Bioconjugate Chem. 1999, 10, 241−245
241
Design, Synthesis, and Spectroscopic Properties of Peptide-Bridged Fluorescence Energy-Transfer Cassettes Yanlong Li† and Alexander N. Glazer* Departments of Chemistry and Molecular and Cell Biology, University of California, Berkeley, California 94720. Received July 6, 1998; Revised Manuscript Received December 1, 1998
A general partial solid-phase synthetic scheme was developed for the synthesis of energy-transfer cassettes with the donor and acceptor dyes bridged by a peptide. In these cassettes, 6-carboxyfluorescein (Fam) served as a donor. For the second dye, 6-carboxy-X-rhodamine (Rox) was used as a fluorescent acceptor or erythrosin B as a quencher. Different peptides bearing Rox at the amino terminus and Fam linked through different diamines to the carboxyl terminus were synthesized to examine the effects of the chain length and rigidity on energy-transfer efficiency. The ratio of emission intensities at 605 nm of the acceptor dye (ROX) in the cassette Rox-GPPPEPPP-p-xylylenediamine-Fam versus free ROX with 488 nm excitation was ∼14 and is similar to that obtained for optimized oligonucleotide primers bearing the same dyes [Ju, J., Ruan, C., Fuller, C. W., Glazer, A. N., and Mathies, R. A. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 4347-4351].
INTRODUCTION
In recent years, we have developed sets of fluorescence energy-transfer (ET)1 primers for DNA sequencing and mapping (reviewed in refs 1 and 2). In these primers, a donor dye is attached to the oligonucleotide at the 5′ end, and the acceptor is attached to a modified thymidine internal in the primer sequence. ET cassettes are superior to single dye-labeled probes because the large acceptor-dependent Stokes shifts allow excellent rejection of adventitious autofluorescence, Raleigh and Raman scattering at the emission wavelength of the acceptor, and because multiplex assays can be carried out by using sets of labels with a common dye as a donor and different dyes as acceptors. In addition, we have developed a general method to tag primers of any sequence with ETcoupled fluorophores using a universal ET cassette which contains a polydideoxyribose phosphate spacer and can be incorporated at the 5′ end of an oligonucleotide primer by conventional automated synthesis (3). Other universal ET cassettes would be useful; ones that could be coupled to any molecule of interest. Such general ET cassettes would need to embody the following * To whom correspondence should be addressed, Department of Molecular and Cell Biology. Phone: (510) 642-3126. Fax (510) 643-9290. E-mail:
[email protected]. † Department of Chemistry. Present address: Gull Laboratories, 1001 East 4800 South, Salt Lake City, UT 84117. 1 Abbreviations: Fam, 5-carboxyfluorescein; Rox, 5-carboxyX-rhodamine; DIPEA, N,N-diisopropylethylamine; DMSO, dimethyl sulfoxide; DMF, N,N-dimethylformamide; ET, energy transfer; NHS, N-hydroxysuccinimide; TEAA, triethylamine acetate; tBoc, tert-butyloxycarbonyl; tBu, tert-butyl; TFA, trifluoroacetic acid; MMT, monomethoxytrityl. 1× TBE buffer, 89 mM Tris/89 mM boric acid/2 mM EDTA, pH 8.0. The standard one-letter amino acid abbreviations used are E, L-Glu; F, L-Phe; G, Gly; P, L-Pro. Dye-labeled peptides are named systematically by the abbreviation of the first dye (at the N terminus) followed by one letter abbreviations for the amino acid residues, the abbreviation for the diamine and the abbreviation for the second dye (at the C terminus). The ethylenediamine linker was abbreviated as HN-C2-NH.
components: (i) a donor dye; (ii) an acceptor dye; (iii) a conformationally constrained bridge of readily varied length connecting the donor and acceptor dyes; and (iv) a functional group for attachment of the cassette to target molecules. We describe here a preliminary examination of alternative ET cassettes which employ 9-phenylxanthene dyes as donor and acceptors (or quenchers) and conformationally constrained peptide derivatives as the bridging moieties. Peptides are particularly attractive bridge candidates because of the convenience of solid-phase synthesis and the availability of a wide range of building blocks. Indeed, peptides bearing donor dyes and quenchers are widely used for proteinase assay (e.g., refs 4-13). EXPERIMENTAL PROCEDURES
All chemicals were purchased from Aldrich (Milwaukee, WI) except where otherwise indicated. Erythrosin B-5-isothiocyanate and the NHS esters of the 5-carboxy isomers of Fam and Rox were purchased from Molecular Probes (Eugene, OR). Peptides on Merrifield resin were obtained through custom synthesis performed by SynPep (Dublin, CA). Chymotrypsin was purchased from Sigma (St. Louis, MO). Synthesis of Erythrosin B-Ala-Phe-Ala-Gly-NHC2-NH-Fam. Erythrosin B isothiocyanate (∼4 mg) was dissolved in DMSO (0.2 mL) and added to the peptide AFAG-Merrifield resin (∼2 mg of peptide) in an Eppendorf tube. DIPEA (5 µL) was added to this mixture. After vortexing for 2 h, a few resin beads were removed and checked by the Kaiser test for the completeness of dye coupling (14). When the reaction was complete, the supernatant was removed by centrifugation. After three sets of alternating washes with DMSO (1.0 mL) and methanol (1.0 mL), the resin beads were added to 30% (v/v) ethylenediamine in methanol (0.2 mL). After vortexing for 2 h, the supernatant was transferred to a fresh Eppendorf tube. Ether (2 mL) was added to precipitate the labeled peptide, erythrosin B-AFAG-NH-C2-NH2. The precipitate was redissolved in DMSO (0.2 mL) and the
10.1021/bc980080k CCC: $18.00 © 1999 American Chemical Society Published on Web 02/26/1999
242 Bioconjugate Chem., Vol. 10, No. 2, 1999
peptide derivative reprecipitated to remove residual ethylenediamine. The NHS ester of Fam (∼2 mg) was dissolved in DMSO (0.2 mL) and added to erythrosin B-AFAG-NH-C2-NH2 followed by DIPEA (5 µL). After vortexing for 2 h, ethyl acetate (2 mL) was added to precipitate the doubly labeled peptide, erythrosin B-AFAGNH-C2-NH-Fam. The precipitate was dissolved in methanol, and the doubly labeled peptide purified by reversedphase HPLC. In the HPLC solvent, the erythrosin B-AFAG-NH-C2-NH-Fam peak showed two visible absorbances at 529 and 495 nm with a A529nm:A495nm ratio of 0.755. In 1× TBE, the erythrosin B absorbance was negligible relative to that of Fam. Rox-Gly-Pro-Pro-Glu-Pro-Pro-NH-C2-NH-Fam.Ethylenediamine in methanol (30% v/v; 0.2 mL) was added to tBoc-GPPE(-tBu)PP-Merrifield resin (∼5 mg of peptide) to give tBoc-GPPE(-tBu)PP-NH-C2-NH2. This product was then labeled with NHS ester of FAM (∼8 mg) to give tBoc-GPPE(-tBu)PP-NH-C2-NH-Fam. After precipitation with ethyl acetate, tBoc-GPPE(-tBu)PP-NH-C2NH-Fam was added to 75% (v/v) TFA in dichloromethane (0.3 mL) and allowed to deprotect for 1 h. The product, GPPEPP-NH-C2-NH-Fam, was precipitated with ether (2 mL). Addition of the NHS ester of Rox (∼5 mg) in DMF (0.1 mL) and DIPEA (5 µL) to GPPEPP-NH-C2-NH-Fam produced Rox-GPPEPP-NH-C2-NH-Fam. Alternatively, 4-methoxytrityl chloride (∼0.5 g) was dissolved in dichloromethane (0.5 mL) and added to GPPEGG-Merrifield resin (∼5 mg of peptide). Pyridine (0.5 mL) was added to the above mixture. While stirring, the mixture was heated at 45 °C for 48 h. After completion of the reaction, the peptide was treated as described above to give MMT-GPPEPP-NH-C2-NH2. The rest of the synthesis was performed as described above except that deprotection was performed with 3% (v/v) TFA in dichloromethane for 5 min. Mass spectrometry of Rox-GPPEPP-NH-C2-NH-Fam gave (M + 1)+ of 1510 (corresponding to the calculated mass of 1509) and a ratio of A495nm (FAM):A585nm (Rox) of 0.816. Rox-Gly-Pro-Pro-Glu-Pro-Pro-p-xylylenediamineFam. This compound was synthesized following the scheme for Rox-GPPEPPNH-C2-NH-Fam except that p-xylylenediamine, C6H4(CH2NH2)2, was used for cleavage. Rox-GPPEPP-p-xylylenediamine-Fam purified by HPLC had a ratio of A495nm (FAM):A585nm (Rox) of 0.873. Rox-Gly-Pro-Pro-Pro-Glu-Pro-Pro-Pro-p-xylylenediamine-Fam. This compound synthesized as described for Rox-GPPEPP-p-xylylenediamine-Fam except that GPPPEPPP-Merrifield resin was used as the starting material. Rox-GPPPEPPP-p-xylylenediamine-Fam purified by HPLC had a ratio of A495nm (FAM):A585nm (Rox) of 0.896. Hydrolysis of Erythrosin B-AFAG-NH-C2-NHFam by Chymotrypsin. Chymotrypsin (0.25 mg) was added to a solution of erythrosin B-AFAG-NH-C2-NHFam (1.0 mL, A498 ) 0.12/cm) in 0.1 mM CaCl2, 50 mM Tris-HCl, pH 8. A control solution was prepared leaving out chymotrypsin. The fluorescence emission spectra of both the sample and the control solutions were measured after 24 h incubation at 37 °C. Chromatography. HPLC was performed on a Waters 991 system with a photodiode array detector. All runs were carried on a Bio-Rad C18 column (4.6 × 250 mm) with a flow rate of 1.0 mL/min. Purification was performed with a linear gradient from 50% methanol and 50% 0.05 M TEAA to 100% methanol in 10 min. Peaks were collected manually and the solvent removed by vacuum-drying.
Li and Glazer
Mass Spectrometry. Low-resolution fast-atom bombardment mass spectrometry was performed by Mass Spectrometry Center of University of California, Berkeley. Absorbance and Fluorescence Spectroscopy. All absorbance spectra were measured in 1× TBE buffer (unless otherwise specified) with a Perkin-Elmer Lambda 6 UV-vis spectrophotometer. The ratio of the extinction coefficients of free Fam at 495 nm to that of free Rox at 585 nm is 1.01. The absorbance of Rox at 495 nm is 7.5% of its absorbance at 585 nm, whereas Fam contributes no absorbance at 585 nm. The ratios of Fam-to-Rox absorbance given for the ET cassettes are corrected for the Rox contribution at 495 nm. Fluorescence emission spectra were collected on a Perkin-Elmer MPF-44B spectrofluorimeter with excitation at 488 nm. The sample absorbance at Amax in 1× TBE ranged between 0.010 and 0.012/cm. The bandwidth of the excitation and emission slits was set to 3.0 nm. RESULTS
In their classic test of the Fo¨rster prediction (15) that the rate constant for energy transfer is proportional to the inverse sixth power of the distance between the donor and acceptor, Stryer and Haugland (16) synthesized oligomers of L-proline as spacers of defined length carrying an R-naphthyl group at the carboxyl end as a donor and a dimethylaminonaphthalenesulfonyl group at the imino end as an acceptor. The synthesis was carried out by the Merrifield solid-phase method, and the acceptor was attached to the imino end of the peptide before release from the resin. Treatment with hydrazine was then used to release the peptide derivative and to provide a reactive group at the carboxyl terminus for the attachment of the donor. However, we found that 9-phenylxanthene dyes subjected to this synthetic procedure were unstable under the conditions of hydrazinolysis. To address this problem, we examined the use of diamines in place of hydrazine to introduce the attachment site for a dye. Since, in principle, different diamines could be used to effect the release of the peptide, this allowed examination of the effect of the portion of the bridge immediately proximal to one of the dyes on the efficiency of the energy-transfer process. Preliminary experiments showed that primary amines that are liquids or are readily soluble in organic solvents, such as ethylenediamine, 1,3-diaminopropane, and p-xylylenediamine, all cleaved 9-phenylxanthene dye-peptide conjugates efficiently from the resin without adverse effect on the dye. In an initial test of the procedure, we synthesized erythrosin B-AFAG-NH-C2-NH-Fam (Figure 1). This compound was designed with a donor (Fam) and a quencher (erythrosin B) and a chymotrypsin-sensitive Phe-Ala bond to show that this synthetic procedure would be readily applicable to the synthesis of energytransfer peptides of the type widely used for proteinase assays. The fluorescence emission of Fam at 525 nm was quenched >95% in erythrosin B-AFAG-NH-C2-NH-Fam. A 24 h incubation with chymotrypsin restored the fluorescence emission to 90% that of free 6-carboxyfluorescein (Figure 2). Figure 3 presents the synthetic scheme for the preparation of peptides which carry Fam as a donor and Rox as acceptor and in which the γ-carboxyl group on a glutamyl residue is available for the conjugation of the ET cassette to a target molecule. With tBoc-GPPE(-tBu)PP-Merrifield resin as starting material, ethylenediamine
Properties of Fluorescence Energy-Transfer Cassettes
Bioconjugate Chem., Vol. 10, No. 2, 1999 243
Figure 1. Synthetic scheme for erythrosin B-AFAG-NH-C2NH-Fam.
Figure 3. Synthetic schemes for Rox-GPPEPP-NH-C2-NHFam.
most pronounced increase of Rox emission on 488 nm excitation, over 13 times that of equimolar free Rox (Figure 4c). DISCUSSION
Figure 2. Fluorescence emission with 488 nm excitation of Erythrosin-AFAG-NH-C2-NH-Fam before (curve 1) and after (curve 3) hydrolysis by chymotrypsin. Curve 2 shows the fluorescence emission of an equimolar solution of free Fam. See the Experimental Procedures for details.
cleavage introduced a free amino group at the C-terminus. After conjugation to Fam, the peptide was deprotected with 75% TFA for 1 h to remove the tBoc from the N-terminus. The free N-terminal amine then readily reacted with Rox NHS ester. When a monomethoxytrityl (MMT) group was used to protect the N-terminal amine, deprotection was achieved with 3% TFA in 5 min. The latter procedure is more compatible with syntheses that may involve dyes unstable under strongly acidic conditions. The product Rox-GPPEPP-NH-C2-NH-Fam was obtained with both synthetic schemes (Figure 3). The mass of Rox-GPPEPP-NH-C2-NH-Fam matched the proposed structure. Rox-GPPEPP-p-xylylenediamine-Fam and GPPPEPPP-p-xylylenediamine-Fam were synthesized following the same procedure with p-xylylenediamine for cleavage from the resin and GPPEPP or GPPPEPPP as the peptide sequences. When excited at 488 nm, the compound Rox-GPPEPPNH-C2-NH-Fam showed strong emission from Rox at 605 nm, but also significant residual fluorescence emission from the donor Fam at 525 nm (Figure 4a). In contrast, Rox-GPPEPP-p-xylylenediamine-Fam offered a negligible donor residue emission (Figure 4b). Increase in donoracceptor separation by two additional prolyl residues in Rox-GPPPEPPP-p-xylylenediamine-Fam gave the
We present here a straightforward general procedure which depends largely on solid-phase synthesis for the production of ET peptide cassettes. The novel feature of this procedure is the use of diamines both to release the peptide from the resin and to provide a primary amino group at the C-terminus of the peptide for facile addition of a dye. This procedure allows the synthesis of ET peptide cassettes varying only in the nature of the diamine at the C-terminus. Surprisingly, different diamines have a substantial impact on the fluorescence emission properties of the ET cassette. With 488 nm excitation, RoxGPPEPP-NH-C2-NH-Fam and Rox-GPPEPP-p-xylylenediamine-Fam (Figure 4, panels a and b) show similar enhancement of Rox fluorescence emission at 605 nm (relative to equimolar free Rox). However, Rox-GPPEPPNH-C2-NH-Fam shows significant emission from the Fam donor, whereas the donor emission in Rox-GPPEPPp-xylylenediamine-Fam is virtually completely quenched. Thus, the structure (and possibly the rigidity) of the portion of the ET cassette proximal to a dye has a disproportionately large effect on the spectroscopic properties of the cassette. It is customary to link dyes to oligonucleotides (1, 2) and the like through long aliphatic tethers. The result reported here suggests that systematic studies of the influence of the structure of these tethers on the fluorescence properties of the ET cassettes would be worthwhile. Studies of ET cassettes with oligonucleotide and oligodideoxyribose phosphate spacers (3, 17-20) have yielded the unexpected result that both the efficiency of energy transfer, as measured by quenching of donor emission, and the intensity of fluorescence emission from the acceptor increase with longer donor-acceptor spacing,
244 Bioconjugate Chem., Vol. 10, No. 2, 1999
Figure 4. Comparison the fluorescence emission on 488 nm excitation of peptide ET-cassettes with that of an equimolar solution of free Rox. (a) Rox-GPPEPP-NH-C2-NH-Fam versus Rox. (b) Rox-GPPEPP-p-xylylenediamine-Fam versus Rox. (c) Rox-GPPPEPPP-p-xylylenediamine-Fam versus Rox.
reaching a maximum at a chain length of 8-10 monomer units (17-19). The inverse result is predicted by the Fo¨rster equation (15), and such expectation is validated by studies of donor-acceptor pairs bridged by a rigid backbone (16). The anomalous behavior of cassettes with oligonucleotide and polydideoxyribose phosphate spacers is attributable to their conformational flexibility which may allow for close contact of donor and acceptor in some conformers with consequent quenching of the emission. In this report, we examine the dependence of energy transfer in Fam/Rox oligopeptide cassettes on the structure of the spacer separating the dyes. Here, as in oligonucleotide and polydideoxyribose phosphate spacers studied earlier, peptide length-dependent variation in the orientation factor (κ2) is not likely to be significant because the 9-phenylxanthenes are attached to the oligopeptides through bonds that do not hinder their mobility. Experimental support for this assertion is provided by Stryer and Haugland (16), who concluded that a dansyl group attached to the imino end of proline oligopeptides had a fast rotational mobility independent of that of the oligopeptide moiety. They therefore used
Li and Glazer
the average value of κ2, i.e., 2/3, in the energy-transfer calculations for oligoproline peptides varying in length (16). Eisinger and co-workers (21, 22) reached the same conclusion for chromophores attached to polypeptide chains that have little or no secondary or tertiary structure. The two peptides Rox-GPPEPP-p-xylylenediamineFam and Rox-GPPPEPPP-p-xylylenediamine-Fam differ only in the length of the Pro-containing segments. Both of these ET peptides show near-complete quenching of donor emission. However, the longer peptide, Rox-GPPPEPPP-p-xylylenediamine-Fam, gave significantly greater enhancement of Rox fluorescence emission (Figure 4c). The ratio of emissions for 488 nm excitation for this ET cassette versus free ROX approaches 14 and is similar to the ratio of 14 obtained for optimized oligonucleotide primers bearing the same dyes (2). The difference in the absorbance spectra of Rox-GPPEPP-pxylylenediamine-Fam (A495nm:A585nm ) 0.873) and RoxGPPPEPPP-p-xylylenediamine-Fam (A495nm:A585nm ) 0.896) is consistent with lesser dye-dye interaction in the longer ET cassette. The conformational aspects of the Pro-Pro or Pro-ProPro segments of the oligopeptides deserve a brief comment. Pro is unique among amino acids in that the side chain is bonded covalently to the nitrogen atom of the peptide group. Therefore, the backbone at a Pro residue within an oligopeptide chain has no amide hydrogen for participation in hydrogen bonding or in resonance stabilization of the peptide bond of which it is a part. The cyclic five-membered ring also imposes rigid constraints on rotation about the N-CR bond of the backbone. Pro residues, therefore, do place major constraints on the conformation of the oligopeptide backbone. However, even the Pro-Pro and Pro-Pro-Pro segments of these oligopeptides most likely exist in alternative conformations since the trans form of imide bond involving Pro is only slightly favored (4:1) over the cis form (23). These segments are too short to assume a helical conformation. The oligopeptide portion of each of the two ET cassettes thus exists as a population of conformers each varying in the separation between the donor and acceptor dyes. In conclusion, as for ET primers with oligonucleotide spacers, for ET peptide-based cassettes with partially flexible regions, the optimum donor-acceptor separation will need to be established empirically. ACKNOWLEDGMENT
We thank Richard A. Mathies for many valuable discussions. This research was supported by Amersham Life Science Inc. and by the Director, Office of Energy Research, Office of Health and Environmental Research of the U.S. Department of Energy under Contract DEFG-91ER61125. Financial support from the W. M. Keck Foundation is also gratefully acknowledged. LITERATURE CITED (1) Glazer, A. N., and Mathies, R. A. (1997) Energy transfer fluorescent reagents for DNA analyses. Curr. Opin. Biotechnol. 8, 94-102. (2) Ju, J., Glazer, A. N., and Mathies, R. A. (1996) Nat. Med. 2, 246-249. (3) Ju, J., Glazer, A. N., and Mathies, R. A. (1996) Cassette labeling for facile construction of energy transfer fluorescent primers. Nucleic Acids Res. 24, 1144-1148. (4) Gershkovich, A., and Kholodovych, V. V. (1996) Fluorogenic substrates for proteases based on intramolecular fluorescence energy transfer (IFETS) J. Biochem. Biophys. Methods 33, 135-162.
Properties of Fluorescence Energy-Transfer Cassettes (5) Matayoshi, E., Wang, G. T., Krafft, G. A., and Erickson, J. (1990) Novel fluorogenic substrates for assaying retroviral proteases by resonance energy transfer. Science 247, 954958. (6) Wang, G. T., Matayoshi, E., Huffaker, H. J., and Krafft, G. A. (1990) Design and synthesis of new fluorogenic HIV protease substrates based on resonance energy transfer. Tetrahedron Lett. 31, 6493-6496. (7) Garcia-Echeveria, C., and Rich, D. H. (1992) New intramolecularly quenched fluorogenic peptide substrates for the study of the kinetic specificity of papain. FEBS Lett. 297, 100-102. (8) Pennington, M. W. and Thornberry, N. A. (1994) Synthesis of a fluorogenic interleukin-1β converting enzyme substrate based on resonance energy transfer. Pept. Res. 7, 72-76. (9) Geoghegan, K. F. (1996) Improved method for converting an unmodified peptide to an energy-transfer substrate for a proteinase. Bioconjugate Chem. 7, 385-391. (10) Maggiora, L. L., Smith, C. W., and Zhang, Z. Y. (1992) A general method for the preparation of internally quenched fluorogenic protease substrates using solid-phase peptide synthesis. J. Med. Chem. 35, 3727-3730. (11) Contillo, L. G., Singleton, D. H., Andrews, G. C., Spencer, R. W., Faraci, W. S., Martin, W. H., and Stock, I. A. (1994) A general strategy for the synthesis of eosin- fluorescein energy transfer substrates for high sensitivity screening of protease inhibitors. Techniques in Protein Chemistry V, pp 493-500, Academic Press, San Diego. (12) Handa, B. K., Keech, E., Conway, E. A., Broadhurst, A., and Ritchie, A. (1995) Design and synthesis of a quenched fluorogenic peptide substrate for human cytomegalovirus proteinase. Antiviral Chem. Chemother. 6, 255-261. (13) Kornelyuk, A. I., Terentiev, A. G., Fisher, S., and Porter, T. (1995) Synthesis and characterization of fluorogenic peptide substrate of HIV-1 protease based on fluorescence resonance energy transfer. Biopol. Kletka 11, 57-61.
Bioconjugate Chem., Vol. 10, No. 2, 1999 245 (14) Kaiser, E., Colescott, R. L., Bossinger, C. D., and Cook, P. I. (1970) Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides. Anal. Biochem. 34, 595-598. (15) Fo¨rster, T. (1948) Zwischenmolekulare energiewanderung und fluoreszenz. Ann. Physik. 2, 55-75. (16) Stryer, L. and Haugland, R. P. (1967) Energy transfer: A spectroscopic ruler. Proc. Natl. Acad. Sci. U.S.A. 58, 719726. (17) Ju, J., Ruan, C., Fuller, C. W., Glazer, A. N., and Mathies, R. A. (1995) Fluorescence energy transfer dye-labeled primers for DNA sequencing and analysis. Proc. Natl. Acad. Sci. U.S.A. 92, 4347-4351. (18) Ju, J., Kheterpal, I., Scherer, J. R., Ruan, C., Fuller, C. W., Glazer, A. N., and Mathies, R. A. (1995) Design and synthesis of fluorescence energy transfer dye-labeled primers and their application for DNA sequencing and analysis. Anal. Biochem. 231, 131-140. (19) Hung, S.-C., Mathies, R. A., and Glazer, A. N. (1997) Optimization of spectroscopic and electrophoretic properties of energy transfer primers. Anal. Biochem. 252, 78-88. (20) Hung, S.-C., Mathies, R. A., and Glazer, A. N. (1998) Comparison of fluorescence energy transfer primers with different donor-acceptor dye combinations. Anal. Biochem. 255, 32-38. (21) Eisinger, J. (1969) Intramolecular energy transfer in adrenocorticotropin. Biochemistry 8, 3902-3908. (22) Eisinger, J., Feuer, B., and Lamola, A. A. (1969) Intramolecular singlet excitation transfer. Application to polypeptides. Biochemistry 8, 3908-3915. (23) Creighton, T. E. (1984) Proteins. Structures and Molecular Principles, pp 163-164, W. H. Freeman & Co., New York.
BC980080K
