Homogeneous enzyme cleavage assays using FRET in a Fluorescent Imaging System *J.A. Alcock, J. George, M.J. Hatcher, C.G. Norey, P.M. Baxendale and D.D. Burns * to whom all correspondence should be addressed. Amersham plc, Cardiff Laboratories, Forest Farm Estate, Whitchurch, Cardiff, CF14 7YT, Wales, UK. Telephone: +44 (0)29 2052 6435, Fax: +44 (0)29 2052 6230,e-mail: [email protected] Protease assay A peptide substrate is labelled at the N-terminus with a Cy3 (donor) and at the C-terminus with a Cy5 (acceptor) as shown in Figure 3. Excitation at Cy3 wavelength in the intact substrate results in efficient energy transfer to Cy5 which can give rise to either the quenching of the Cy3 emission signal or generation of Cy5 emission signal. However, upon enzyme cleavage the Cy3 donor and Cy5 acceptor are no longer in close proximity, so FRET does not occur. The removal of the quencher and loss of FRET results in a decrease in Cy5 signal and the significant increase in Cy3 signal. Loss of FRET and Cy5 signal Efficient FRET and Cy5 signal cleavage Efficient FRET and Cy5 signal Loss of FRET and Cy5 signal Nuclease cleavage Cy5 acceptor Cy3 donor Quenching removed Generation of Cy3 signal Efficient quenching of Cy3 signal Fluor/Fluor pairs The use of CyDye technology has great potential for ratiometric determination. This example (Figure 4) of a fluor/fluor labelled peptide demonstrates single excitation and dual emission enabling monitoring of protease activity in real time. Figure 4. FRET assay : Asp-N digestion of Cy3-YVA↑ ↑ DAPVK-Cy5 70000 Cy5 acceptor 60000 The nuclease assay shown in Figure 2 illustrates this principle, whilst comparing the data obtained from BMG POLARstar plate reader and LEADseeker Imager. The graph shows a signal decrease with corresponding increase in enzyme concentration. Both instruments produce very similar results. Fluorescence intensity Cy3 donor % Signal Change 20 0 -20 0.0×10 -00 5.0×10 -05 1.0×10 -04 1.5×10 -04 2.0×10 -04 DNase I Units/µ µl DNA substrate (50nM) was incubated with increasing concentrations of active bovine pancreatic Dnase I in assay buffer (50mM Tris, pH 7.5,10mM MnCl2 and 0.005% Tween 0 20) for 60 minutes at 37 C. Assays were run in 384 well plates with a final reaction volume of 50µl and then read in BMG POLARstar (  -) and LEADseeker (  ). Values are mean ! SEM (n=3). 45 60 A Cy3B-peptide-Cy5Q substrate was incubated at room temperature with/without Asp-N enzyme. Assays were run in 96 and 384 well plates (total assay volumes =110µl and 55µl respectively). Images at 530nm excitation and 590nm emission wavelengths show signal from (Top row) substrate digested over time (Middle row) undigested substrate = no enzyme (Bottom row) completely digested substrate = control for both 96 and 384 well assays. Exposure time = 15 seconds. Fluor/Quencher pairs Several fluor/fluor and fluor/quencher pairs were evaluated from the CyDye range but the § optimum pairing observed was a novel Cy3B donor with a Cy5Q acceptor. An improved signal:noise value in the range of 10:1 illustrates the benefit of using this pairing. Figure 6. FRET assay : Asp-N digestion of Cy3B-YVA↑ ↑ DAPVK-Cy5Q 75 + enzyme 50 25 - enzyme 10 20 30 40 50 60 70 30000 Cy3B-YVA↑DAPVK-Cy5Q(100nM ) was incubated at room temperature in Tris buffer (50mM, pH 8.0, 0.005% Tween 20 with or without 5ng/well Asp-N enzyme. Assays were run in 96 (  ) and 384(  ) well plates with 110µl and 55µl reaction volumes respectively. Signal increase was measured over time at 530nm excitation and 590nm emission wavelengths. Values are mean ! SEM (n=3). 20000 180 360 540 720 900 Time (secs) 40 30 Time in minutes 0 60 20 384-well 0 0 80 10 0 40000 10000 100 T=0 50000 Figure 2. DNA Nuclease assay 120 96-well Figure 3. Protease Schematic Nuclease assay A dsDNA substrate is labelled at opposite ends and strands with Cy3 and Cy5 as shown in Figure 1. When the DNA molecule is intact the fluors are close enough to efficiently transfer energy (FRET). Excitation at Cy3 (donor) wavelength results in a Cy5 (acceptor) emission. Digestion of the substrate results in the fluors no longer being in close proximity, therefore, FRET does not occur and there is a decrease in the Cy5 signal. Figure 1. Nuclease Schematic Figure 5. LEADseeker Image of FRET assay : Asp-N digestion of Cy3B-YVA↑ ↑ DAPVK-Cy5Q Cleaved peptide (nM) Introduction The application of FRET (fluorescence resonance energy transfer) molecules used as substrates in (1) enzyme assays has attracted much recent interest . Homogeneous enzyme cleavage assays using FRET in a fluorescent imaging system have the potential for miniaturisation and so increase throughput in enzyme studies. The recent introduction of new fluorescent dyes with better photophysical properties have also (2) helped to advance this particular use of FRET . FRET occurs when a suitable fluorescent energy donor and an energy acceptor molecule are in close proximity (<100 Œ ) to one another. The excitation energy absorbed by the donor is transferred non-radiatively to the acceptor which can then further dissipate this energy either by fluorescent emission (if a fluorophore) (3) or by non-fluorescent means (if a quencher) . Enzyme assays have been developed using fluorescent donors based on Cy™3 and acceptors based on Cy5 to show their beneficial properties. CONCLUSION Cy3-YVA↑DAPVK-Cy5 (100nM) was incubated at room temperature in Tris buffer (50mM, pH8.0, 0.005% Tween 20 with 10ng/well Asp-N enzyme, total assay volume for a 96 well plate was 110µl. Signal measured at 530nm excitation: increase in Cy3 emission at 570nm (  ), decrease in energy transfer (Cy3 to Cy5) at 670nm (  ). LEADseeker Imaging The LEADseeker image in Figure 5 shows an example of an enzyme cleavage assay using a fluor/quencher pair in both 96 and 384 well plates. The wells containing enzyme digested substrate over time (top row of each plate) illustrate the comparable data obtained in the assays. This is confirmed when the fluorescent values are plotted as shown on the graph in Figure 6. * Cy, CyDye and LEADseeker are trademarks of Amersham Biosciences Limited or its subsidiaries. Tween is a trademark of ICI Americas, Inc. POLARstar is a trademark of BMG LabTechnologies, Inc. CyDye or portions thereof is manufactured under licence from Carnegie Mellon University, US patent number 5268486. § Patent application number WO 99/31181. Patent application pending. ©Amersham Biosciences UK Limited, 1998 - All rights reserved. Amersham Biosciences UK Limited Amersham Place Little Chalfont Buckinghamshire England U.K. HP7 9NA. Amersham Biosciences AB SE-751 84 Uppsala Sweden. Amersham Biosciences Inc 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USA. Amersham Biosciences Europe GmbH Munzinger Strasse 9 D-79111 Freiburg Germany. This poster was presented at SBS '99,Edinburgh September 1999. • Enzyme cleavage events illustrating the use of FRET have been demonstrated in a Fluorescent Imaging System with encouraging results. • Recently introduced CyDye pairs that are brighter (Cy3B) and have superior quenching properties (Cy5Q) have produced higher signal:noise values. • FRET offers the capability of homogeneous enzyme cleavage assays for use in HTS, with the option of signal increase or signal decrease. References 1. Abriola, L. et al, J. Biomolecular Screening Vol.4, No.3 : 121-127, 1999. 2. Szöllsi ,J. et al, Cytometry 34 : 159-179, 1998. 3. Gershkovich, A. A. et al, J. Biochem. Biophys. Methods 33 : 135-162, 1996. Amersham Biosciences is a trademark of Amersham plc. 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