Bioconjugate Chem. 1990, I , 82-88
a2
Synthesesand melanoma avidities of 1-125derivativesof ellipticine and m-Amsa. J. Nucl. Med. 27, 1076. (22) Bernstein, A., Hurwitz, E., Maron, R., Arnon, R., Sela, M., and Wilchek, M. (1978)Higher antitumor efficacy of daunomycin linked to dextran. J. Natl. Cancer Znst. 60, 379-383. (23) A reviewer has suggested another plausible explanation for the biphasic cleavage rates. An Amadori rearrangement, occurring during the syntheses of the hydrazide conjugates, might generate ketone hydrazones or other rearranged product(s) with different hydrolysis kinetics.
Registry No. I, 124536-15-8;11,124536-16-9;111, 124536-17-
0; IV, 124536-18-1;V, 124536-19-2;VI, 124536-20-5;VII, 124536-
21-6; VIII, 124536-22-7;IX, 124536-23-8;tert-butyl carbazate, 870-46-2; N-(4-amino-3-methoxyphenyl)methanesulfonamide, 57165-06-7;5-methyl-9-oxoacridan-4-carboxylic acid, 24782-669; 9-methoxyellipticine,10371-86-5;ellipticine,519-23-3;methyl 5-bromopentanoate,5454-83-1;hydrazine, 302-01-2;2,3-dialdehydodextran, 37317-99-0; IV dextran dialdehyde derivative, 124820-50-4;VI11 dextran dialdehyde derivative, 124820-49-1; IX dextran dialdehyde derivative, 124820-48-0.
Photo- Cross- Linking of Psoralen- Derivatized Oligonucleoside Methylphosphonates to Single-Stranded DNA Purshotam Bhan and Paul S. Miller’ Department of Biochemistry, School of Hygiene and Public Health, The Johns Hopkins University, 615 N. Wolfe St., Baltimore, Maryland 21205. Received August 21, 1989
The preparation of oligodeoxyribonucleoside methylphosphonates derivatized with 3-[(2-aminoethyl)carbamoyl]psoralen [ (ae)CP] is described. These derivatized oligomers are capable of cross-linking with single-stranded DNA via formation of a photoadduct between the furan side of the psoralen ring and a thymidine of the target DNA when the oligomer-target duplex is irradiated with 365-nm light. The photoreactions of (ae)CP-derivatized methylphosphonate oligomers with single-stranded DNA targets in which the position of the psoralen-linking site is varied are characterized and compared to results obtained with oligomers derivatized with 4’-[[N-(aminoethyl)amino]methyl]-4,5’,8trimethylpsoralen [(ae)AMT]. It appears that the psoralen ring can stack on the terminal base pair formed between the oligomer and its target DNA or can intercalate between the last two base pairs of the oligomer-target duplex. Oligomers derivatized with (ae)CP cross-link efficiently to a thymidine located in the last base pair ( n position) or 3’to the last base pair ( n + 1 position) of the target, whereas the (ae)AMT-derivatized oligomers cross-link most efficiently to a thymidine located in the n + 1 position. The results show that both the extent and kinetics of cross-linking are influenced by the location of the psoralen-linking site in the oligomer-target duplex.
Psoralens, a class of naturally occurring photoreactive furocoumarins found in a variety of plants, were used as medicinal agents by the ancient Egyptians to treat the skin disorder vitiligo (1). In more recent times, these compounds have been used clinically in the treatment of a number of skin diseases including psoriasis ( I ) and cutaneous T-cell lymphoma (2). Psoralens are planar molecules which are able to intercalate into double-stranded regions of DNA and RNA. Upon irradiation with long-wavelength ultraviolet light, the 3,4 and 4‘,5‘ double bonds of the pyrone or furan ring, respectively, can undergo 2+2 cycloadditions with the 5,6 double bond of pyrimidine nucleosides to form cyclobutane type monoadducts (3). If the psoralen has intercalated into a suitable site, the furan-side monoadducts can undergo a further cycloaddition reaction to form a cross-link between the two strands of the doublestranded nucleic acid. Thus psoralens have been found to be useful in mapping the secondary structure of large RNA molecules ( 4 ) . Psoralens, such as 8-methoxypsoralen, show a preference for cross-linking with -TpA- sequences in doublestranded DNA ( 5 ) ;however, their ability to specifically recognize long, unique nucleic acid sequences is limited. When conjugated to oligonucleotides or oligonucleotide analogues, either by attachment through linker arms or
by monoadduct formation with thymine bases of the oligomer, the psoralens can be targeted to specific sites in the nucleic acid (6-10). We have used this approach to prepare oligonucleoside methylphosphonates which are capable of cross-linking to complementary sequences on single-stranded DNA and RNA in a sequence-specific manner (11-13). The oligomer is conjugated through its 5’phosphoryl group with 4’-[ [N-(aminoethy1)aminolmethyl]-4,5’,8-trimethylpsoralen[ (ae)AMT], l which, upon irradiation at 365 nm, forms a cyclobutane adduct between the pyrone ring of the (ae)AMTand a thymine or a cytosine base of the targeted nucleic acid. The oligonucleoside methylphosphonates themselves are nuclease-resistant oligonucleotide analogues which are Abbreviations used: (ae)AMT, 4‘- [ [N-(2-aminoethyl)ami-
no]methyl]-4,5’,8-trimethylpsoralen;(ae)CP, 3-[(2-aminoeth-
yl)carbamoyl]psoralen;3-CP, 3-carboxypsoralen;CDI, l-ethyl3- [3-(dimethylamino)propyl]carbodiimide; DEAE, diethylaminoethyl; EDA, ethylenediamine; EDTA, ethylenediaminetetraacetic acid; PAGE, polyacrylamide gel electrophoresis; Tris-HC1, tris(hydroxymethy1)aminomethane hydrochloride; TLC, thin-layer chromatography; HPLC, highperformance liquid chromatography; TBE, 0.089 M Trisborate, 0.002 M EDTA (pH 8.0);TEAB,triethylammonium bicarbonate buffer, CPG, control pore glass.
1043-1802/90/2901 -0082$02.50/0 0 1990 American Chemical Society
Psoralen-Derivatized Oligonucleoside Methylphosphonates
capable of entering mammalian cells intact and binding to complementary nucleic acid sequences (14, 15). It appears that psoralen-derivatized oligonucleoside methylphosphonates would be useful sequence-specificantisense reagents to study gene expression in cell culture. In addition, such oligomers could be used to study the mechanism of action of the antisense oligomers and to determine if the oligomers bind exclusively to their targeted nucleic acids within the cells. In this paper we describe the synthesis and photoreactions of novel 3-[(2-aminoethyl)carbamoyl]psoralen [ (ae)CP] derivatized oligonucleoside methylphosphonates. In contrast to (ae)AMT, the (ae)CP derivatives react via formation of furan-side photoadducts. The interactions of these oligomers with single-stranded DNA targets in which the position of the psoralen cross-linking site is varied are characterized and compared to results obtained with (ae)AMT-derivatized oligomers. EXPERIMENTAL PROCEDURES
T-[~’P]ATPwas purchased from Amersham Inc. and T4 polynucleotide kinase was purchased from United States Biochemical Corp. Reagents for the synthesis of oligodeoxyribonucleotide methylphosphonates and oligodeoxyribonucleotides were purchased from American Bionetics Inc. 1-Ethyl-3-[3-(dimethy1amino)propyllcarbodiimide (CDI) was obtained from Sigma Chemicals and 4,5’&trimethylpsoralen was purchased from Aldrich Chemicals. SEP-PAK (2-18 reversed-phase cartridges were obtained from Waters Associates. Wherever necessary, reactions were monitored by TLC on precoated thin layer (0.25 mm) silica gel 60 F-254 plates purchased from EM Reagents. Polyacrylamide gel electrophoresis was carried out on 20 cm X 20 cm X 0.75 mm gels containing 0.089 M Tris, 0.089 M boric acid, 0.2 mM EDTA, and 7 M urea (16). The gels were autoradiographed at -80 OC and the film was scanned on a LKB Ultroscan XL Densitometer. Analytical and preparative HPLC was carried out on a Varian 5000 LC instrument a t 254-nm detection with a Whatman Partisil ODS3-RAC I1 reversedphase column. The column was eluted for 20 min with a linear gradient of 1%-50% acetonitrile in 0.1 M sodium phosphate buffer (pH 5.8) at a flow rate of 1.5 mL/min. All the reactions involving psoralens were performed in subdued light. Synthesis of Oligodeoxyribonucleotides. Oligodeoxyribonucleotides were synthesized on CPG supports using P-cyanoethyl phosphoramidite chemistry (2 7). After the cleavage from the support and deprotection, the crude oligonucleotides were phosphorylated with [32P]ATPand T4 polynucleotide kinase (18). The oligomers were next purified by polyacrylamide gel electrophoresis, extracted with 1 M TEAB, and desalted on a SEP-PAK cartridge (19). These purified 32P-labeled phosphodiester targets were used for cross-linking experiments (vide infra). Synthesis of Oligodeoxyribonucleoside Methylphosphonates. The methylphosphonate oligomers were synthesized on CPG supports with 5’-(dimethoxytrity1)nucleoside 3’- [ (N,N-diisopropylamino)methyl] phosphonamidite monomers (20). The oligomers were deprotected, purified, and phosphorylated as previously described (18, 22). Synthesis of 3-Carboxypsoralen. 3-Carboxypsoralen was synthesized from 2-hydroxy-4-methoxybenzaldehyde in eight steps according to the procedure of Worden et al. (22) and was obtained in 14% overall yield as a yellow powder: mp 258-261 “C; 300-MHz ‘H NMR (CDClJ 6.95 (d, J = 2.4 Hz, 4’-H), 7.63 (s, 5-H), 7.80 (d, J = 2.4 Hz, 5’-H), 7.98 (s, 8-H), 9.05 ppm (s, 4-H).
Bioconjugafe Chem., Vol. 1, No. 1, 1990
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Synthesis of 4’-[ [N-(2-Aminoethyl)amino]met hyl]-4,5’,8-trimethylpsoralenDerivatives of Oligodeoxyribonucleoside Methylphosphonates. 4,5’,8Trimethylpsoralen was aminomethylated a t the 4’position (23) and was then converted to (ae)AMT as described by Lee et al. (11). The 5’-phosphorylated methylphosphonate oligomer (20 OD) was dissolved in 346 p L of 0.1 M imidazole buffer (pH 6.0). A solution of 1 M CDI (39 pL) was added and the reaction mixture was incubated at room temperature for 4 h. The mixture was diluted to 5 mL with 25 mM TEAB (pH 9.0) and purified with a SEP-PAK C-18 cartridge. The SEP-PAK was washed with 20 mL of 25 mM TEAB (pH 9.0) and the product was eluted with 50% acetonitrile in 100 mM TEAB (pH 9.0). The buffer was removed by evaporation a t 37 “C and the residue was lyophilized from water. The resulting 5’-imidazolide adduct was dissolved in 408 pL of 0.75 M lutidine hydrochloride buffer (pH 7.5) and a solution containing 51 pL of acetonitrile and 102 pL of 0.05 M (ae)AMT was added. The reaction was incubated at 37 “C for 48 h and after dilution with 5 mL of 50% aqueous acetonitrile was chromatographed on DEAEcellulose. The column was first washed with 50% acetonitrile (15 mL) and the product was eluted with 50% acetonitrile in 330 mM TEAB buffer. The product was further purified by preparative HPLC on a ODS 3-RAC I1 (4.5 mm X 100 mm) column and was obtained in a 58% overall yield. Synthesis of 3-Carboxypsoralen-DerivatizedOligodeoxyribonucleotide Methylphosphonates. The 5’imidazolide adduct of the methylphosphonate oligomer (vide supra) was first converted to its 5’-aminoethyl amidate derivative (24). Typically, the 5’-imidazolide (10 OD) was dissolved in 160 FL of 0.75 M lutidine hydrochloride buffer and 70 pL of 1 M EDA in 0.4 M lutidine hydrochloride buffer (pH 7.5) was added. The reaction mixture was incubated at room temperature for 20 h, diluted to 5 mL with 25 mM TEAB, and loaded onto a SEPPAK cartridge. The column was washed with 10 mL of 25 mM TEAB and the derivatized oligomer was eluted with 50% acetonitrile in 100 mM TEAB. Evaporation followed by lyophilization from water gave the 5’aminoethyl amidate of the methylphosphonate oligomer. This adduct was dissolved in a mixture of 200 pL of 0.4 M lutidine hydrochloride buffer (pH 7.5) and 100 NL of acetonitrile. A solution containing 2 M CDI and 0.13 M 3-carboxypsoralen in 300 pL of 0.4 M lutidine hydrochloride was added and reaction was incubated a t room temperature for 24 h. The mixture was diluted with 5 mL of 25 mM TEAB and applied to a SEP-PAK. The cartridge was washed with 10 mL of 25 mM TEAB and two 10-mL portions of 5% acetonitrile in 25 mM TEAB. The psoralen-derivatized oligomer was eluted with 3 mL of 50% acetonitrile in 100 mM TEAB and lyophilized. Final purification was done by polyacrylamide gel electrophoresis. The residue was dissolved in 10 pL of gel loading buffer which contained 80% formamide and 0.2% each of bromophenol blue and xylene cyano1 in TBE buffer. The mixture was electrophoresed on a 15% polyacrylamide slab gel. The yellow area of the gel corresponding to the adduct was excised, crushed, and extracted with 1M TEAB (5 x 1 mL) at room temperature. The extract was diluted to 20 mL with water and passed through a SEP-PAK cartridge. The cartridge was washed with two 10-mL portions of 25 mM TEAB; the product was eluted with 50% acetonitrile in 100 mM TEAB and lyophilized from water. Cross-Linking of Psoralen-Derivatized Oligodeoxyribonucleoside Methylphosphonates with Oli-
84
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B/ocon/ugate Chem., Vol. 1, No. 1, 1990
0 0.P-0
[ow-]. R
O=!TH,
w OH
in either the R or S , configuration and thus each oligomer consists oP2" diastereoisomers, where n is the number of methylphosphonate linkages. Previous experiments with (ae)AMT-derivatized methylphosphonates of the type shown in Figure l b demonstrated that the methylphosphonate and phosphoramidate linkages are totally resistant to hydrolysis by endonucleases and by nucleases found in fetal bovine serum. The phosphodiester linkage is also quite resistant to endonuclease hydrolysis and has a half-life of approximately 48 h in serum-containing medium (12). The single-stranded oligodeoxyribonucleotide targets and their complementary psoralen-derivatized methylphosphonate oligomers are shown a t the bottom of Figure 2. The targets are 21 nucleotides in length and the psoralen-derivatized methylphosphonate oligomers are 9 nucleotides long. The binding site for the oligomer occupies positions 3-11 of the target. The position of the psoralen-linking site, which is a T residue in each target, varies for each of the targets. Thus the psoralen-linking site is located at n 1 (nucleotide residue 12 of the target), n (nucleotide residue 11of the target) or n - 1 (nucleotide residue 10 of the target), where n is defined as the position of the last base pair formed between the oligomer and the target. The oligodeoxyribonucleotide targets were synthesized by using standard phosphoramidite chemistry on controlled pore glass supports and were end labeled with 32Pwith polynucleotide kinase and [32P]ATP. The methylphosphonate oligomers were synthesized on controlled pore glass supports using protected 5'-0-(dimethoxytrity1)nucleoside 3'-0-[ (N,N-diisopropylamino)methyl]phosphonamidite synthons (20). After deprotection and purification (21),the oligomers were phosphorylated with polynucleotide kinase and ATP. The 5'-phosphorylated oligomers were converted to their imidazolide derivatives by reaction with l-ethyl-3-[3-(dimethylamino)propyllcarbodiimide in imidazole buffer at pH 6 (24). The imidazolide, which was obtained in quantitative yield, was purified by reversed-phase chromatography on a SEPPAK C-18 cartridge. Oligomers derivatized with (ae)CP were prepared by first reacting the imidazolide derivative of the 5'-phosphorylated oligomer with ethylenediamine in lutidine buffer at pH 7.5 (11). The resulting 5'-(aminoethy1)phosphoramide oligomer was purified by reversed-phase chromatography and then reacted with 3-[(2-aminoethy1)carbamoyllpsoralen in t h e presence of l-ethyl-3-[3(dimethylamino)propyl]carbodiimide. The (ae)CPoligomer was purified by preparative gel electrophoresis and was obtained in 25% overall yield. Oligomers derivatized with (ae)AMT were prepared by reaction of the 5'-imidazolide derivative with 4'-[ [N(aminoethyl)amino]methyl]-4,5',8-trimethylpsoralenin lutidine buffer. The (ae)AMT-oligomers were then purified by DEAE-cellulose chromatography and by preparative HPLC on a (2-18 reversed-phase column. The overall yield of the (ae)AMT-oligomer, based on the amount of starting oligonucleoside methylphosphonate, was approximately 50%. Cross-Linking Experiments with PsoralenDerivatized Methylphosphonate Oligomers. A solution containing 0.1 pM 32P-labeledtarget and 10 pM psoralen-derivatized oligomer was irradiated a t 365 nm and the reaction mixture was subjected to polyacrylamide gel electrophoresis under denaturing conditions. As shown in Figure 3, target DNA cross-linked with oligomer has a lower mobility on the gel than the non-cross-linked target. Further irradiation of the reaction mixture at 254
+
OM
b Figure 1. Structures of oligonucleoside methylphosphonates derivatized with (a) 3-[ (2-aminoethyl)carbamoyl]psoralenor (b) 4'- [ [ N -(aminoethy1)amino]methyl]- 4,5',8-tr imethylpsor alen .
godeoxyribonucleotide Targets. A solution of 0.1 pM 32P-labeled21-mer target DNA and 10 pM of the psoralen-derivatized oligonucleoside methylphosphonate in 10 pL of water was preincubated in a borosilicate glass tube (Corning) at 4 "C for 10 min. The solution was then irradiated at 365 nm for 0-30 min a t an intensity of 0.83 J cm-2 min-' in a thermostated water bath using an UltraViolet Products Inc. long-wavelength ultraviolet lamp. The reaction mixture was lyophilized; the residue dissolved in loading buffer and electrophoresed on a 15% acrylamide gel containing 7 M urea. The wet gel was autoradiographed. The extent of cross-linking was determined by scanning densitometry of the autoradiogram. RESULTS
Psoralen-DerivatizedOligonucleosideMethylphosphonates and Their Oligodeoxyribonucleotide Targets. The structures of the psoralen-derivatized oligodeoxyribonucleoside methylphosphonates are shown in Figure 1. The oligomers are derivatized with either 3carbamoylpsoralen (Figure l a ) or (aminomethyl)trimethylpsoralen (Figure l b ) through ethylphosphoramide linker arms. The first internucleotide bond a t the 5' end of the oligomer is a phosphodiester linkage whereas the remaining internucleotide bonds are methylphosphonate linkages. Each methylphosphonate linkage can occur
Psoralen-Derivatired Oligonucleoside Methylphosphonates largo1
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Bioconjugate Chem., Vol. 1, No. 1, 1990
30
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1 10 20 D - G G G A G G A C G A A T G C A C A C G A G C T C C T G C T T *
n
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D - G G G A G G A C G T A A G C A C A C G A G
CTCCTGCAT* Figure 2. Kinetics of cross-linking of (ae)CP- or (ae)AMT-derivatized oligonucleoside methylphosphonates a t 4 or 30 "C. The
sequences of the oligomers and their single-stranded DNA targets are shown below the graphs. The asterisk represents the (ae)CP or (ae)AMT group.
nm results in disappearance of the cross-linked target and regeneration of the original target DNA (data not shown). The extent of cross-linking was determined from the autoradiogram by densitometry. The kinetics of cross-linking at 4 "C and 30 "C were determined. Figure 2 shows the results when (ae)CPor (ae)AMT-oligomers are irradiated with targets having the psoralen-linking site located at n + 1 (Figure 2A,B), n (Figure 2C,D), or n - 1 (Figure 2E,F). In the case of the n + 1 target, the cross-linking reaction is approximately linear over the first 10 min and does not increase beyond that point. The extent of cross-linking is the same at 4 "C for both the (ae)CP- and (ae)AMT-oligomers. At 30 "C the extent of cross-linking by d-(ae)AMTpTpTCGTCCTC is approximately 4-fold greater than that of d-(ae)CPpTpTCGTCCTC. . -
Unlike the n + 1 target, cross-linking with the n and n - 1targets increases continuously over the 30-min observation period. In the case of the n target, cross-linking by (ae)CPpApTCGTCCTC is approximately 2.5-fold greater than that of (ae)AMTpApTCGTCCTC. The extent of cross-linking by each oligomer is essentially the same at both 4 and 30 "C. For the n - 1 target system, (ae)AMTpTpACGTCCTC cross-links about 1.5-foldmore extensively than d-(ae)CPpTpACGTCCTC. Again the extent of cross-linking by both oligomers is essentially independent of the temperature of the cross-linking reaction. DISCUSSION
Previous studies from our laboratory have shown that (ae)AMT-derivatized oligonucleoside methylphospho-
86 Bioconjwte Chem., Vol. 1, No. 1, 1990 1
2
3
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Bhan and Miller
5 4
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xc
(BPB
Figure 3. Cross-linking reactions between the n singlestranded DNA target and (ae)CPpApTCGTCCTCor (ae)AMTpApTCGTCCTC after irradiation for 30 min at 30 OC: lane 1, 32P-labeledtarget; lane 2, target plus (ae)AMT-oligomer not irradiated; lane 3, target plus (ae)AMT-oligomerirradiated for 30 min; lane 4, target plus (ae)CP-oligomer not irradiated; lane 5, target plus (ae)CP-oligomer irradiated for 30 min. The reaction mixtures were electrophoresed on a 15% polyacrylamide gel containing 7 M urea and the gel was autoradiographed. The top of the gel and the positions of the tracking dyes bromophenol blue (BPB) and xylene cyano1 (XC) are indicated. nates of the type shown in Figure l b are capable of crosslinking with single-strandedDNA and RNA target nucleic acids. In this system cross-linking occurs via a photoinduced 2+2 cycloaddition reaction resulting in the formation of a cyclobutane adduct between the pyrone ring of the (ae)AMT group and a pyrimidine base of the target nucleic acid. Because the trimethylpsoralen moiety is tethered to the oligomer by the aminoethyl linker, furan side adduct formation with the target strand is not sterically feasible. Studies on monoadduct formation between 4’(hydroxyethyl)-4,5’,8-trimethylpsoralen,4,5’,8-trimethylpsoralen, or 8-methoxypsoralen and double-stranded DNA have shown that adduct formation occurs mostly with the furan ring of the psoralen (25-27). Although theoretical calculations suggest that the pyrone double bond should be more reactive than the furan double bond (28), the preference for furan side adduct formation may result in part from unfavorable steric interactions between substituents a t the 3- or 4-position of the pyrone ring and the 5-methyl group of thymine (26, 29). Thus it seemed possible that oligomers derivatized with a psorden moiety restricted to forming furan monoaddudsmight be capable of efficiently forming cross-links with appropriate target nucleic acids. To test this possibility, we have prepared methylphosphonate oligomers derivatized with 3-carbamoylpsoralen(3-CP). The 3-CP group is linked via an aminoethyl linker arm to the 5’ end of the oligomer through a phosphoramide linkage as shown in Figure la. Unlike 8-methoxypsoralen and the trimethylpsordens, 3-carbethoxypsoralenforms only monoadducts with DNA (30). The furan-side adduct has the cis-syn configuration (31) which is the same configuration as the furan-side adducts formed by 8-methoxypsoralen (26). It appears that the 3-carbethoxy group sterically hinders formation of pyrone-side adducts. T h u s 3carbethoxypsoralen is not capable of forming interstrand cross-linkswith DNA. Examination of computer.
generated molecular models, suggested that the aminoethyl linker arm should enable the furan ring of the psoralen moiety to form a cis-syn type cyclobutane adduct with suitably positioned pyrimidine nucleosides in the target strand. The cross-linkingstudies were carried out with singlestranded 21-mer DNA targets. The site for adduct formation between the psoralen and the target, which we will call the psoralen-linking site, is a thymidine residue. Thymidine was chosen as the linking site because thymidine and uridine nucleosides show approximately 1015-fold greater reactivity toward photoadduct formation with 8-methoxypsoralen, 3-carbethoxypsoralen, and oligomers derivatized with (ae)AMT than does cytosine (12,31, 32). In addition to e x v i n i n g the relative reactivities of pyrone versus furan adduct forming psoralen derivatives, we also wished to examine the effect of the position of the psoralen-linking site in the target on the kinetics and extent of cross-linking. Our previous studies with (ae)AMT-derivatized oligomers showed that the crosslinking reaction was mainly dependent upon the ability of the oligomer to bind to its complementary target sequence and upon the fidelity of the binding interaction (11-13). However, it also appeared that the location of the psoralen-linking site in the target and the sequence of the nucleotides surrounding the psoralenlinking site were important determinants of the extent of the cross-linking reaction. In order to further investigate these possibilities, we have prepared the DNA target systems shown in Figure 2. In these systems, the psoralen-linking site is a T residue which is located a t the n + 1,n,or n - 1position of the target strand. Each oligomer should form equally stable duplexes with the target having five G-C base pairs and four A-T base pairs. Therefore any differences in the extent of cross-linking should reflect the change in the psoralen-linking site and not the stability of the oligomer-target duplex. Cross-linking reactions were convenientlyfollowed using polyacrylamide gel electrophoresisunder denaturing conditions by observing the shift in mobility of the 32P-labeled target DNA (see Figure 3). The rates and yields of cross-linking of the (ae)CP- and (ae)AMT-oligomers after 30 min of irradiation are summarized in Figure 2. Both (ae)CP- and (ae)AMT-oligomers undergo extensive photoadduct formation when bound to their complementary targets. The highest yields of cross-linking for both oligomers are observed to the n + 1target a t 4 “C. Thus for these two psoralens, photoadduct formation through the pyrone side or the furan side occurs with equal facility in this target system. Comparative studies with 8-methoxypsoralen, 5-methoxypsoralen, 5methylisopsoralen, and 3-carbethoxypsoralen have shown that the extent of photoadduct formation is lowest for 3-carbethoxypsoralen (33).This low reactivity may result from the negligible association constant of the 3-carbethoxypsoralen with DNA and to the rapid photodegradation of the compound (31).It appears that these problems are overcome when 3-CP is attached to the oligomer. In this arrangement,formation of a duplex between the oligomer and its target brings the psoralen ring into close proximity with its binding site, thus enabling the psoralen to productively bind and react with a thymidine residue in the linking site. Although the yield of photoadduct formation is high a t 4 O C , it decreases dramatically for both oligomers when the temperature of the reaction is raised to 30 OC. The melting temperature of the duplex formed between d-
Bioconjugafe Chem., Vol. 1, No. 1, 1990 87
Psoralen-Derivatized Oligonucleoside Methylphosphonates ,
-
cc
c
&
d-PGGGAGGACGNN nNGCACACGAG
7
t
-
d-pGGGAGGACGi NNGCACACGAG CTCCTGCX Y
Figure 4. Schematic representation of the partial (upper)and
full (lower) intercalation modes of the psoralen ring (represented by the rectangle) when psoralen-derivatized oligomers interact with their target DNAs. TpTCGTCCTC and the target 21-mer is 33 “C at a total strand concentration of 2 pM. A somewhat higher melting temperature would be expected for the oligomertarget duplex under conditions of the cross-linking experiments due to the higher concentration of the oligomer in the reaction mixtures. Thus this oligomer-target duplex would be expected to begin to melt at 30 “C. Examination of molecular models suggests that upon oligomertarget duplex formation, the psoralen ring can assume two different types of intercalated conformations as illustrated schematically in Figure 4. In order for crosslinking to occur when the psoralen linking site is in the n + 1 position, the psoralen ring must stack on the terminal base pair and intercalate between this base pair and the T residue at the n 1 position. In this partial intercalation mode the alignment of the psoralen ring with the T residue would be expected to be sensitive to thermal perturbation and the effects of “end fraying” when the temperature of the duplex is raised. Under these conditions, the increased thermal motions imparted in the psoralen ring and the target would lead to reduced cross-linking at higher temperatures. The greater reduction in cross-linking for d-(ae)CPpTpTCGTCCTC versus d-(ae)AMTpTpTCGTCCTC may reflect the steric constraints imposed by the amide linkage on partial intercalation by the carbamoylpsoralen group. In contrast to cross-linking at the n + 1 position, the rates and yields of cross-linking at the n and n - 1 positions are essentially the same at both 4 and 30 “C for both types of derivatized oligomers. The cross-linking reaction for both the n and n - 1 targets continues over the 30-min irradiation period. This behavior is in sharp contrast to that seen with the n + 1target. In this case photoadduct formation increases linearly for the first 10 min of irradiation and then stops. In the case of the n type target, cross-linking could occur when the psoralen is in either the partial-intercalation mode or in the full-intercalation mode, whereas cross-linking to the n - 1 target must occur via the fullintercalation mode. Duplexes formed when the psoralen is in the full-intercalation conformation would be expected to be less subject to thermal “end fraying” due to the added stability provided by the intercalated psoralen. The observation that cross-linking is not affected by increases in temperature for either the n and n - 1 target systems suggests that the psoralen cross-links when it is in the full-intercalation mode. The greater extent of cross-linking by d-(ae)CPpApTCGTCCTC with the n target versus that of d(ae)CPpTpACGTCCTC with the n - 1 target suggests that photoadduct formation occurs more readily to the 5’-face of the thymine in the n position than from the 3’-face of thymidine in the n - 1 position. This preference may result from restrictions on the conformational
+
flexibility of the intercalated psoralen ring imposed by the rigid carboxamide group of the linker arm of the (ae)CP-oligomers. It appears that such restrictions are less serious for the (ae)AMT-oligomers, which crosslink to the thymidine with equal efficiency in both the n and n - 1 targets. Such behavior is consistent with the expected greater flexibility of the aminoethyl linker arm of the (ae)AMT-oligomers. It is interesting to note that the psoralens when attached to the oligomers appear to show a preference for 3‘-ApT binding sites in contrast to the behavior of free psoralens. The extended kinetics of cross-linking observed for both the (ae)CP- and (ae)AMT-oligomers in the n and n - 1 targets is also consistent with the full-intercalation mode of psoralen binding. Free-psoralen derivatives undergo photodegradation in solution which leads to a loss of their ability to form photoadducts (5,23,32). It appears that psoralen is protected from such photodegradation when it is intercalated with nucleic acid bases (5,23). The ability of the oligomers to continue to photocross-link with the n and n - 1 targets over a prolonged period of irradiation suggests that the psoralen rings of the oligomer are protected from photodegradation as a result of intercalation. On the other hand, partial intercalation of the psoralen ring results in less protection from photodegradation and thus the cross-linking reaction is over after 10 min of irradiation. Furthermore, the results suggest that psoralen-derivatized oligomers, once bound to their targets, do not exchange freely with unbound oligomer. The results of our experiments show that both (ae)CPand (ae)AMT-oligomers can cross-link efficiently with target single-strand DNA and that the extent of crosslinking can be controlled by selecting suitable psoralenlinking sites. In the case of (ae)CP-oligomers, efficient cross-linking occurs when the psoralen-linking site is located at either the n or n + 1 position of the target DNA. For (ae)AMT-oligomers the most efficient crosslinking occurs when the psoralen-linking site is located a t the n 1 position. Experiments are currently underway with single-stranded oligoribonucleotide targets in order to assess the parameters which affect cross-linking of psoralen-derivatized methylphosphonate oligomers to cellular RNAs. A better understanding of the factors which affect the interactions of photoreactive methylphosphonate oligomers with target nucleic acids are essential to designing antisense oligomers which can be used to study gene expression in mammalian cells.
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ACKNOWLEDGMENT This work was supported by grants from the National Institutes of Health (Grant GM 39127), the National Cancer Institute (Grant CA 42762), and the Department of Energy (Grant DE-FG02-88ER60636). LITERATURE CITED (1) Ben-Hur, E. (1984) The photochemistry and photobiology
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