even deep in scattering and absorbing tissue. Point scanning multiphoton microscope with TCSPC detection is the prevalent instrumentation for FLIM-FRET measurements [76].
Conjugation and Other Methods in Polymeric Vaccines
Mesut Karahan in Synthetic Peptide Vaccine Models, 2021
Fluorescence life is the time the induced level molecule passes, before it passes the basic electronic level. Most aromatic molecules have a fluorescence lifetime of 10 ns. The maximum wavelengths that aromatic amino acids absorb and fluorescence will depend on the fluorescent lifetime. In the studies, the synthesis of peptide epitopes is carried out in the structure of tryptophan and other aromatic amino acids, especially in foot and mouth disease, hepatitis B, and other diseases (Budama 2006). On synthetic polyelectrolytes and their structures (Budama et al. 2008), using different wavelengths is determined by the degree of peptide-polymer conjugation reaction using biomolecules (peptide, protein, etc.) with the same type of electric charge and, lastly, the FRET method is used to investigate the metal binding mechanism of the biopolymer (Acar et al. 2019; Karahan, Mustafaeva, and Ozer 2007). With the help of the FRET method, the intermolecular distance relationships can be examined in the distance measurement between the two places in the macromolecules (Acar et al. 2019). In addition, this method is considered to be a powerful technique to study molecular interactions in living cells with improved spatial (angstrom) and temporal (nanosecond) resolution, distance range, and sensitivity, and a wider range of biological applications (Sekar and Periasamy 2003; Carmona, Juliano, and Juliano 2009). Investigation of protein fragments in terms of these properties is thought to be of great benefit in peptide vaccine synthesis.
Discovery of RNA-targeted small molecules through the merging of experimental and computational technologies
Published in Expert Opinion on Drug Discovery, 2023
The three fluorescence-based assays that are often employed for screening of RNA binders are: (1) fluorescence resonance energy transfer (FRET)-based assay (Figure 3(a)), (2) time-resolved FRET (TR-FRET) assay, and fluorescent indicator displacement (FID). FRET refers to the transfer of energy from a donor fluorophore to an acceptor fluorophore conjugated to the target biomolecule. FRET-based assays are convenient and extremely sensitive and have become popular for screening small-molecule libraries against RNA targets. Simone et al. [92] used a FRET-based assay to screen for small-molecule stabilizers of the C9orf72 (G4C2)4 G-quadruplex RNA, which is a known cause of frontotemporal dementia and amyotrophic lateral sclerosis [116]. The authors monitored the changes in melting temperature of the 5’-FAM and 3’-TAMRA labeled (G4C2)4 RNA upon heating in the presence of small molecules and identified three structurally similar small molecules that stabilize the RNA. The small molecules were subsequently shown to reduce the frequency of RNA foci and the levels of dipeptide repeat protein in C9orf72 patient neurons. Furthermore, the most effective small molecule, DB1273, was found to improve survival and reduce levels of toxic poly-(glycine-arginine) in C9orf72 flies.
A clinical role for Förster resonance energy transfer in molecular diagnostics of disease
Published in Expert Review of Molecular Diagnostics, 2019
The large popularity of FRET-based applications started in the early 90s, driven by significant advances in new fluorophores, detection methods, and instrumentation [1]. Implementation of FRET techniques into molecular diagnostics has started synchronously, with a continuous increase over the last 30 years. Owing to its strong distance dependence in the biological interaction range (circa 1 to 20 nm), FRET can quantify almost any target of interest (proteins, nucleic acids, metabolites, drugs, toxins, human cells, microbes, and other pathogens) from various types of clinical specimen (body fluids, cells, tissues) [2,3]. FRET can also monitor molecular dynamics in biophysics and molecular biology, such as DNA-DNA, protein–protein, or protein–DNA interactions, and protein conformational changes [4]. FRET-based biosensors have been utilized to monitor cellular dynamics not only in heterogeneous cellular populations, but also at the single-cell level in real time.
Protein interactions study through proximity-labeling
Published in Expert Review of Proteomics, 2019
Benoît Béganton, Isabelle Solassol, Alain Mangé, Jérôme Solassol
Like PLA, the FRET technique makes it possible to detect in vivo the direct interactions between two proteins by fluorescence emission. This approach requires, however, a particular molecular engineering. The studied cells need to express the interest proteins fused to a fluorophore donor for one and an acceptor fluorophore for the other. When the two fused proteins that are expressed in the cells interact, the donor fluorophore is excited by a light stimulus; it transfers its energy by resonance to the acceptor fluorophore, which, in turn, emits a fluorescent signal at a specific wavelength [68] (Figure 1). The advantage of this technique is to carry a dynamic analysis of in vivo interactions of cells in culture. The specificity of energy transfer between donor and acceptor also significantly reduces the false-positive rate. Nevertheless, one of the limitations of this approach is that it is not applicable to clinical samples that cannot be transfected to express fused proteins such as tissue sections (Table 1).
Related Knowledge Centers
- Absorption Spectroscopy
- Fluorescence
- Fluorophore
- Microscopy
- Quantum Yield
- Refractive Index
- Near & Far Field
- Radiative Transfer
- Emission Spectrum
- Molar Absorption Coefficient