Fluorescent techniques are widely used in biological research, which are related to their high sensitivity, selectivity, fast response time, flexibility and experimental simplicity. Fluorescent tags or probes are ideal for many imaging and diagnostic applications, also are be used for biological sensors. In many cases, biological sensors consist of fluorescent versions of a particular protein. However, the production and purification of labeled recombinant proteins is labor-intensive and not very efficient. Peptides, owing to their modular nature, synthetic accessibility and biomolecular recognition potential, are efficient and selective fluorescent sensors. Here we offer a guide to help researchers add suitable fluorescent modifications to your experimental repertoire.
Table 1 Advantages and limitations of different fluorescent probes (Pazos, Elena, et al).
For Fluorescent peptides, the fluorescent dye can be attached to the N- or C- terminus or side-chain of amino acids such as lysine, glutamic. Peptides labelled with a single dye as tracers can be assayed by fluorescence. Below is a list of dyes or fluorescence:
Table 2 Available dyes or fluorescence in GenScript (include but not limited)
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FRET (Förster resonance energy transfer) is a powerful research tool for the study of biochemical reaction, and offers a safer alternative to the use of radiolabeled isotopes and methods are quick, sensitive and easily automated.
In the case FRET peptides, FRET pairs consist of an acceptor and quencher and can either be attached internally or externally. Internal attachment of FRET pairs is recommended for longer sequences. In general the donor and acceptor moieties are different, in which case FRET can be detected by the appearance of fluorescence of the acceptors or by quenching of donor fluorescence. The donor probe is always a fluorescent molecule, and not all acceptor moieties are fluorescent (Table 3).
Appropriate donor-acceptor pairs should have enough spectral overlap for efficient energy transfer to take place, yet have enough of a difference in spectra as to be distinguishable from one another. For example, EDANS (Ex/Em=340 nm/490 nm) is often used as a donor in conjunction with the DABCYL in the development of FRET probes, as is Mca (Ex/Em=328 nm/393 nm) and the N-2,4-dinotrophenyl quencher.
Figure 1. Overlap of donor emission (Em.) and acceptor absorption (Abs.) spectra (normalized) and the resulting spectral overlap integrand (red curve) (Algar, W. Russ, et al.).
Table 3 Common FRET pairs in GenScript (include but not limited)
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Applications for fluorescent peptides range from the study of peptide-protein interactions, enzyme activity assays, immunoassay, in vivo imaging, or development of novel disease models.
For subcellular imaging and tracking
Many discoveries in cell biology rely on making specific proteins visible within their native cellular environment. Combined with confocal or fluorescent microscopy, fluorescent-labelled peptides are used to identify specific targets. For example, cell penetrating peptides (CPPs) modified with FITC and photoactivatable probes have been used to track binding patterns and dynamic behavior over time (Figure 2). Similarly, Kirkham et al. found that FITC-labeled CPPs are uptaken by fibroblasts with low risk of cytotoxicity.
Figure 2. Structures of constructed CPP probes (Left) and the intracellular distribution of fluorescent-labeled CPPs in live BSC-1 cells (Middle and Right) (Pan et al.).
Molecules that absorb in the near-infrared (NIR) region, 700–1000 nm, can be efficiently used to visualize and investigate in vivo molecular targets because most tissues generate little NIR fluorescence. Many novel peptide-based near infrared red (NIR) fluorescent probes have been developed for in vivo biomedical imaging, for instance, figure 3 shows typical NIR fluorescence images of athymic nude mice bearing subcutaneous U87MG glioblastoma tumor after intravenous injection of 3 nmol of RGD-Cy5.5. Hara et al. found that using Cy7 labeled fibrin-targeting peptides, CT scanning and confocal microscopy are used to differentiate between acute and subacute murine DVT (deep vein thrombosis). Similar techniques have also been used for detecting apoptosis as a symptom for glaucoma. Fluorophore-labeled peptides that are activated by caspases can be imaged in vivo to track glaucoma progression.
Figure 3. In-vivo image of a rat injected with 3nmol of RGD-Cy5.5 (Chen, et al).
enzyme activity detection
Proteolytic enzymes play important roles in infectious diseases and are ideal targets for developing novel therapeutics. Protease assays usually involve a fluorescent donor moiety and a quenching acceptor molecule, separated by a peptide containing the protease cleavage sequence. As shown in figure 4, a donor molecule, such as Abz or Lucifer Yellow is covalently attached to the C-terminus, while acceptor molecules (ex. Dabsyl, DNP, EDDnp) are coupled to the N-terminus. If the peptide is cleaved by protease, the peptide will no longer be quenched by FRET and fluorescence will be released. Since protease studies are most effective when FRET is combined with shorter peptides, peptide libraries are recommended for these experiments.
Figure 4 Schematic representation of the FRET peptide mechanism with Abz/EDDnp donor/acceptor pair. Fluorescence is released upon cleavage of any peptide bond within the amino-acid sequence (Carmona, Adriana K., et al).
GenScript’s peptide services have been trusted by 10,000+ scientists since 2002, more than 300 peptide modifications are available. The design and synthesis work at GenScript for fluorescent peptide include modification of sequences, selection of donor/acceptor pairs, improvement of FRET substrate solubility and quenching efficiency.
Further information, please visit https://www.genscript.com/peptide-services.html
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1. Algar, W. Russ, et al. "FRET as a biomolecular research tool—understanding its potential while avoiding pitfalls." Nature methods 16.9 (2019): 815-829.
2. Johnson, Oleta T., Tanpreet Kaur, and Amanda L. Garner. "A conditionally fluorescent peptide reporter of secondary structure modulation." ChemBioChem 20.1 (2019): 40-45.
3. Pan, Deng, et al. "A general strategy for developing cell-permeable photo-modulatable organic fluorescent probes for live-cell super-resolution imaging." Nature communications 5.1 (2014): 1-8.
4. Kirkham, Steven, et al. "A self-assembling fluorescent dipeptide conjugate for cell labelling." Colloids and Surfaces B: Biointerfaces 137 (2016): 104-108.
5. Hara, Tetsuya, et al. "Molecular imaging of fibrin deposition in deep vein thrombosis using fibrin-targeted near-infrared fluorescence." JACC: Cardiovascular Imaging 5.6 (2012): 607-615.
6. Qiu, Xudong, et al. "Single-cell resolution imaging of retinal ganglion cell apoptosis in vivo using a cell-penetrating caspase-activatable peptide probe." PLoS One 9.2 (2014).
7. Marcondes, M. F. M., et al. "Substrate specificity of mitochondrial intermediate peptidase analysed by a support-bound peptide library." FEBS open bio 5 (2015): 429-436.
8. Rossé, Gérard, et al. "Rapid identification of substrates for novel proteases using a combinatorial peptide library." Journal of combinatorial chemistry 2.5 (2000): 461-466.
9. Pazos, Elena, et al. "Peptide-based fluorescent biosensors." Chemical Society Reviews 38.12 (2009): 3348-3359.
10. Held, Paul. "An introduction to fluorescence resonance energy transfer (FRET) technology and its application in bioscience." Bio-Tek Application Note (2005).
11. Rao, Jianghong, Anca Dragulescu-Andrasi, and Hequan Yao. "Fluorescence imaging in vivo: recent advances." Current opinion in biotechnology 18.1 (2007): 17-25.
12. Chen, Xiaoyuan, Peter S. Conti, and Rex A. Moats. "In vivo near-infrared fluorescence imaging of integrin αvβ3 in brain tumor xenografts." Cancer research 64.21 (2004): 8009-8014.
13. Carmona, Adriana K., Maria Aparecida Juliano, and Luiz Juliano. "The use of Fluorescence Resonance Energy Transfer (FRET) peptidesfor measurement of clinically important proteolytic enzymes." Anais da Academia Brasileira de Ciencias 81.3 (2009): 381-392.