Bioconlugate Chem. 1002, 3,554-558
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Synthesis and Properties of an Oligodeoxynucleotide Modified with a Pyrene Derivative at the 5’-Phosphate Jeffry S. Mann, Yoko Shibata, and Thomas Meehan’ Division of Toxicology and Department of Pharmacy, University of California, San Francisco, California 94143. Received March 23,1992 The synthesis of an oligonucleotide (ODN) modified with pyrene (pyr) on the 5’-phosphate is described. The ODN and pyrene are joined through a linker composed of four methylene groups. Modification of the oligonucleotidewas effected via condensation of the 2-cyanoethylNfl-diisopropylphosphoramidite of 4-(l-pyreny1)butanol (pyr-mdOPAm, 2) with the 5’-OH of an ODN. This derivative is suitable for incorporation into automated solid-phase DNA synthesis and was attached to the 5’ terminus of the DNA chain through a phosphodiester linkage. The properties of the 5’-(pyr-m4)d(T)15(3) and the duplex it formed with d(A)15 were investigated by fluorescence and absorbance spectroscopy. The pyrene fluorescence in the modified duplex was quenched 96.3% relative to an identical concentration of free 4-(l-pyrenyl)butanol. The ultraviolet spectrum of the 5’-(pyr-m4)-d(T)15and 5 ’ - ( p y r - ~ ) - d ( T ) ~ ~ d (A)15 modified duplex, in the 320-360-nm region, was red-shifted 6 nm relative to the free 4-(l-pyrenyl)butanol. The T, values of the unmodified and modified duplexes at 0.1 M NaCl were 34.9 and 41.9 OC, respectively. The pyrene-induced stabilization corresponds to a free energy change (AAGO) of -2.6 kcal/mol.
INTRODUCTION
Antisense oligodeoxynucleotides(ODN) are widely used for sequence-specific, down-regulation of gene expression ( I ) . The replication of vesicular stomatitis virus (21,herpes simplex virus (3),influenza virus (41, and HIV ( 5 , 6 )has been arrested by synthetic oligonucleotides complementary to sequences within the viral genome. Antisense oligonucleotides have also been used to inhibit leukemia cell proliferation (7) and the production of gene products from a number of oncogenes such as ras (8),c-myc (9), c-myb (IO),and N-ras (11). The development of clinically useful antisense drugs is limited in part by the expense of synthesizing ODNs. This problem is magnified by the low cellular uptake of ODNs and their susceptibility to degradation by nucleases. Our goal is to introduce chemical modifications into ODNs that will increase cellular uptake, nuclease resistance, and the binding affinity of the modified ODNs to their target sequences. Derivatization of ODNs with a neutral polycyclic aromatic hydrocarbon such as pyrene is clearly a promising approach. Pyrene is nonmutagenic and has a high LD50 (250mg/kg, mice), which makes it an attractive candidate for in vivo applications (12).Analogous to ODNs derivatized with cholesterol (13) or phospholipids (141, an ODN derivatized with the lipophilic pyrene moiety is expected to show enhanced cell association, which may lead to increased transport of the ODNs across cell membranes. Recent work has indicated that placement of a polymethylene tail on an acridine-modified ODN led to a substantial increase in antisense activity, that may be due in part to increased ODN transport (15). Further, pyrene intercalates between the base pairs of both single-stranded and double-stranded nucleic acids (16) and, therefore, should stabilize the duplex between the modified ODN and its target sequence. Charged intercalator-derivatized oligonucleotides are known to exhibit enhanced binding affinity for complementary sequences. For example, the melting temperature (T,) of the duplex formed between an octathymidylate derivatized with the intercalator acridine and ita complementary sequence was found to be twice
that of the unmodified duplex (17). Charged intercalating groups attached at the 3’ and/or 5’ ends of the ODN also impart nuclease resistance to the modified ODN. Asseline, et al. (18) reported that an octa-8-D-thymidylate derivatized at both the 3‘ and 5‘ ends with acridine was resistant to degradation by exonucleases. Compared to ODN derivatization with positively charged intercalators, such as acridine, little attention has been paid to the rational conjugation of pyrene to synthetic oligonucleotides (1921). As yet pyrene-modified ODNs have not been evaluated for antisense activity. Since neutral intercalators would be expected to enhance the transport of ODNs and they undergo significantly different interactions with nucleic acids compared to charged intercalators (161, we have embarked upon a program to evaluate the utility of neutral intercalator-modified ODN. In this paper we describe the synthesis of a pyrene derivative (2) tethered to a phosphoramidite via a linker arm composed of four methylene groups. The pyrene phosphoramidite was incorporated into the solid-phase synthetic cycle and attached to the 5’ end of d(T)15. Spectroscopic and thermodynamic data indicate that the pyrene interacts strongly with both single strand and duplex DNA, most likely in an intercalative or stacking manner. RESULTS
Synthesis of Pyr-m4OPAm (2). The 2-cyanoethyl NJV-diisopropylphosphoramiditeof 4-(l-pyrenyl)butanol, 2, was synthesized as shown in Scheme I. The commercially available pyrenebutyric acid was reduced to the corresponding alcohol 1in 87 % yield with LiAlH4 in THF at 25 OC. Alcohol 1was treated with diisopropylethylamine and 2-cyanoethylNfl-diisopropylphosphoramidochloridite in THF. The desired product 2 was isolated by silica gel chromatography in 78% yield. The structure of phosphoramidite 2 was confirmed by ‘H and 31PNMR spectroscopy and chemical ionization (CI) mass spectrometry. Synthesis of Oligonucleotide-Pyrene Conjugate 5’( p y r - m ( ) d ( T ) ~(3). ~ Oligonucleotides were synthesized using automated solid-phase phosphoramidite method0 1992 American Chemical Societv
Pyrene-Derlvatlred OllgodeoxynucleotMes Scheme I *
Bkmnjugete Chem., Vol. 3, No. 8, lQ92 IS5 (8)
a b
1 (
’50
E1: 1Z r l H E2: R = CHzOP,,CCH,CH&N N-iPr2
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(a) LiAlH,, THF; (b) ClPN-(iPr)dOCHzCHZCN),EtN(iPr)Z.
ology starting with 5’-DMT-dT attached to a CPG support. Phosphoramidite 2 was introduced in the 15th cycle using a procedure which incorporated lengthened reaction times and increased addition of activator and phosphoramidite relative to the standard coupling. The overall yield of coupling reactions 1-14 was estimated to be 86-89 % from the height of the peak at X = 504 nm in the absorbance spectrum of the DMT cation produced in the deblocking step. The extent of 5’ derivatization with pyrene was estimated at approximately 85 % from HPLC analysis of the crude oligonucleotide mixture. The oligonucleotides were purified by reverse-phase HPLC using 0.1 M triethylammonium acetate (pH 7) and a 40-min linear acetonitrile gradient (040% CH3CN) at a flow rate of 1.0 mllmin. The major peak ( t =~28.4 min) was collected and lyophilized,affording 28 Azm units of purified oligomer 3. The purified oligonucleotide was digested using snake venom phosphodiesterase and analyzed by reverse-phase HPLC using 0.1 M triethylammonium acetate (pH 7) and a 50-min linear acetonitrile gradient (&loo% CHsCN) at a flow rate of 1.0 mL/min. The column effluent was monitored by UV (260 nm) and fluorescence (245-nm excitation and >320-nm emission). The resulting chromatogram showed a UV peak which coeluted with deoxythymidine 5’-monophosphate ( t =~9.2 min) and a peak which exhibited both fluorescence and UV absorbance with the same retention time as an authentic sample of 4-(l-pyrenyl)butanol ( t =~41.2 min). The identity of the fluorescent peak was confirmed by CI mass spectrometry (ml2 = 274.4). Physical Properties of 5’-(pyr-m*)d(T)ls(3). The properties of the 5’-pyrene derivatized ODN, 5’-(pyr-m4)d(T)16 and the duplex it formed with d(A)15 were investigated by fluorescence and UV spectroscopies. Relative to the free 4-(l-pyrenyl)butanol, the fluorescence yield of pyrene in the single- and double-stranded ODN was approximately 9.0% and 3.7%, respectively (Figure 1). The maxima in the UV spectra of the modified single- and double-stranded oligomers were red-shifted by 6 nm (346 nm vs 340 nm) relative to those of an aqueous solution of 4-(l-pyrenyl)butanol (Figure 2). The thermal stability of the modified duplex relative to the native duplex was measured over a range of NaCl concentrations (0,024.52 M). Both the modified and native duplexes displayed cooperative transitions over the range of salt concentrations employed. The modified duplex exhibited a sharper melting transition than the unmodified duplex indicating an enhanced cooperativity (Figure 3). A plot of T, versus log [Na+l was linear and displayed a steady increase in transition temperature (T,) for both the native and modified duplexes (Figure 4). Cooperativity was confirmed by the linearity exhibited in the UT, vs In Ct plot, which was constructed from data obtained at single-strand concentrations (Ct) between 1.2 and 4.8 rM (Figure 5).
0 340
460
400 Wavelength (nm)
L
1
340
400
Wavelength
460
(nm)
Figure
1. (a) Fluorescence emission spectrum of a 0.12 NM solution of 441-pyreny1)butanol. (b) Fluorescence emission spectra of the pyrene-modified dTl5 single-strand (-) and the pyrene-modified duplex dT15-dA15 (- -1. Both spectra were measured on solutions which were 0.12 rM in pyrene-modified dTI5. All samples were dissolved in 0.1 M NaC1,O.Ol M sodium phosphate (pH 7) and recorded with the excitation monochromator at 276 nm. Although fluorescence intensity is in arbitrary units, the relative intensities of the spectra in a and b are as shown.
DISCUSSION
Synthesis of Pyrene-Oligonucleotide Conjugate 5’(pyr-mr)d(T)~~ (3). The synthesis of derivatives which can be incorporated into the solid-phase synthetic cycle is central to our strategy for preparing pyrene-labeled oligonucleotides. This approach avoids the difficulties of solution-phase oligonucleotidesynthesis and the structural ambiguity encountered in the course of postsynthetic modification of oligonucleotides. Additionally, the synthesis of pyrene phosphoramidites, such as 2, provides a versatile and convenient avenue in order to vary linkerarm length and composition without prior attachment to anucleotide monomer. This strategy places no restrictions on oligonucleotide length or base sequence. It will also allow a systematic investigation into the linker-length parameter and its effect on antisense activity and the physical properties of pyrene-modified ODNs. Duplex Stability. Model (CPK) building studies indicate that pyrene attached to an oligonucelotide via a linker arm composed of four methylene groups is fairly constrained yet has three possible modes of interaction with the duplex: intercalation, end stacking, and, less likely, groove binding. Stacking and intercalation are shown in Figure 6. The quenching of the pyrene fluo-
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Mann et al.
658 Bioconlugete Chem., Vol. 3, No. 6, 1992 (a)
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-2
Figure 4. Dependence of the duplex melting temperature (T,) on sodium ion concentration [Na+] for pyrene-modified dT15dA15 (+) and unmodified dT15-dA15( 0 )duplexes at a total strand concentration of 4.5 r M in 0.01 M sodium phosphate (pH 7).
0.2
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Figure 2. (a) Comparison of the UV absorbance spectra of the pyrene-modified dT16 single-strand (0) and pyrene-modified dT15-dA15duplex (+). (b) Comparison of the UV absorbance and the red-shifted (6 nm) spectra of 4-(l-pyrenyl)butanol (0) pyrene-modified duplex dT15-dAl5(+),in the 300-360-nm region. All samples were dissolved in 0.1 M NaCl, 0.01 M sodium phosphate (pH 7 ) .
10
25
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55
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Temp ( " C )
Figure 3. Melting curve8for unmodified (0) and pyrenemodified (+) d(T)15-d(A)15in 0.3 M NaC1,O.Ol M sodium phosphate (pH 7).
rescence, increase in duplex stability, and the magnitude of the red-shift in the UV spectrum of the duplex indicate that the pyrene interacta strongly with one or more nucleobases; possibly stacking on the exterior of the ultimate base pair or intercalating between the ultimate and penultimate base pairs. Both models might be used to explain the enhanced stability of the modified duplex relative to ita unmodified counterpart. Groove binding would be expected to lead to less significant duplex stabilization and quenching of the pyrene fluorescence
-14
-13
-12
lnCt
Figure 5. Dependence of duplex melting temperature (T,)on single-strand concentration (C,)for the pyrene-modified (+) and duplexes in 0.10M NaC1,O.l M sodium phosphate unmodified (0) (PH 7 ) .
Figure 6. Two possible modes of pyrene-duplex interaction: (a) stacking on the ultimate base pair and (b) intercalation between duplex base pairs.
(I7). Pyrene fluorescence is also quenched in the single strand, but to a lesser degree than in the double-stranded oligomer. Pyrene fluorescence in the double-stranded sample is reduced by a factor of 96.3 5% relative to the free pyrenebutanol and 60 5% compared to the pyrene-modified single-stranded sample. Previous work in this laboratory has shown that benzo[alpyrene derivatives and pyrene interact strongly with single-stranded DNA at high salt concentrations (16). This binding probably resulta from intercalation into loosely ordered intrastrand duplexes which can be more easily unwound to accomodate the hydrocarbon than the native duplex. Inspection of the thermodynamic parameters calculated from plots of 1/T, vs In Ct for both the modified and unmodified duplexes reveals a free energy difference (AAGO) of -2.6 kcal/mol
PyreneDerivatlzed OIigodeoxynucleotMes
Bioconlugete Chem., Vol. 3, No. 0, 1992 557
pmol scale with a Biosearch 8600 DNA synthesizer using phosphoramidite chemistry (23). The standard coupling program (6.5 min) was used for the synthesis of d(A)rs and d(T)15. The coupling program was modified to include a 60-min coupling cycle and two extra additions of pyrene phosphoramidite 2 and tetrazole a t the final step of the synthesis. The oligonucleotides were deprotected by EXPERIMENTAL SECTION treatment with concentrated NH40H for 5 h a t 55 "C. Materials and Methods. Pyrenebutyric acid, lithium The deprotected oligonucleotides were evaporated to aluminumhydride (l.OMinTHF),and2-cyanoethylN,N- dryness and taken up in 400 pL of 100 mM triethylamdiisopropylphosphoramidochloriditewere purchased from monium acetate, pH 7.0, and purified by reverse-phase Aldrich. Diisopropylethylamine (Aldrich) was distilled HPLC eluting with 100 mM triethylammonium acetate from NaOH under Nz and stored over molecular sieves (4 with agradient of acetonitrile. The gradient was developed A). THF (Fisher) was distilled from sodium metal under linearly from 0 %-BO % acetonitrile over 40 min at a flow Nz immediately prior to use. Column chromatography rate of 1 mL/min. and TLC were performed on 23&400 mesh, 60-A silica gel T, Determination. T,,, values were determined using (Aldrich), and Rediplates, 250 pm (Fisher), respectively. a Gilford 2600 spectrophotometer. The temperature was Reverse-phase HPLC was performed on a CUZorbax ODS controlled by a Gilford 2527 thermal programmer. Data column (4.6 mm X 25 cm) on an HPLC composed of an points were collected every 0.5 min. Single-strand conAltex Model llOA pump and a Kratos Spectroflow 430 centration was determined from the calculated UV exgradient programmer. The column effluent was monitored tinction coefficients of the individual strands. The spectral at 260 nm with a Hitachi 100-40 UV detector linked in overlap contributions from the pyrene in the modified tandem with a SchoeffelFS 970 LC fluorometer. 'H NMR strand were determined and found to be negligible. spectra were recorded on a G.E. QE 300 spectrometer. Thermodynamic parameters were calculated from plots The samples were dissolved in CDCl3, and the absorbance of l/Tm vs In Ct, where Ct is the total single-strand of the residual solvent protons was used as a reference. 31P concentration, using a previously published procedure (24). NMR spectra were measured on a Bruker 300 MHz Nuclease Digestion of Modified and Unmodified instrument and were referenced to an external standard Oligonucleotides. The oligonucleotide (2 OD units) was Of 85% &Pod. diluted to 3.0 mL with a buffer solution composed of 0.11 Synthesis of 4 4 l-Pyreny1)butanol(1). PyrenebuM Tris-HC1,O.ll M NaC1, and 0.15 mM MgClz (pH 8.9). tyric acid (888.1mg, 3.08 mmol) was dissolved in anhydrous Snake venom phosphodiesterase (0.1 Kunitz unit) was THF (20 mL) with stirring under argon. To this solution added, the mixture was vortexed and left overnight at 23 was carefully added LiAlH4 (1.0 M in THF, 4.0 mL). The "C. In order to precipitate the protein prior to HPLC resulting solution was stirred for 4 h, then EtOAc (15 mL) analysis, the digest mixture was reduced in volume to 250 was added dropwise and the solution was poured into icepL followed by the addition of 750 pL of acetonitrile. The cold 10% HzS04 giving a bright yellow mixture. The mixture was vortexed and then centrifuged and the mixture was transferred to a separatory funnel and the aqueous acetonitrile supernatant containing the monoorganic layer was removed. The aqueous layer was nucleotide and 4-(l-pyreny1)butanol was drawn off. The extracted with EtOAc (3 X 20 mL). The organic layers protein was redissolved and the precipitation carried out were pooled, washed with 10% KzC03 (1X 20 mL) and two more times. The three aqueous acetonitrile superH20 (1 X 20 mL), dried (NazSOd), and evaporated to natants were combined, evaporated to dryness, and dryness, giving 739 mg (87%) of the alcohol as a pale yelreconstituted in 2W400 pL of 0.1 M triethylammonium low solid. TLC (CHZClz): Rf0.26. NMR (CDC13): 6 1.62acetate before HPLC analysis. 1.88 (m, 4 H), 3.34 (t,J = 7.8 Hz, 2 H), 3.64 (t,J = 6.5 Hz, 2 H), 7.84-8.28 (m, 9 H). ACKNOWLEDGMENT 4 4 l-Pyrenyl)butanol, 2-CyanoethylN,N-Diisopropylphosphoramidite (2). 4-(l-Pyrenyl)butanol (I; 161.5 This work was supported in part by NIH Grant 40598 mg, 0.60 mmol) was dissolved in anhydrous T H F (500 and the Elsa U. Pardee Foundation. We thank Alan R. pL) under Ar with stirring. Diisopropylethylamine (152 Wolfe for helpful discussions and his generous assistance mg, 1.2 mmol) was added via syringe followed by 2-cyain the preparation of the manuscript. CIMS was carried noethyl N,N-diisopropylphosphoramidochloridite (142 out a t the Bioorganic and Biomedical Mass Spectrometry mg, 0.60 mmol). Within one min a voluminous white Facility of the University of California, San Francisco. precipitate had formed. The reaction mixture was stirred for 40 min then the precipitate was removed by filtration. LITERATURE CITED The filtrate was diluted to 25 mL with EtOAc and washed (1) For areview, see: Helene, C., and Toulme, J.-J. (1990) Specific with 0.1 M NaHzP04, pH 7.0 (1 X 10 mL). The organic regulation of gene expression by antisense, sense and antigene layer was removed, dried (NazS04) and rotary evaporated nucleic acids. Biochim. Biophys. Acta 1049, 99-125. to dryness affording 304 mg of a pale yellow oil. The crude (2) Leonetti, J. P.,Rayner, B., LeMaitre, M., Gagnor,C., Milhaud, product was chromatographed on silica (1 cm X 10 cm) P. G., Imbach, J. L., and LeBleu, B. (1988) Antiviral activity eluted with EtOAc/hexanes (1:3). A fast-moving fraction of conjugates between poly-(L-lysine) and synthetic oligodewas isolated and evaporated to dryness, affording 213 mg oxynucleotides. Gene 72,323-31. (75%) of the desired product as a fluorescent pale yellow (3) Miller, P.S.,and Ts'O, P. 0. P. (1987) A new approach to oil. 1H NMR (CDC13): 6 1.15 (t, J = 6.9 Hz, 12 H), 1.82 chemotherapy based on molecular biology and nucleic acid (m, 2 H), 1.96 (m, 2 H), 2.56 (t, J = 6.3 Hz, 2 HI, 3.38 (t, chemistry: Matagen (Masking tape for gene expression). J = 7.5 Hz, 2 H), 3.68 (m, 6 HI, 8.08 (m, 9 H).31PNMR Anticancer Drug Des. 2, 117-27. (CDCl3): 6 147.85. Mass spectrum (ammonia CI): calcd (4) Zerial, A., Thuong, N. T., and Helene, C. (1987) Selective for C Z ~ H ~ ~ N474.6, Z O ~ found P 475.0. inhibition of the cytopathic effect of type A influenza viruses DNA Synthesis and Purification. The unmodified by oligodeoxynucleotidescovalently linked to an intercalating agent. Nucleic Acids Res. 15, 9909-19. and modified oligonucleotides were synthesized on a 1.0at 25 "C. The stabilization of the pyrene-modified duplex results from a reduction of the transition enthalpy ( A N o = -16.5 kcal/mol) and in the transition entropy (AAS" = -46.8 &mol K) of the modified duplex relative to the unmodified duplex.
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( 5 ) Vickers, T., Baker, B. F., Cook, P. D., and Zomes, M. (1991)
Inhibition of HIV-LTR gene expression by oligonucleotides tageted to the TAR element. Nucleic Acids Res. 19,3359-68. (6) Laurence, J., Sikder, S. K., Kulkosky, Miller, P., and Ts’O, P. 0. P. (1991)Induction of chronic human immunodeficiency virus infection is blocked in vitro by a methylphosphonate oligodeoxynucleoside targeted to a U3 enhancer element. J. Virol. 65, 213-9. (7) Szczylic, C., Skorski, T., Nicolaides, N. C., Manzella, L., Malaguarnera, L., Venturelli, D., Gewirtz, A. M., and Calabretta, B. (1991) Selective inhibition of leukemia cell proliferation by BCR-ABL antisense oligonucleotides. Science 253, 562-5. (8) Chang, E. H., Miller, P. S., Cushman, C., Devadas, K., Pirollo, P. 0. P., and Yu, Z. P. (1991)Antisense inhibition K. F., Ts’O, of ras-pal expression that is sensitive to a point mutation. Biochemistry 30,8283-6. (9) Degols, G., Leonetti, J.-P.,Meehti, N., and LeBleu, B. (1991) Antiproliferative effects of antisense oligonucleotidesdirected to the RNA of the c-myc oncogene. Nucleic Acids Res. 19, 945-8. (10) Gewirtz, A. M., and Calabretta, B. (1988)A c-myb antisense oligodoxynucleotide inhibits normal human hematopoiesas. Science 242, 1303-6. (11) Tidd, D. M., Hawley, P., Warenius, H. M., and Gibson, I. (1988) Evaluation of N-ras oncogene antisense, sense and nonsense sequence methylphosphonate oligonucleotide analogues. Anticancer Drug Des. 3, 117-27. (12) Karcher, W., Fordham, R. J., Dubois, J. J., Glaude, P. G. J.M.,andLigthart,J. A.M. (1985)SpectralAtlusofPolycyclic Aromatic Compounds, p 91, D. Reidel Publishing Co., Dordrecht. (13) Letsinger, R. L., Zhang, G., Sun, D. K., Ikeuchi, T., and Sarin, P. S. (1989) Cholesteryl-conjugated oligonucleotides: Synthesis properties and activity as inhibitors of replication of human immunodeficiencyvirus in cell culture. h o c . Natl. Acad. Sci. U.S.A. 86,6553-6. (14) Shea, R. G., Marsters, J. C., and Bischofberger, N. (1990) Synthesis, hybridization properties and antiviral activity of lipid-oligodeoxynucleotideconjugates. Nucleic Acids Res. 18, 3777-83. (15) Saison-Behmoaras, T., TocquB, B., Rey, I., Chassignol, M., Thuong, N. T., and Helene, C. (1991)Short modified antisense
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oligonucleotidesdirected against Ha-ras point mutation induce selective cleavage of the mRNA and inhibit T25 cells proliferation. EMBO J. 10, 1111-8. (16) Wolfe, A., Shimer, G. H., Jr., and Meehan, T. (1987) Polycyclic aromatic hydrocarbons physically intercalate into duplex regions of denatured DNA. Biochemistry 26,6392-6. (17) Thuong, N. T., Asseline, U., and Montenay-Garestier, T. (1989) Oligodeoxynucleotidescovalently linked to intercalating and reactive substances: synthesis, characterization and physicochemical studies. In Oligodeonynucleotides: Antisense inhibitors of gene expression (Cohen, J. s.,Ed.) pp 2547, CRC Press Boca Raton, FL. (18) Asseline, U., and Thuong, N. T. (1988) Oligothymidylates substitues par un derive de l’acridine en position 5’ a la fois en position 5’ et 3’ on sur un phosphate internucleotidique. Nucleosides Nucleotides 7,431-55. (19) Casale, R., and McLaughlin, L. W. (1990) Synthesis and Properties of an oligodeoxynucleotidecontaining a polycyclic aromatic hydrocarbon site specifically bound to the N2amino group of a 2’-deoxyguanosine residue. J.Am. Chem. SOC.111, 5264-71. (20) Yamana, K., and Letsinger, R. L. (1985) Synthesis and properties of oligonucleotides bearing a pendant pyrene. Nucleic Acids Res. 16, 169-72. (21) Yamana, K., Ohashi,Y., Nunota, K., Kitamura, M.,Nakano, H., Sangen, O., and Shimidzu, T. (1991) Synthesis of oligonucleotide derivatives with pyrene group at sugar fragment. Tetrahedron Lett. 32, 6347-50. (22) Lee, H., Hinz, M., Stezowski,J. J., and Harvey, R. G. (1990) Synthesis of polycyclic aromatic hydrocarbon-nucleoside and nucleotide adducts specifically alkylated on the amino functions of deoxyguanosine and deoxyadenosine. Tetrahedron Lett. 31, 6773-6. (23) McBride, L. J., and Carruthers, M. H. (1983) An investigation of several deoxynucleoside phosphoramidites useful for synthesizingdeoxyoligonucleotides. Tetrahedron Lett. 24, 245-8. (24) (a) Marky, L. A., and Breslauer, K. J. (1987) Calculating thermodynamic data for transitions of any molecularity from equilibrium melting curves. Biopolymers 26, 1601-20. Albergo, D. D., Marky, L. A., Breslauer, K. J., and Turner, D. H. (1981)Thermodyanmica of (dC-dG), double-helixformation in water and deuterium oxide. Biochemistry 20, 1409-13.
