Guidelines in Designing FRET Peptide Substrates

What is FRET?
Fluorescence or Förster resonance energy transfer (FRET) briefly, is a distance-dependent transfer of excited state energy from an initially excited donor to an acceptor, with the donor molecule typically emitting at shorter wavelengths that overlap with the absorption of an acceptor (1-3). FRET occurs when a donor (fluorophore) and an acceptor (another fluorophore or quencher) are within a specified distance, usually within 10-100 Å. The donor-acceptor distance at which the energy transfer is 50% is called the Förster radius (Ro). Within this distance, when a donor transfers its resonance energy to a quencher, a decrease in the donor fluorescence is seen. FRET efficiency falls dramatically as the donor-acceptor distance exceeds the Förster radius. Figure 1 shows a schematic representation of a FRET peptide where the fluorescence of the donor is quenched through resonance energy transfer. Enzyme hydrolysis of the peptide results in spatial separation of the donor and acceptor, which leads to the recovery of the donor’s fluorescence.

Figure 1. Schematic representation of a FRET peptide proteolytic cleavage.

Guidelines
1. Choose a donor/acceptor pair where the absorption spectrum of the quencher overlaps with the emission spectrum of the donor (see Table 1 for recommendations). We generally use a fluorescent donor and a non-fluorescent acceptor (quencher) to make protease peptide substrates (AnaSpec’s QXL^™ 520 has been proven to be an efficient quencher for FAM and HiLyte Fluor^™ 488). The choice of donor/acceptor pair may be limited by the kind of fluorometer filter on hand.

Table 1. Chemical reactivities and spectral properties of FRET building blocks.

Quencher (Acceptor)	max (nm)	Amine-Reactive	Thiol-Reactive	Carbonyl-Reactive (Amine-Containing)	Recommended FRET Donor
Dnp	348	Dnp-X, acid; Dnp-X, SE	Dnp C2 maleimide	Dnp C2 amine	Trp, Abz, Abz(N-Me), Mca
DABCYL	428	DABCYL, acid; DABCYL, SE	DABCYL C2 maleimide	DABCYL C2 amine DABCYL hydrazide	EDANS, AMCA
DABCYL Plus™	437	DABCYL Plus™ acid; DABCYL Plus™, SE	DABCYL Plus™ C2 maleimide	DABCYL Plus™ C2 amine DABCYL Plus™ hydrazide	EDANS, AMCA
QXL™ 490	488	QXL™ 490, acid; QXL™ 490, SE	QXL™ 490 C2 maleimide	QXL™ 490 C2 amine QXL™ 490 hydrazide	EDANS, AMCA
QXL™ 520	508 & 530	QXL™ 520, acid; QXL™ 520, SE	QXL™ 520 C2 maleimide	QXL™ 520 C2 amine QXL™ 520 hydrazide	FAM, FITC, Rh6G HiLyte Fluor™ 488
QXL™ 570	578	QXL™ 570, acid; QXL™ 570, SE	QXL™ 570 C2 maleimide	QXL™ 570 C2 amine QXL™ 570 hydrazide	HiLytePlus™ 555, HiLyte Fluor™ 555, Cy3®, TAMRA, ROX, Alexa Fluor® 555
QXL™ 610	594 & 628	QXL™ 610, acid; QXL™ 610, SE	QXL™ 610 vinyl sulfone	QXL™ 610 C2 amine QXL™ 610 hydrazide	ROX, Texas Red®, Sulforhodamine 101 HiLyte Fluor™ TR
QXL™ 670	668	QXL™ 670, acid; QXL™ 670, SE	QXL™ 670 C2 maleimide	QXL™ 670 C2 amine QXL™ 670 hydrazide	HiLytePlus™ 647, HiLyte Fluor™ 647, Cy5® Alexa Fluor® 647
QXL™ 680	679	QXL™ 680, acid; QXL™ 680, SE	QXL™ 680 C2 maleimide	QXL™ 680 C2 amine QXL™ 680 hydrazide	HiLytePlus™ 647, HiLyte Fluor™ 647, Cy5® Alexa Fluor® 647

Trademarks of other companies: Alexa Fluor^®, Texas Red-Molecular Probes (Invitrogen); Cy^® dyes-GE Healthcare.

2. Within the same peptide sequence, the donor and acceptor molecules must be in close proximity (typically 10-100 Å) in order to get good quenching. Once an active protease recognizes and cleaves the substrate into two separate fragments, the increase in the donor-acceptor distance causes FRET efficiency to decrease, resulting in the recovery of the donor’s fluorescence. The time-dependent increase in fluorescence intensity is related to the extent of substrate hydrolysis.

3. Beside using the native sequence, sequences containing unnatural amino acid or modified bonds other than a regular amide bond can be used to increase efficiency of cleavage or to protect the peptide from degradation or increase solubility. For example, Ac-DE-Dap(QXL^™ 520)-EE-Abu- [COO]AS-C(5-FAMsp)-NH₂, where an ester bond is used in place of an amide bond to increase cleavage efficiency.

4. Most fluorophores are amino reactive, which means they can be conjugated to the -amino group or the -amino group of Lysine.

5. Thiol reactive dyes can be used to conjugate to Cys-containing peptides. This is an economical way to utilize the dyes since the peptides can be HPLC purified first before reacting with the dyes.

6. For hydrophobic sequences, Lysines or Arginines may be added to increase solubility. These amino acids must be added at the appropriate positions without adversely affecting the protease recognition site.

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