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FRET Peptide Substrates

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.

PEG chains for custom peptide synthesis

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)

Custom FRET substrate FAM CPQ2

5-FAM-GGPLGVRGKK(CPQ2)-PEG2-C-CO2H Kwong, Gabriel A., et al. "Mathematical framework for activity-based cancer biomarkers." Proceedings of the National Academy of Sciences 112.41 (2015): 12627-12632.

FRET Custom Peptide Citations

"All peptides were synthesized by CPC Scientific. For recombinant enzyme studies and ABNz, intramoleculary quenched peptides were used: MMP substrate, 5-FAM–GGPLGVRGKK(CPQ2)–PEG2–C; thrombin substrate, 5-FAM–GGfPRSGGGK(CPQ2)–PEG2–C; where 5-FAM is the 5-carboxyfluorescein fluorophore, CPQ2 is the quencher, PEG2 is the linker polyethylene glycol, and lower case letters indicate the d-stereoisomer of the residue. In vivo protease-sensitive substrates were synthesized to contain a urinary reporter comprised of a protease-resistant d-stereoisomer of glutamatefibrinopeptide B with a near-infrared dye for urinary detection (biotin–CGPLGVRGKK(Cy7)eGvndneeGffsar; Cy7 is cyanine7). Targeting peptides were synthesized and cyclized: LyP1, C–K(5-FAM)–C6–CGNKRTRGC; iRGD, C–PEG2–CRGDKGPDC; where C6 is the 6-aminohexanoic acid linker and there is a disulfide bridge between Cysteines 2 and 3."

1. Kwon, Ester J., Jaideep S. Dudani, and Sangeeta N. Bhatia. "Ultrasensitive tumour-penetrating nanosensors of protease activity." Nature Biomedical Engineering 1 (2017): 0054.Learn More »

"Protease activity in regurgitant from three replicate tanks were compared using a mixture of 7 internally quenched fluorescent substrates available from CPC Scientific, Sunnyvale, California (Table 1) [AMYD-112, AMYD-114, AMYD-109, MMPS-024, SUBS-017, AMYD-111, CASP-060]. These substrates were chosen based on their diverse sequence composition to enable detection of multiple protease classes."

2. Goupil, Louise S., et al. "Cysteine and Aspartyl Proteases Contribute to Protein Digestion in the Gut of Freshwater Planaria." PLoS Negl Trop Dis 10.8 (2016): e0004893.Learn More »

"Internally quenched peptides of the sequences of IFFDTWDNE, TWDNEAYVH, EAYVHDAPV, and HDAPVRSLN, corresponding to amino acids 103 to 111, 107 to 115, 111 to 119, and 115 to 123 of the reference human pro–IL-1b sequence (UniProt: P01584), were labeled on the N terminus with Mca and on the C terminus with Lys-Dnp (CPC Scientific)."

3. LaRock, Christopher N., et al. "IL-1b is an innate immune sensor of microbial proteolysis." Science Immunology 100.200: 300 (2016).Learn More »

"Thrombin-sensitive peptide (ThS-P) azidoacetyl alanine-K(5FAM)GALVPRGSAGK(CPQ2) was custom synthesized (2143 MW, > 95% purity; CPC scientific, Sunnyale, CA, USA) and dissolved in DMSO (20 mm ThS-P)."

4. Welsh, J. D., et al. "Platelet-targeting sensor reveals thrombin gradients within blood clots forming in microfluidic assays and in mouse." Journal of Thrombosis and Haemostasis 10.11 (2012): 2344-2353.Learn More »

"Fusolisin’s restriction specificity was verified using the FRET substrate CPQ2-Gly-Phe-Ile-Thr-Ala-Phe-Pro-Lys-(5FAM)-Arg-Arg-NH2 that was custom synthesized by CPC scientific (Sunnyvale, CA, USA)."

5. Doron, Lior, et al. "Identification and Characterization of Fusolisin, the Fusobacterium nucleatum Autotransporter Serine Protease." PloS One 9.10 (2014): e111329.Learn More »

Subsequently, 5FAM-Abu-Gly-Asp-Asp-Asp-Lys-Ile-Val-Gly-Gly-Lys(CPQ2)-Lys-Lys-NH2 (purity: 97.2%, CPC Scientific, Inc.) produced according to a synthesis method known per se was diluted with an assay buffer to prepare a 2.1 uM (“u” represents “micro”) substrate solution."

6. Ikeda, Zenichi, et al. "Fused heterocyclic compound." U.S. Patent Application No. 14/619,489.Learn More »

"Cysteine-terminated peptides (Q1 = 5FAM-GGPLGVRGKK(CPQ-2)-PEG2-C, CPC Scientific;"

7. Kwong, Gabriel A., et al. "Mathematical framework for activity-based cancer biomarkers." Proceedings of the National Academy of Sciences 112.41 (2015): 12627-12632.Learn More »

"Peptides corresponding to the substrate sequences L/VP4.1 and non-hydrolysable linker (NHL) were synthesized between a 5-carboxyfluorescein (5-FAM) fluorophore/CPC Quencher (CPQ2) quencher pair (CPC Scientific). Additional lysines were added for increased solubility giving final peptide sequences of CPQ2-IVYELQGP-K(5FAM)-KK-NH2 and CPQ2-GGSGGS-K(5FAM)-KK-NH2 for L/VP4.1 and NHL, respectively. The identity and sequence of the L/VP4.1 and NHL peptides were confirmed by CPC Scientific via LC-MS and determined to be 96.7% and 95.1% pure, respectively."

8. Miles, Linde A., et al. "Seneca Valley Virus 3C pro Substrate Optimization Yields Efficient Substrates for Use in Peptide-Prodrug Therapy." PloS One 10.6 (2015): e0129103.Learn More »

"The custom-made thrombin-sensitive peptide azidoacetyl-AK(5FAM)-GALVPRGSAGK(CPQ2)-NH2 was obtained from CPC Scientific (Sunnyvale, CA) for click reactions to anti-CD61, as previously described. "

9. Zhu, Shu, et al. "FXIa and platelet polyphosphate as therapeutic targets during human blood clotting on collagen/tissue factor surfaces under flow." Blood 126.12 (2015): 1494-1502.Learn More »

"Materials Customized peptides AGCGAK(CPQ2) and azidoacetyl-AGCGAK(CP488) (CPC scientific, Sunnyale, CA, USA) are conjugated with a quencher (CPQ2, peptide Q) or a fluorophore (CP488, peptide F) (MW 996.1 and 944.9, respectively, purity > 90%)…"

10. Zhu, S., et al. "Platelet-targeting thiol reduction sensor detects thiol isomerase activity on activated platelets in mouse and human blood under flow." Journal of Thrombosis and Haemostasis (2016), 14: 1070–81.Learn More »

"Fusolisin’s restriction specificity was verified using the FRET substrate CPQ2-Gly-Phe-Ile-Thr-Ala-Phe-Pro-Lys-(5FAM)-Arg-Arg-NH2 that was custom synthesized by CPC scientific (Sunnyvale, CA, USA)."

11. Hershey, David M., et al. "Magnetite biomineralization in Magnetospirillum magneticum is regulated by a switch-like behavior in the HtrA protease MamE." BioRxiv (2016): 047555.Learn More »

"The cleavage preference of AGP was tested using an E(Edans)-AAXAAK-(Dabcyl)-NH 2 fluorescent substrate kit (CPC Scientific Inc., Calif., USA) with “A” representing alanine residues, “K” lysine residues, and “X” different individual amino acid residues as specified by their one ..."

12. Shi, Jing, et al. "Properties of Hemoglobin Decolorized with a Histidine-Specific Protease." Journal of Food Science 80.6 (2015): E1202-E1208.Learn More »

"... Dabcyl-HPHPHLSFMAIPK(5-FAM)KK-NH 2 (98% purity; Figure 1) was purchased from CPC Scientific (Sunnyvale, CA). ..."

13. Jensen, Jesper Langholm, et al. "The function of the milk-clotting enzymes bovine and camel chymosin studied by a fluorescence resonance energy transfer assay." Journal of Dairy Science 98.5 (2015): 2853-2860.Learn More »

"ANP FAP and aP NIRF were synthesized by CPC Scientific (Sunnyvale, CA)… Another internally-quenched FRET peptide substrate (ANPFAP) for FAP has recently been reported and demonstrated for use as an activity-based, in vivo imaging tool. The peptide sequence contains two internal Gly-Pro dipeptide motifs, susceptible to FAP cleavage and a Cy5.5/QSY21, quenched-FRET pair."

14. Bainbridge, Travis W., et al. "Selective Homogeneous Assay for Circulating Endopeptidase Fibroblast Activation Protein (FAP)." Scientific Reports 7.1 (2017): 12524.Learn More »

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:

  • Aggrecanase
  • ADAMs
  • ACE-2
  • APCE
  • 2A protease
  • BACE1
  • Calpains
  • Capases
  • Carboxypeptidases
  • CaspasesCathepsins
  • Chymopapain
  • Complement component C1s
  • CMV protease
  • ECE-1
  • Factor Xa
  • Furin
  • Granzyme K
  • HCV protease
  • HIV proteaseHRV1
  • Kallikreins
  • Interferon alpha A
  • Lethal Factor Protease
  • Malaria Aspartyl Proteinase
  • MMPs
  • Pepsin
  • Plasmin
  • Plasmepsin II
  • Proteinases Protein Tyrosin Phosphatase
  • Renin
  • SARS
  • TACE
  • Thrombin
  • TEV protease
  • Trypsin
  • West Nile Virus Protease

TABLE OF COMMON FRET PAIRS

DONOR (FLUOROPHORE) EXCITATION λ EMISSION λ 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
Cy5.5 678 nm 701 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)
Eu(III) Chelate 340 nm 613 nm QSY-7

Time-resolved FRET (TR-FRET) peptides

Time-resolved FRET (TR-FRET) has emerged as a method that utilizes long-lived fluorophores (characteristic of lanthanide elements) to delay measurements by 50–150 µs. Consequently, TR-FRET peptides are labeled with a well-defined fluorescent donor (a fluorophore) that delays the measurements by this timeframe. This time delay allows the signal to be cleared of most nonspecific short-lived emissions. TR-FRET eliminates background fluorescence resulting in better data quality. Eu(III) Chelate and QSY-7 (Ex/Em = 340/613 nm) is an ideal FRET pair for TR-FRET HTS assays.

TR-FRET peptide Eu(III)

Elshan, NGR Dayan, et al. Analytical Biochemistry 464 (2014): 24-29.