Photochemistry and Photobiology Vol. 53. No. 1. Printed in Great Britain. All rights reserved

pp. 41-56, 1991

003 I-%SS/91 503.00+0.00

Copyright 0 19Yl Pcrgamon Prcss plc

THE ROLE OF GROUND STATE COMPLEXATION IN THE ELECTRON TRANSFER QUENCHING OF METHYLENE BLUE FLUORESCENCE BY PURINE NUCLEOTIDES DAVIDA. DUNN,VIVIAN H. LIN and IRENEE. KOCHEVAR* Wellman Laboratories, Department of Dermatology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA

(Received 14 May 1990; accepted 2 July 1990) Abstract-The effect of three purine nucleotides on the fluorescence of methylene blue in aqueous (GMP) and xanthosine-5’-monophosphate buffer has been investigated. Guanosine-5’-monophosphate cause fluorescence quenching while adenosine-5‘-monophosphatecauses a red shift in the fluorescence maximum. All three nucleotides form ground state complexes with the nucleotides as indicated by absorption spectroscopy. The fluorescence changes at nucleotide concentrations < 30 mM are best described by a static mechanism involving the formation of non-fluorescent binary and ternary complexes in competition with dimerization of the dye. Quenching of the fluorescence decay ( ~ 3 6 ps) 8 at high GMP concentrations (10-100mM) occurs at the rate of diffusion. The mechanism of fluorescence quenching may involve electron transfer within the singlet excited dye-nucleotide complex although published values of the oxidation potentials of various purine derivatives would suggest that all three nucleotides should cause quenching. Evidence for electron transfer was obtained from flash photolysis experiments in which 100 mM GMP was found to cause the appearance of a long lived transient species absorbing in the region expected for semimethylene blue.

1984) and direct reaction between the sensitizer and DNA either by hydrogen abstraction or electron The term “photonuclease” describes a potential transfer. The radical cations of the‘ bases in DNA class of molecules which are capable of photoinitiating the sequence-specific cleavage of DNA have been shown to lead to single strand breaks or alkali-labile sites (Sevilla et al., 1979; Croke et al., (Perrouault et al., 1990; Saito et a l . , 1989). Many compounds have been shown to photosensitize the 1988; Opitz and Shulte-Frohlinde, 1987). The phenothiazinium dye, methylene blue (MB)t formation of single strand breaks or alkali-labile is a potential photonuclease. Methylene blue sites in DNA (reviewed in Kochevar and Dunn, intercalates with DNA preferentially at G-C sites 1990). Although some of these photosensitizers are (Muller and Crothers. 1975) although evidence for site-selective (Bowler et a l . , 1984; Blau et al., 1987; Slama-Schwok et al., 1989; Blacker et al., 1986; external binding has also been reported (Norden Chang and Meares, 1984; Subramanian and Meares, and Tjerneld, 1982). Early work indicated that MB 1986; Kelly ef al., 1985; Barton and Raphael, 1984, photosensitizes the destruction of DNA by preferen1985), none are sequence specific unless attached tially reacting with guanine (Simon and Van to a complimentary polynucleotide (Perrouault et Vunakis, 1962; Waskell et al., 1966; Friedman and al., 1990; LeDoan et al., 1987; Praseuth et al., 1987; Brown, 1978). Blau er al. (1987) and OhUigin ef Benimetskaya et al., 1983). A fundamental under- al. (1987) demonstrated that MB photosensitized standing of the mechanisms by which photo- cleavage of DNA specifically at guanine in a reacsensitized strand cleavage occurs would aid in devel- tion which did not require oxygen. The fluorescence of MB is quenched upon binding to double-stranded opment of efficient photonucleases. Photosensitization mechanisms include the gener- DNA and to poly[d(G-C)] but not to poly[d(A-T)]. ation of singlet oxygen [which reacts with guanine In addition to fluorescence quenching, a decrease to yield alkali-labile sites in DNA (Simon and Van in the yield of triplet MB was also observed. It was Vunakis, 1962)], formation of radicals including the suggested that fluorescence quenching occurs by hydroxy radical, capable of initiating cleavage by electron transfer from guanine to the excited singlet hydrogen abstraction from the sugar phosphate state of bound MB. This interpretation is consistent backbone (Von Sonntag et al., 1981; Von Sonntag, with the report of Lober and Kittler (1978) who showed that the fluorescence quenching or enhancement of molecules bound to DNA could be corre‘To whom correspondence should be addressed. lated with the ability of the excited singlet state to tAbbreviarions: AMP, adenosine-5‘-monophosphate; GMP, oxidize the DNA bases. guanosine-5’-monophosphate; HITC, 1,1’,3,3,3’,3‘Methylene blue photosensitizes the destruction of hexamethylindotricarbocyanine perchlorate; MB, methylene blue; XMP, xanthosine-S’monophosphate. purine bases and their derivatives. Zenda el al. INTRODUCTION

47

DAVIDA. DUNNet al.

48

(1965) compared the reactivity of 31 purine bases and other related compounds with photoexcited MB. The most reactive compounds were those most closely related in structure to guanine. Nucleotides photoreduced MB to leucomethylene blue (Knowles, 1971). The most reactive nucleotide was found to be xanthosine monophosphate (XMP) followed by guanosine monophosphate (GMP) and adenosine monophosphate (AMP). Only XMP and GMP were significantly oxidized in the reaction. The major fraction (55%) of the products of the photosensitized degradation of 3‘,5’-di-O-acetyl-2deoxyguanosine in methanol-water (1: 1) has been attributed to a mechanism which involves singlet oxygen (Cadet et al., 1983). The excited state of M B responsible for the reaction with D N A or the derivatives of guanine is not clear. The production of singlet oxygen derived products in the reactions with diacetyl-deoxyguanosine suggests the participation of ’MB*. The triplet state lifetime of MB is unaffected by nucleotides (Knowles, 1971). The MB photosensitized formation of 8-hydroxyguanine in D N A has recently been shown to occur by a mechanism which appears to involve the production of singlet oxygen from 3MB* (Floyd et al., 1989). The quenching of MB fluorescence and the lower yield of triplet upon binding to DNA (Kelly et al., 1987) suggests, however, that photosensitized cleavage may occur by a mechanism which includes the direct reaction with ‘MB*. However, the quantum yield for cleavage is extremely low (lo-’; Blau et al., 1987) and, therefore, even small amounts of unquenched triplet dye could also participate. This report focuses on fundamental aspects of the reaction of the excited singlet state of MB with nucleotides. In particular we have examined the role of ground state complexation on the efficiency of fluorescence quenching. To determine whether the reaction with the M B singlet can be described as a classic electron transfer reaction between the photoexcited acceptor and the ground state donor, we chose three nucleotides which vary in their oxidation potential and, therefore, may vary in their ability to reduce the M B singlet. Results of these studies should provide basic information relevant to the MB-photosensitized reaction which leads to guanine specific cleavage of DNA. MATERIALS AND METHODS

Materials. Guanosine-5’-monophosphate, disodium salt

(GMP). adenosine-5’-monophosphate, sodium salt (AMP), xanthosine-5’-monophosphate(XMP), disodium salt, and ribose-S‘-monophosphate,disodium salt were obtained from Sigma (St. Louis, MO) and used without further purification. Methylene blue [Fluka (Puriss); Ronkonkoma, NY] was purified by column chromatography on silica gel using ethanol and increasing amounts of methanol (up to 75%) as the eluting solvent. The purified material appeared as one spot on TLC with (R, 0.13; silica gel; 40 : 40 : 20, methyl ethyl ketone, acetic acid, ethanol). The absorption spectrum of the purified dye was

found to be identical with that of the unpurified material. In both cases, the ratio of absorbances of 3 x 10-“ M solutions in water at 664 : 610 nm was 2.1 indicating the absence of significant amounts of methylene azure B in the commercial grade dye (Bergmann and O‘Konski. 1963). In the fluorescence quenching experiments, we found no significant difference in the results if purified or unpurified dye was used. Unpurified dye was used in the fluorescence lifetime and transient absorption experiments. All solutions were prepared in 100 mM sodium phosphate buffer (50 mM monobasic sodium phosphate; Sigma, St. Louis, MO: and 50 mM dibasic sodium phosphate; Baker, Philipsburgh, PA) at a pH of 6.9 in doubly distilled water. The 1,1’,3,3,3’,3’-hexamethylindotricarbocyanineperchlorate (HITC) quantum counter was purchased from Exciton (Dayton, OH) and used as received. Absorption spectra. A Cary 2300 (Varian) was used to measure absorbance spectra with baseline correction using buffer. In most cases the reference was buffer; however, in measuring the change in MB absorbance upon the addition of nucleotides, the reference buffer contained an appropriate amount of sodium phosphate. Methylene blue concentrations were estimated by the absorbance measured at 664 nm using a value of 95000 M-l cm-I for the extinction coefficient in acetate buffer (Bergmann and O’Konski, 1963). Fluorescence spectra. A Spex Fluorolog 11 was used to measure fluorescence spectra. The signal was measured as the ratio of the output from the sample photomultiplier to that from a reference photomultiplier containing either HITC or Rhodamine B. All measurements were made in the right-angle geometry using a 1 cmz quartz cuvette. For the fluorescence excitation spectra, the Rhodamine B was replaced with a solution of HITC according to the procedure outlined by Nothnagle (1987). This procedure allows for corrected excitation spectra to be measured over the excitation range of 500-730 nm, with the emission monochromator set at 740 nm without the use of filters. The corrected fluorescence spectrum, shown in Fig. 2,was measured in the ratio mode vs Rhodamine B. Fluorescence lifetimes. Measurements were made by time correlated single photon counting at the Regional Laser and Biotechnology Laboratory at the University of Pennsylvania (Philadelphia, PA). Excitation from a modelocked argon pumped dye laser (pulse duration, 10 ps) was set at 601 nm. The monochromator was a McPherson Model 375 (0.2m) equipped with a single holographic grating. The emission at 680 nm was detected with a microchannel plate photomultiplier tube (Hamamatsu model R2809-VO7). A polarizing filter (54.7 deg) was used to minimize transient effects due to rotational orientations of the molecules and a long-pass filter was used to eliminate scattered excitation light from reaching the detector. The instrument response function (-30 ps) is shown in Fig. 3. Flash photolysis. Measurements were made in the laboratory of Professor Henry Linschitz at Brandeis University (Waltham, MA) using the 694.3 nm output from a 0switched ruby laser (-1.0 J/pulse; pulse duration, 30 ns). The transients were monitored at a right angle to the excitation beam as described previously (Andrews et al., 1978). The limiting time response of this system is 30 ns. RESULTS

Ground state association of methylene blue with nucleorides Addition of AMP, GMP, o r XMP to a solution of M B causes an increase in absorbance to the red of the 664 nm absorbance maximum of the dye.

Methylene blue fluorescence quenching

49

' 1.250 1.000 0.750 0.500 0.250 0.000 490

Wavelength (nm)

540

590

640

690

740

790

Wavelength (nm)

Figure 1. Absorption spectra of methylene blue in the Figure 2. Absorption, fluorescence excitation and fluorpresence and absence of GMP. [MB] = 6.4 x lo-" M ; escence spectra of methylene blue in phosphate buffer. [GMP]= 0 to 31 m M ;100 m M phosphate buffer; pH 6.9. Absorption spectrum (triangles; 1.5 x lo-' M). Fluorescence excitation spectra (Acm = 740 nm) at high (1.5 x M; circles) and low (7.9 x M; squares) Solutions were prepared in 100 mM phosphate Concentration. Corrected fluorescence spectrum M; Acxc = 610 nm; solid line). HITC used as buffer (pH 6.9) containing MB at a concentration (7.9 x a quantum counter. less than M and the nucleotides at concentrations ranging from 0 to approx. 30 mM. Constant ionic strength (120 mM) was maintained by adding was investigated by measuring the corrected fluorsodium phosphate (dibasic for GMP and XMP and escence excitation spectrum of MB at concenmonobasic for AMP). The pH of the solutions trations where the dimer is a substantial fraction changed from a value of 7.2 in the absence of nucle- of the ground state species. At concentrations of otide and 30 mM Na2HP04to 6.9 at 30 mM GMP > lo-' M , more than 20% of the MB is in the or XMP and no Na2HP04. Solutions of AMP did dimeric form, assuming Kd = 1.2 x lo4 M - l . Since not vary from pH 6.9. Figure 1 shows a typical set the extinction coefficient of the dimer is 1.5 times of absorption spectra obtained upon varying the greater than that of the monomer at 610 nm concentration of nucleotide. The spectra in Fig. 1 (Bergmann and O'Konski, 1963), dimer should fail to converge at a single isosbestic point. This account for a significant amount of the absorbance behavior (observed using all three nucleotides) sug- at this wavelength. gests an equilibrium between a multiple number of Figure 2 shows the fluorescence excitation spectra absorbing species. of MB at high (1.5 x M) and low The dimerization of MB could be competitive (7.9 x M) concentrations. The high concenwith complex formation. In aqueous solution, MB tration spectrum has been corrected for inner-filforms dimeric aggregates at relatively low concen- tering of the excitation (Lakowicz, 1983). The spectrations (lo-" M) (Rabinowitch and Epstein, 1941). tra are virtually identical, especially at 610 nm The dimerization constant (Kd) depends on the salt where the excitation of dimer should occur. The are , , , 0.17 ~ ~ and 0.18 for the low concentration and has been found to increase by a ratios E X C , , ~ ~ E X C factor ot 2.5 in 50 mM phosphate buffer compared and high concentration solutions respectively. This to pure water (Kelly et al., 1987). At a MB concen- is in contrast to the significant difference in this tration of 6.5 x lo-" M, 12% of the MB should region for the absorption spectra of these two solbe present as dimeric aggregates assuming Kd = utions [AhlcJAM= 0.43 (7.9 x M) and 0.58 1.2 x M - I . The absorption spectra confirm (1.5 x M)]. Therefore, there is no indication the presence of dimer by the enhanced ratio of that dimer contributes significantly to the fluorabsorbance at 610 and 664 nm. The ratio Ahl,JAM escence at 740 nm. Figure 3 shows a typical fluorescence decay at should be less than 0.5 for pure methylene blue monomer (Bergmann and O'Konski, 1963). Under 680 nm of 4.9 x lo-" M solution of MB (0 rnM our experimental conditions, the ratio is 0.6. GMP) in 100 mM buffer under 601 nm excitation. A fit of the decay to a sum of two exponentials gives the following lifetimes and relative amplitudes Methylene blue fluorescence in buffer (x2 =1.03): (1) 820 (+ 70) ps, 0.09 (+ 0.06); (2) Figure 2 includes the corrected fluorescence spec- 368 ( 2 10) ps, 0.91 ( 2 0.06). trum of MB in 100 mM phosphate buffer (pH 6.9) at a concentration of 7.9 x lo-' M excited at 610 nm. Effect of nucleotides on methylene blue fluorFrom the fluorescence maximum at 686 nm and the escence absorbance maximum at 664 nm, the singlet energy is approx. 42.4 kcal/mol (1.8 V). Figure 4 includes the effect of 30 mM nucleotide The contribution of MB dimer to the fluorescence on the fluorescence of a 6.8 x lo-" M solution of

'

DAVID A . I~

50

':i 0.0

N el Nal.

t

0.3

L O

"i

8

5

4

0.0 -0.5

8

8

a

*

A

0.m

f Sl

onyw

-0.1

om

I f: :

0.0

99% of the triplet state should have decayed, the absorption at 440 nm could indicate that a small amount of semimethylene blue has been formed by electron transfer from GMP according to Scheme 1. Electron transfer to the triplet state

is not energetically feasible. The low yield indicates that back electron transfer is much faster than separation of the radical-radical cation pair. The conspicuous loss of absorbance at 520 nm raises an interesting possibility in regard to the photosensitized oxidation of guanine derivatives by MB. Berg et al. (1978) have shown that thiopyronine (TP) reacts most readily with guanosine through its semioxidized form (TP'). In a series of papers (Berg, 1978; Berg et al., 1978; Kittler er al.. 1980) it was proposed that photosensitized single- and double-strand breaks in DNA occur via a mechanism in which the half-oxidized form of the photosensitizer reacts with guanine in DNA by electron transfer. The absence of the 520 nm absorption in the presence of GMP could, therefore, be due to oxidation of GMP by the semioxidized form of MB. This provides another route to the oxidation of guanine derivatives by MB. MBF

+ GMP e MB' + GMP?

Our results are relevant to the mechanism by which MB photosensitizes the formation of singlestrand breaks in DNA and the destruction of guanine derivatives in solution. Quenching of the MB fluorescence by GMP is accompanied by the production of what may be a small amount of semimethylene blue. The low quantum yield for DNA cleavage may be due to efficient back electron transfer within the guanine-MB complex. Cadet et al. (1983) have shown that MB photosensitizes the destruction of diacetyl-deoxyguanosine in 1 : 1 methanol-water, predominantly by the production of singlet oxygen. Since ground state complexation should be negligible under these conditions these results may not be relevant to experiments carried out in purely aqueous solution. Ground state complexation with GMP and DNA greatly reduces the yield of triplet MB and therefore should inhibit the formation of singlet oxygen. Acknowledgemenrs-This work was supported by the MFEL program under ONR Contract N00014-86-K-0117. The authors gratefully acknowledge the assistance of Dr Gary Holtom of the NIH Regional Laser and Biomedical Laboratories at the University of Pennsylvania for help with the acquisition and interpretation of the fluorescence lifetime data; Dr John Hurley for help with the flash photolysis experiments: and special thanks to Professor Henry Linschitz for the use of his laser and helpful discussions. REFERENCES

Andrews, L. J . , A . Deroulede and H. Linschitz (1978) Photophysical processes of fluorenone. J . Phys. Chem. 82, 2304-2309. Archer, M. D.. I . C. Ferreira. G . Porter and C. J . Tredwell (1977) Picosecond study of Stern-Volmer quenching of thionine by ferrous ions. Now. J . Chim. 1, 9-12. Badea, M. G . and S. Georghiou (1976) Interaction between proflavine and guanosine 5'-phosphate: import-

Methylene blue fluorescence quenching ance of photoexcitation. Phorochem. Phoiobiol. 24, 417-423. Barton, J. K. and A . L. Raphael (1984) Photoactivated stereospecific cleavage of double-helical DNA by cobalt(II1) complexes. J . Am. Chem. SOC. 106, 2466-2468. Barton, J. K. and A . L. Raphael (1985)Site-specific cleavage of left-handed DNA in pBR322 by Atris(diphenylphenanthroline)cobalt(III). Proc. Nail. Acad. Sci., U.S.A. 82, 646c-6464. Benimetskaya, L. Z., N. V. Bulychev. A . L. Kozionov, A. V. Lebedev, Y. E. Nesterkhin, S. Y. Novozhilov, S. G. Rautian and M. I. Stockmann (1983)Two-quantum selective laser scission of polyadenilic acid in the complimentary complex with a dansyl derivative of oligothymidilate. FEBS Leri. 163, 144-149. Berg, H. (1978) Redox processes during photodynamic damage of DNA 11. A new model for electron exchange and strand breaking. Bioeleci. Bioenerg. 5, 347-356. Berg, H., F. A. Gollmick, H . Triebel, E. Bauer, G . Horn, J. Flemming and L. Kittler (1978) Redox processes during photodynamic damage of DNA 1. Results obtained by several physico-chemical methods. Bioeleci. Bioenerg. 5 , 335-346. Bergmann. K. and C. T. O'Konski (1963)A spectroscopic study of methylene blue monomer, dimer, and complexes with montmorillonitc. J . Phys. Chem. 67, 2169-2177. Blacker, A. J., J. Jazwinski. J. M. Lehn and F. X. Wilhelm (1986)Photochemical cleavage of DNA by 2,7-diazapyrenium cations. J . Chem. SOC. Chem. Commun. 1035- 1037. Blau, W., D. T. Croke, J. M. Kelly, D. J . McConnell, C. OhUigin and W. J. M. Van der Putten (1987) Basespecific photocleavage of DNA induced by nanosecond UV pulsed laser radiation or methylene blue sensitisation. J. Chem. SOC. Chem. Commun. 751-752. Bowler, B. E., L. S. Hollis and S . J . Lippard (1984) Synthesis and DNA binding and photonicking properties of acridine orange linked by a polymethylene tether to (1,2-Diaminoethane)dichloroplatinum(lI). J . Am. Chem. SOC. 106, 6102-6104. Brabec, V. and G. Dryhurst (1978) Electrochemical behavior of natural and biosynthetic polynucleotides at the pyrolytic graphite electrode a new probe for studies of polynucleotide structure and reactions. J . Elecrroanal. Chem. 89, 161-173. Cadet, J., C. Decarroz, S . Y. Wang and W. R. Midden (1983) Mechanisms and products of photosensitized degradation of nucleic acids and related model compounds. Isr. J . Chem. 23, 42c429. Chang, C. H. and C. F. Meares (1984)Cobalt-bleomycins and deoxyribonucleic acid: sequence-dependent interactions, action spectrum for nicking, and indifference to oxygen. Biochemisrry 23. 2268-2274. Croke, D . T., W. Blau. C. OhUigin, J. M. Kelly and D. J. McConnell (1988)Photolysis of phosphodiester bonds in plasmid DNA by high intensity UV laser irradiation. Phorochem. Phorobiol. 47. 527-536. Floyd, R. A., M. S. West, K. L. Eneff and J . E. Schneider (1989)Methylene blue plus light mediates 8-hydroxyguanine formation in DNA. Arch. Biochem. Biophys. 273. 106-1 1 1. Friedmann, T. and D . M. Brown (1978) Base-specific reactions useful for DNA sequencing: methylene bluesensitized photooxidation of guanine and osmium tetraoxide modification of thymine. Nuc. Acids Res. 5 , 615-622. Georghiou, S. (1975)O n the nature of interaction between proflavine and DNA. Phoiochem. Phoiobiol. 22, 103-109. Haugen, G. and R. Hardwick (1965)Ionic association in solutions of thionine 11. Fluorescence and solvent

55

effects. J . Phys. Chem. 69, 2988-2996. Kamat, P. V. and N. N. Lichtin (1981) Photoinduced electron ejection from methylene blue in water and acetonitrile. J . Phys. Chem. 85, 3864-3868. Kato, S., M. Morita and M. Koizumi (1964) Studies of the transient intermediates in the photoreduction of methylene blue. Bull. Chem. SOC.Jpn. 37. 117-124. Kavarnos, G. J. and N. J . Turro (1986)Photosensitization by reversible electron transfer: theories, experimental evidence, and examples. Chem. Rev. 86. 401-449. Kelly, J. M., A. B. Tossi, D. J. McConnell and C. OhUigin (1985) A study of the interactions of some polypyridylruthenium(I1) complexes with DNA using fluorescence spectroscopy, topoisomerisation and thermal denaturation. Nuc. Acids Res. 13, 6017-034. Kelly, J. M., W. J. M. Van der Putten and D. J. McConnell (1987)Laser flash spectroscopy of methylene blue with nucleic acids. Phorochem. Phorobiol. 45, 167-175. Kittler. L., G . Lober, F. A. Gollmick and H. Berg (1980) Redox processes during photodynamic damage of DNA 111. Redox mechanism of photosensitization and radical reaction. Bioelecrro. Bioenergei. 7, 503-51 I . Knowles, A. (1971) A mechanism for the methylene blue sensitized oxidation of nucleotides. Phorochem. fhorobiol. 13, 473-487. Kochevar. 1. E. and D. A. Dunn (1990)Photosensitized Reactions of DNA: Cleavage and Addition. In Bioorganic Phoiochemisiry (Edited by H. Morrison), pp. 273-315. Wiley, New York. Kubota, Y., Y. Motoda, Y. Shigemune and Y. Fujisaki ( 1979) Fluorescence quenching of 10-methylacridinium chloride by nucleotides. Phoiochem. Phoiobiol. 29, 1099-1 106. Lakowicz, J. R. (1983) Principles of Fluorescence Specrroscopy, p. 44. Plenum Press, New York. LeDoan, T., L. Perrouault, M. Chassignol, N. T. Thuon and C. Helene (1987)Sequence-targeted chemical modifications of nucleic acids by complementary oligonucleotides covalently linked to porphyrins. Nuc. Acids Res. 15. 8643-8659. Lober, G. and L. Kittler (1978) Redox mechanism involved in the fluorescence quenching of dyes bound to deoxyribonucleic acid (DNA). Siud. Biophys. 73, 25-30. Muller, W. and D . M. Crothers (1975) Interactions of heteroaromatic compounds with nucleic acids. Eur. J. Biochem. 54, 267-277. Norden, B. and F. Tjerneld (1982)Structure of methylene blue-DNA complexes studied by linear and circular dichroism spectroscopy. Biopolymers 21, 1713-1734. Nothnagel, E. A. (1987) Quantum counter for correcting fluorescence excitation spectra at 320- to 800-nm wavelengths. Anal. Biochem. 163, 224-237. Ohno. T., T. L. Osif and N. N. Lichtin (1979)A previously unreported intense absorption band and the pK,, of protonated triplet methylene blue. fhoiochem. Phorobiol. 30, 541-546. OhUigin, C., D. J. McConnell, J. M. Kelly and W. J. M. Van der Putten (1987) Methylene blue photosensitized strand cleavage of DNA: effects of dye binding and oxygen. Nuc. Acids. Res. IS, 7411-7427. Opitz, J. and D. Schulte-Frohlinde (1987) Laser-induced photoionization and single-strand break formation for polynucleotides and single-stranded DNA in aqueous solution: model studies for the direct effect of high energy radiation on DNA. J. Phoiochem. 39, 145-163. Osif, T. L., N. N. Lichtin, M. Z. Hoffman and S. Ray (1980) Evidence for reductive quenching of singlet excited methylene blue by iron (11). J . fhys. Chem. 84, 41 1-414. Perrouault, L., U. Asseline. C. Rivalle, N. T. Thuong. E. Bisagni. C. Giovannangeli, T. Le Doan and C. Helene (1990) Sequence-specific artificial photoinduced endo-

56

DAVIDA. DUNNer a/.

nuclease based on triplet helix-forming oligonucleotides.

Interactions of the dimethyldiazaperopyrenium dication with nucleic acids. 1. Binding to nucleic acid components Praseuth, D., M. Chassignol, M. Takasugi, T. LeDoan, and to single-stranded polynucleotides and photocleavage of single-stranded oligonucleotides. Biochem. N. T. Thuong and C. Helene (1987) Double helices with parallel strands are formed with nuclease-resistant oligo28, 3227-3234. [a]-deoxynucleotides and oligo-[a]-deoxynucleotides Subramanian, R. and C. F. Meares (1986) Photosensitizcovalently linked to an intercalating agent with compliation of cobalt bleomycin. J. Am. Chem. Soc. 108. mentary oligo-[PI-deoxynucleotides.J. Mol. Biol. 196, 6427-6429. 939-942. Von Sonntag, C., (1984) Carbohydrate radicals from ethylRabinowitch, E. and L. F. Epstein (1941) Polymerization ene glycol to DNA strand breakage. Inr. J. Radial. Biol. of dyestuffs in solution. Thionine and methylene blue. 46,507-519. J. Am. Chem. SOC. 63, 69-78. Von Sonntag, C., U. Hagen, A. Schon-Bopp and D. Rehm, D. and A. Weller (1970) Kinetics of fluorescence Schulte-Frohlinde (1981) Radiation-induced strand quenching by electron and H-atom transfer. Isr. 1. breaks in DNA: Chemical and enzymatic analysis of Chem. 8 , 259-271. end groups and mechanistic aspects. Adv. Radiai. Biol. Saito, I., T. Morii, H. Sugiyama, T. Matsuura, C. F. 9, 109-142. Meares and S. M. Hecht (1989) Photoinduced DNA Waskell, L. A., K. S . Sastry and M. P. Gordon (1966) strand scission by cobalt bleomycin green complex. J. Studies on the photosensitized breakdown of guanosine Am. Chem. Soc. 111, 2307-2308. by methylene blue. Biochim. Biophys. Acia 129,49-53. Sevilla, M. D., J. B. D'Arcy, K. M. Morehouse and M. Yao, T., T. Wasa and S . Musha (1978) The anodic voltamL. Englehardt (1979) An EPR study of T-cation radicals metry of deoxyribonucleic acid at a glassy carbon elecin dinucleoside phosphates and DNA produced by trode. Bull. Chem. Soc. Jpn. 51, 1235-1236. photoionization. Phoiochem. Phorobiol. 29, 3742. Zenda, K., M. Saneyoshi and G. Chichara (1965) BiologiSimon, M. I. and H. Van Vunakis (1962) The photocal photochemistry I. The correlation between the dynamic reaction of methylene blue with deoxyribonuphotodynamical behaviors and the chemical structures cleic acid. J. Mol. Biol. 4, 488-499. of nucleic acid-bases, nucleosides. and related comSlama-Schwok, A., J . Jazwinski, A. Bere, T. Montenaypounds in the presence of methylene-blue. Chem. Garestier, M. Rougee, C. Helene and J. M. Lehn (1989) Pharm. Bull. 13, 1108-1113. Nature 344,358-360.

The role of ground state complexation in the electron transfer quenching of methylene blue fluorescence by purine nucleotides.

The effect of three purine nucleotides on the fluorescence of methylene blue in aqueous buffer has been investigated. Guanosine-5'-monophosphate (GMP)...
876KB Sizes 0 Downloads 0 Views