DESIGN & SYNTHESIS OF FRET PEPTIDES AND SUBSTRATES
|FIGURE. FRET caspase-1 substrate, Dabcyl-Tyr-Val-Ala-Asp-Ala-Pro-Val-EDANS (CASP-023). This fluorogenic caspase-1 substrate enables a continuous assay of caspase-1 helpful in the screening of inhibitory compounds (Km = 11.4 µM, kcat = 0.79 s-1)|
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CPC Scientific has extensive knowledge in the design and synthesis of peptide FRET substrates.1-14 We offer a wide range of FRET substrates to suite your research needs as pre-manufactured FRET peptides or as custom FRET sequences. As part of our services, we provide free consulation to help you design your FRET peptide and select the appropriate FRET pair (see Table of Common FRET Pairs). We often recommend our trade-marked highly efficient quencher, CPQ2TM, to pair with the fluorecent donor 5-carboxyfluorescein (5-FAM). This efficient pair, 5-FAM/CPQ2TM, has been cited in a variety of publications in research areas spaning from cancer therapeutics to diabetes.3-12
FRET (Fluorescence Resonance Energy Transfer) is a distancedependent dipole-dipole interaction without the emission of a photon, which results in the transfer of energy from an initially excited donor molecule to an acceptor molecule. It allows the detection of molecular interactions in the nanometer range. FRET peptides are labeled with a donor molecule and an acceptor (quencher) molecule. In most cases, the donor and acceptor pairs are two different dyes. The transferred energy from a fluorescent donor is converted into molecular vibrations if the acceptor is a non-fluorescent dye (quencher). When the FRET is terminated (by separating donor and acceptor), an increase of donor fluorescence can be detected. When both the donor and acceptor dyes are fluorescent, the transferred energy is emitted as light of longer wavelength so that the intensity ratio change of donor and acceptor fluorescence can be measured. In order for efficient FRET quenching to take place, the fluorophore and quencher molecules must be close to each other (approximately 10-100 Å) and the absorption spectrum of the quencher must overlap with the emission spectrum of the fluorophore. While designing a donor-quencher FRET system, a careful comparison of the donor’s fluorescence spectrum with the quencher’s absorption spectrum is required.
The design and synthesis work at CPC for FRET and TR-FRET peptide substrates include modification of sequences, selection of donor/quencher pairs, improvement of FRET substrate solubility and quenching efficiency.
CPC has experience with a wide range of protease peptide substrates including:
TABLE OF COMMON FRET PAIRS.
|Donor (Fluorophore)||Excitation||Emission||Acceptor (Quencher)|
|EDANS (5-[(2-Aminoethyl) amino] naphthalene-1-sulfonic acid)||340 nm||490 nm||Dabcyl (4-(4-Dimethylaminophenylazo)benzoyl)|
|Lucifer Yellow||430 nm||520 nm||Dabsyl (4-(4-Diethylaminophenylazo)benzenesulfonyl)|
|Mca (7-Methoxycoumarin-4-yl)acetyl)||325 nm||392 nm||Dnp (2,4-Dinitrophenyl)|
|Abz (2-Aminobenzoyl)||320 nm||420 nm||pNA (para-Nitroaniline)|
|Abz (2-Aminobenzoyl)||320 nm||420 nm||3-Nitro-Tyr (3-Nitro-tyrosine)|
|Abz (2-Aminobenzoyl)||320 nm||420 nm||4-Nitro-Phe (4-Nitro-phenylalanine)|
|FITC (Fluorescein isothiocyanate)||490 nm||520 nm||Dnp (2,4-Dinitrophenyl)|
|5-TAMRA (Carboxytetramethylrhodamine)||547 nm||573 nm||QSY7|
|CP488||495 nm||519 nm||CPQ2TM (proprietary structure)|
|5-FAM (5-Carboxyfluorescein)||492 nm||518 nm||CPQ2TM (proprietary structure)|
|Cy5||647 nm||665 nm||QSY21|
|Dansyl (5-(Dimethylamino)naphthalene-1-sulfonyl)||342 nm||562 nm||4-Nitro-Phe (4-Nitro-phenylalanine)|
|Trp (Tryptophan)||280 nm||360 nm||Dnp (2,4-Dinitrophenyl)|
|Trp (Tryptophan)||280 nm||360 nm||4-Nitro-Z (4-Nitro-benzyloxycarbonyl)|