J . Am. Chem. SOC.1994, 116, 1131-1132
DNA Sequencing with the Hydroperoxide of Tetrahydrofuran Gangning Liang7.t Peter Gannet1.r Xianglin Shi,* Yi Zhang,' Fa-Xian Chen,' and Barry Gold'.tJ Eppley Insfilule/or Research in Cancer and Allied Diseases and Department of Pharmaceutical Sciences Uniuersiry o/ Nebraska Medical Cenfer Omaha, Nebraska 68198 Department o/ Medicinal Chemistry West Virginia University Morganlown. West Virginia 26506 Laboratory o/ Experimental Pathology National Cancer Institute Roam C301, Building 41 Bethesda. Maryland 20892 Receiued October 25. 1993
The development of new reagents and methods to chemically sequence DNA is of interest for both mechanistic and practical reasons.14 In the present study, we describe conditions under which 2-hydroperoxytetrahydrofuran (THF-OOH), an autoxidation product of THF? reacts with D N A to afford heat-labile sites selectively at either C or G and A. This novel chemistry is discussed along with its use in DNA sequencing. The incubation of THF-OOH'O with j2P-end-labeled DNA in the absence and presence of reducing agents is shown in Figure 1. THF-OOH by itselfor in the presenceof lOmM dithiothreitol ( D M ) or Fe(l1) shows a specificity for C (Figure I, lanes f-h) that is time- (data not shown) and dose-dependent (Figure 2, lanes j-I). T h e intensity of the C cleavage hands is enhanced by themaddition of lOmM DTT (Figure I , lanel),while theaddition of Fe(11) without DTT has a small enhancing affect on the C cleavage (Figure I , lane f us g). The coaddition of Fe(I1) and DTT provides a strong C cleavage pattern; however, the increase is less than that observed with only thecoaddition of DTT (Figure Llanes h us I). Thesubstitution of HzOz for THF-OOH generates a weak cleavage ladder (Figure I , lane e) in the presence of Fe(l1) and D M hut with no C specificity, indicating that HO' is not responsible for the C bands. Above IO mM D M , the cleavage specificity begins to switch from C to G > A (Figure 2, lane 9). and this transition is complete at 100 mM DTT. 7 Eppley
Institute. University of Nebraska Medical Center. Department of PharmaceuticalScience. Univenit y of Nebraska Medical Center. I W u t Virginia University. I National Cancer Institute. ( I ) Maram, A. M.; Gilbcrt. W. P m . Noll. Amd. Sci. U.S.A. 1977, 74, 5W564. (2) Maram. A. M.; Gilbcrl, W. Merhods Emymol. 1980.65.499-560, (3) Johnston. B. H.; Rich. A. Cell 1985.42.113-724. (4) Ambrosc, B. J. B.; Plus. R. C. Biochemirrry 1985. 24. 619k5200. ( 5 ) Ivcncn. B. L.; Dervan. P. B. Nucleic Acids Res. 1987, I S , 7823-7830. (6) Chen. X.; Burrows, C. J.; Rakita, S. E.J . Am. Chem. Soc. 1992. / / 4 , 322-325. (7) Kohwi-Shigcmauu.T.:Kohwi.Y.MerhdsEnrymol. 1992,2/2,I5S180. (8) Johnston. B.
H.Melhodr Enrymol. 1992. 2 2 , 180-194. (9) Rein. H.; Criegce. R. Angev. Chem. 1950.62. 120.
(IO) Over a 45-min period. 2.3-dihydrofuran (26 8.0.37 mal) was slowly addedtoanice-eoldrolutianofHtOt(30%.51 g.0.50mol)andeonccntrated H~SOI(0.1 mL) [based an a method dcrcrikd by Milar. N. A,; Peeler, R. L.,Jr.; Mageli. 0. L. J . Am. Chem. Soe. 1954. 76. 2322-23251. After the
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Fiyre 1. Cleavage pattern induced by THF-OOH in the absencc and presenccofreducingagen~.AS'-["P]-lakled, 85-bprestrictionfragment previouslydescrikd [Church, K. M.; Wurdeman. R.L.;Zhang. Y.;Chcn. F.-X.; Gold, B. Biochemistry 1990.29.68214838] was prepared using standard procedures.2 The "P-lakled DNA and sonicated calf thymus DNA (final eonccntration 100 pM nucleotide) were dissolved in IO mM sodiumcadylate buffer (pH 1.6) eontaining,whenspccified. DTTand/ or Fe(II). The reactions were initiated by the addition of THF-OOH followed by incubation in 10 mM sodium cacodylate buffer (pH 1.6) at 31 'C for the 24 h. Strand breaks in the reacted DNA were generated byheatingat90Tfor 15min (exccptforlanem)followed by treatment with hot piperidine. After purification and in vacuo removal of the piperidine. the DNA was suspended in loading buffer.' denatured. and loaded onto a 12%palyacrylamide (7.8 M urea) denaturing gel that was run at 15 W ( - 5 5 "C): lane a, G , lane b. G A, lane c. controI; lane d. IO mM DTT,lanee, 1 mM H202, lOmM DTT+ 200pM Fe(NH& (SO&; lane f. I M THF-OOH + 200 p M Fc(NH,)~(SO,)~;lane g. 1 M THF-OOH; lane h, 1 M THF-OOH + 200 pM F~(NHI)I(SOI)I IO mM DTT,lanes i-I. 1.6,71,230, and loo0 mM THF-OOH with IO mM D l T lane m, 1 M THF-OOH with IO mM DTT without neutral thermal treatment but with piperidine treatment.
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Neither 2-hydroxy-THF nor y-butyrolactone(decomposition products of THF-OOH" ) shows any DNA cleaving activity under a variety of conditions (data not shown). The damage induced by THF-OOH at C or G A is only exposed as strand breaks
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afterheatingtheDNAat9OoCatpH7.0followedbytreatment with base. If the DNA is either not heated at pH 7.0 (data not shown) or not treated with piperidine (Figure 1, lane m). no cleavage is observed. This implies that the initial damage must first be transformed intoan ahasic site, which in turn is converted to a strand break by alkali. ESR analysis of the decomposition of THF-OOH in the presence of the spin trap 5,5-dimethyl-l-pyrroline N-oxide (DMPO) shows the presence of three radical species (Figure 3,
addition,thcsolutian wasstirred for an additional45mi" withthetemocraturc mainlainti below 10°C. Thercactian miiturcuasihcnsaiuraied~~thralid NHLI and ertracied vith CH,CI, The combined CH,CI, C I I ~ B E I Swere then cxlraaed wth 20% BOUMUS NaOH and thc liver ~ was ~ ~ ~ ~ U~U M U~ . .. w~i r h e d~ ~ ~ ~ ~ ~ ~ ~ l ~ ~ ~ ~ ~ ~ . with CHKh and neutralized to slightly above pH 1 . 2 with HOAc while the temperaturewasmaintainedklow IO'C. Thisaqumussolutionwassaturated uilh NHLI and crlraclcd w i t h CH2CI,, and the combined ~ x t r a c t were i uarhedwtheold. 10% UaHCO,.dr~edover~aSO,.~ltcred.aodcon~ntnted in vacuo Io ydd II 1g 087 yield1 of a clear liquid ' H U M R (CDCI,) 6 1.83 (m. 2. C I I d I 95 (m.2. CH,r. 3.91 (m. 2. O-CH,). 5 55 tm. I . O C H OOHIand8.69 (s. LOOH): "C U M R (CDCI,)6 2 3 92and 29 13 (CH;rl. (11) Murai,S.; Sonoda,N.;Tsuuumi, S.Bull. Chrm. Soc. Jpn. 1%3,36, 61 1 3 (O-CHl]. 101.9 ~O.CH-OOWJ 527-530.
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0 1994 American Chemical Society
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Communications to the Editor
I 1 32 J. Am. Chem. Soc., Vol. 116, No. 3, 1994
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Figure 2. Effectof DTT wnantration of the cleavage pattern induced by THF-OOH (reaction wnditions the same as described in Figure I):
lanea,G,laneb.G+ A;lanec,wntroklaned, 1.0MTHF-OOH,lanes c-n. 1.0 M THF-OOH with 0.001, 0.005, 0.01, 0.02. 0.05, 0.10. 0.25. 0.50. 0.75. and 1.0 M DTT. respetively; lane 0, 1.0 M DTT. top). The magnitude of the coupling constants obtained by computer simulationallows tentativeassignment of these radicals as follows: carbon-centered, R'(a(N) = 15.9 G, a(H) = 24.9 C); alkoxy, RO' (a(N) = a(H) = 15.2 G ) ; and hydroperoxy, ROO'(a(N) = I4.0G,a(H) = ll.OG).12 Thespeciesidentified as RO' is not HO' because the line intensities attributed to it are insensitive to the addition of H202or EtOH. The intensities of thelinesassignedtoROO'increaseas theconcentrationof DMPO increases and are absent when the reaction is run under Ar. The additionofTHF-OOH toDTTinthepresenceofDMPO(Figure 3, bottom) caused an increase in the intensity of R' relative to RO', although the spectrum is complicated by the trapping of D T T (a(N) = 15.02 G, a(H) = 16.6 G).13 At high ratios of DTTtoTHF-OOH (>>I),noEPRsignalisobserved,presumably because of rapid reduction of RO' by DTT. Based on these data and a previous product study." we suggest the following scheme for the formation of the different species:
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Figure 3. ESR spectra of the DMPO-trappcd THF-OOH degradation products (*, R'; 0, RO'; ROO': A, DTT'): (top) THF-OOH (130 mM); (bottom) THF-OOH (I30 mM) + DTT (IO mM). ESR spectra were remrdcd on a Varian E-3 I min after reaction initiation. All reactions wntainedDMPO(60mM)andcalfthymusDNA (300~Lfinalvolume) (20 pg/mL) in 10 mM sodium cawdylate buffer (pH 7.4) and were initiated by additionof 50pLofanaquU)usIOlutionofTHF-OOH(final concentration, 130 mM). Instrument settings: racivcr gain (a) 2.5 X 10'. (b) 1.25 X IO3;time constant, I s; modulation amplitude. 1 G,scan time. 16 min; magnetic field. 3470 100 G.
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dominant species trapped during the decomposition of THFOOH in the presence of D I T (Figure 3b), we rentatiuely suggest THF-OOH -, THF-O' HC(=O)O(CH,),' that a lesion at N3-C is involved in the formation of the apyrimidinic site, although the specificity for C differentiates HC(=O)O(CH,),O-O' THF-OOH from other radical precursors. The nature of the THF-OOH-mediated C-lesion that affords In conclusion, because of the uniform band intensities for all a beat-labile product is not known, but both 0'- and N3-aLylC C with no trace ofstrand breaks at T, THF-OOH in the presence are released from DNA upon neutral thermal hydrolysis.14J~It of low concentrations of reducing agent is suitable as a C has also been reported that carbon-centered free radicals, e.&, sequencing reagent. CHI', react with nucleosides to afford N4- and N 3 - m e t h ~ l C . ~ ~ Wnnrinp: the autoxidation products of THF can undergo Since the ESR results show that the Cantered radical is the detonation upon heating. In our hands, THF-OOH, even at > I M concentrations, undergoes smooth and nomiolent decompo(12) Buettner. 0 . R. Free R o d i d s Biol. Med. 1987.3.259-303. (13) Dana. M.J.; F0rni.L. G.;Shuter.S. L.Chem.-Biol.Inleracl. 1981, sition in the presence of reducing agents.
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61, 177-188. (14) Singer. 6.;Krager. M.;Camno. M. Bioehrmirrry 1978.17, 12461250. ( I S ) Beranek, D.T.;Weis, C. C.; Swcnson, D.H. Corcimgenesis 1980, I , 595-606. (16) Zady, M. F.; Won& J. L.J. Org. Chrm. 1980.45.2373-2377.
AcknowMgment. This workwassupportedbyGrant CA29088 and Laboratory Cancer Grant CA36727 from the National Cancer Institute and American Cancer Center Core Grant SIG16.
