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PNA-Nitrogen Mustard Conjugates Are Effective Suppressors of HER-2/neu and Biological Tools for Recognition of PNA/DNA Interactions Zhanna V. Zhilina,† Amy J. Ziemba,† Peter E. Nielsen,‡ and Scot W. Ebbinghaus†,* Arizona Cancer Center, University of Arizona, 1515 North Campbell, 85724, Tucson, Arizona, and Department of Medical Biochemistry and Genetics, The Panum Institute, University of Copenhagen, Blegdamsvej 3c, 2200 Copenhagen N, Denmark . Received October 7, 2005
Peptide nucleic acids (PNAs) are promising tools for gene regulation. One of the challenges of using PNAs as gene regulators is the need to optimize the efficiency of interaction with critical sequences of DNA. To improve the efficiency of binding between PNAs and the HER-2/neu promoter, mono- and bis-pyrimidine-rich PNAs were conjugated to a nitrogen mustard at either the amino or carboxy terminus. Gel shift analysis demonstrated that conjugation to an alkylating agent slowed PNA binding and favored PNA:DNA:DNA triplex helix formation while preserving a high binding affinity. Sites of DNA alkylation were visualized by piperidine cleavage and showed PNA binding first by Hoogsteen bond formation with the target duplex to form a stable PNA:DNA:DNA triplex structure which is later converted to a PNA:DNA:PNA triple helix by strand invasion and Watson-Crick base pairing by a second PNA molecule. In this way, PNA-directed DNA alkylation was used to deduce the mode of PNA binding. Transient transfection experiments demonstrated that the PNA-nitrogen mustard conjugates suppressed HER-2/neu expression by up to 80%. In comparison with an unmodified mono-PNA or a bis-PNA, these results indicate that the covalent adducts stabilized PNA binding in cells and suggest that the conjugation of PNAs to nitrogen mustards is a robust strategy for developing antigene PNA oligonucleotides to prevent transcription.
INTRODUCTION Antigene and antisense strategies with peptide nucleic acids (PNAs)1 have attracted a great deal of attention in medicinal and biological chemistry (1-5). The ability of oligonucleotides to recognize specific DNA or RNA sequences affords a versatile approach to control gene expression, but native DNA or RNA oligomers are susceptible to endogenous nucleases and lack the membrane permeability necessary for many in vivo applications. As a result, there has been considerable interest in the development of synthetic structures that specifically recognize and bind selectively to DNA, possess increased resistance to nuclease digestion, and optimize membrane permeability (6-8). Peptide nucleic acids are DNA mimics in which the sugar-phosphate backbone has been substituted by N-(2-aminoethyl)glycine units; therefore, PNAs possess a neutral backbone, which provides a unique advantage for hybridization relative to DNA or RNA oligomers (9-11). PNAs can bind to complementary sequences by Watson-Crick base pairing, and are able to discriminate single base mismatches. Binding is characterized by high affinity and high rates of association, together giving PNAs the ability to disrupt double stranded DNA by forming a DNA-PNA strand-invasion complex. PNAs are not substrates for endogenous nucleases or proteases, suggesting that they will be highly active in vivo (12-14). The inhibition of gene transcription by PNAs presents a series of technical challenges. One of them is the need to optimize the efficiency of DNA binding to critical sequences within gene * To whom correspondence should be addressed: E-mail:
[email protected]; phone (520) 626-3424; fax (520) 6265462. † University of Arizona. ‡ University of Copenhagen. 1 Abbreviations. PNA, peptide nucleic acid; TFO, triplex forming oligonucleotide, PAM, phenyl acetic acid mustard; Chl, chlorambucil.
promoters, which might be accomplished by modifying the PNA via terminal conjugation of a small molecule. Different chelating and intercalating agents were successfully conjugated to PNAs in order to enhance the duplex invasion potency of the PNAs and increase the stability of the PNA-DNA complex (15-20). However, there are no reports on PNA terminal modification with small molecules resulting in more effective downregulation of gene expression. We postulated that PNA conjugation to a DNA alkylating agent would significantly improve gene regulation by stabilizing PNA binding to a target sequence. In this report we present the synthesis of a PNA-alkylator conjugate, using a short, symmetric PNA and a nitrogen mustard as an alkylating agent. The HER-2/neu oncogene is overexpressed in a number of human cancers, and its overexpression correlates with poor clinical outcome (21-23). The results of clinical studies with HER-2/neu-directed therapies demonstrate that preventing the expression or function of the HER-2/neu gene product can induce the regression of metastatic tumors that overexpresses HER-2/neu (24, 25). The HER-2/neu oncogene is a good target for antigene oligonucleotide strategies because of the presence of polypurine tracts capable of triplex formation with triplexforming oligonucloetides (TFOs) that can prevent in vitro transcription (26, 27) and transcription factor binding (28), and reduce HER-2/neu mRNA levels in a breast cancer cell line (29). The HER-2/neu promoter was successfully used for evaluating triplex formation with TFO-alkylator or TFO-minor groove binder conjugates (30-32) and strand invasion complex formation with PNAs (33). The polypurine tract is located from -218 to -245 upstream of the first codon (+1) in a region of the HER-2/neu promoter (Figure 1) that serves as an alternate transcription initiator that can function independently of the TATA box (34). Nitrogen mustard derivatives are widely used in chemotherapy (35-37). The biological activity of these agents is usually
10.1021/bc0502964 CCC: $33.50 © 2006 American Chemical Society Published on Web 01/04/2006
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PNA−Nitrogen Mustard Conjugates for HER-2/neu
Figure 1. Schematic representation of the HER-2/neu promoter target sequence and two PNA molecules forming a strand invasion complex. The target sequence is a 9 bp purine/pyrimidine tract from -220 to -228 upstream of the first codon. Dots represent Hoogsteen hydrogen bonds and vertical lines represent Watson-Crick bonds.
mediated through alkylation at N-7 of guanine and subsequent DNA interstrand cross-linking (38-40). However, covalent binding can occur at multiple DNA sites, bringing nonspecific toxicity; moreover, the efficiency of the nitrogen mustards is limited because of deactivation by hydrolysis or due to reaction with nucleophilic molecules other than DNA. Nitrogen mustards have been conjugated to TFOs in order to direct highly specific DNA alkylation (41, 42). Because PNAs are highly resistant to degradation by a variety of enzymes, we began investigating them as candidate antigene molecules for suppression of HER2/neu expression. We initially postulated that PNA modification with an alkylating agent would not only stabilize the DNA/ PNA complex but might also facilitate strand invasion by stabilizing Hoogsteen strand during the process of disrupting the duplex by a second PNA strand. Furthermore, PNAalkylator conjugates can be used as tools to study the binding mechanism between DNA and PNA by positioning the alkylating moiety at different PNA ends and analyzing the sites of interaction with the target duplex. In this report we describe the design and synthesis of novel bioconjugates of the nitrogen mustards (phenylacetic acid mustard (PAM) and chlorambucil (Chl)) to a PNA, demonstrate site-specific DNA alkylation with the target site in the HER2/neu promoter, and show strong downregulation of HER-2/ neu promoter activity in reporter gene assays.
EXPERIMENTAL PROCEDURES Materials. Solvents and chemicals obtained from commercial sources were analytical grade or better and were used without further purification. Solvents for HPLC analysis were obtained as HPLC grade and were filtered (0.2 µM) prior to use. PNA VII (see Table 1 for sequence information) was synthesized according to published procedures (43, 44). PNAs I, III, V, and IX were purchased from BioSynthesis, Inc. (Lewisville, TX) on a 2 µM scale and obtained on solid support prior to final deprotection for further modification. A small portion of the PNAs (about 1/10 of the provided PNAs on resin) was cleaved from the solid support before modification for analysis. PNAs were cleaved from the solid support by mixing the resin with 300 µL of cleavage cocktail (95% TFA, 5% m-cresol). The mixture was agitated for 90 min, filtered off, and precipitated with cold diethyl ether, followed by HPLC purification. All PNAs were 95% pure or greater by C18 HPLC. Pentafluo-
rophenyl esters of phenylacetate mustard (PFP-PAM) and chlorambucil (PFP-Chl) were synthesized in our group as previously described (45). Spectral Analysis. Matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectra were acquired using a Micromass (Manchester, UK) MALDI-LR TOF mass spectrometer and Applied Biosystems Voyager DE-STR (Framingham, MA), operating a 337 nm nitrogen laser. Samples were mixed with an equal volume of a saturated R-cyano solution in 50% acetonitrile/50% water containing 0.1% trifluoroacetic acid (TFA), and 1 µL was spotted on the target plate and allowed to air-dry prior to mass analysis. Mass spectra acquired using the Micromass mass spectrometer were collected in Reflection mode with a 15000 V source voltage using a 0.5 ns sample period. The reflection voltage was set to 2000 V. The detector was held at 1800 V and 5 laser shots at 3 Hz were combined per mass spectrum recorded. Mass spectra acquired using the Applied Biosystems MALDI-TOF mass spectrometer were collected in positive reflection mode with an acceleratory voltage of 20000 V. The grid voltage was set at 66% with an extraction delay time of 275 ns. 400 laser shots at 20 Hz were combined per mass spectrum recorded. All modified and unmodified PNAs were analyzed by reverse phase HPLC using a VYDAC C18 250 × 10 mm column with a gradient of 0 to 80% solvent B over 40 min. Solvent B was 0.1% TFA in acetonitrile (flow rate 4 mL/min), solvent A was 0.1% TFA in water. Detection was carried out by UV absorbance at 260 nm. Preparative HPLC purification was performed on Varian proStar system equipped with a photodiode array (PDA) detector. Synthesis of PNA-Mustard Conjugates. Mono-PNA5′PAM Conjugate (PNA II). The mono-PNA (I) was obtained on solid support with the free 5′ (N-terminal) amino group but all other side chain protecting groups in place and was incubated in 300 µL of dry dichloromethane (DCM) for 2 h, then the resin was filtered and washed with DCM (2 × 2 mL). PFP-PAM (2.5 mg, 6 µmol) was dissolved in 150 µL of DCM and mixed with 150 µL of dry dimethylformamide (DMF) and 50 µL of triethylamine (Et3N). The resulting mixture was added to the resin and bubbled through with argon for 3 h. The resin was filtered, washed with DMF (2 × 2 mL), DCM (2 × 2 mL), and dried under vacuum for 2 h and then overnight in a desiccator. The resulting PNA-5′PAM conjugate was then cleaved from the resin by using 500 µL of TFA/m-cresol cleavage cocktail as described for unmodified PNAs, HPLC purified (tR ) 19.4 min) and lyophilized. MALDI-TOF MS analysis for PNA II (C123H170Cl2N48O37): calculated 2982.9; found 2983.5. Mono-PNA-3′Chl Conjugate (PNA IV). The mono-PNA (III) was obtained on the solid support with standard protecting groups on the 5′ terminus and side chains except for a lysine at the 3′ end that was protected with methoxytrityl (Mmt) for selective deprotection. PNA IV had a cysteine (S) at the C-terminus to separate the target lysine from the solid support and provide a potential site for additional conjugation. The resin was incubated and washed with DCM as described for PNA I.
Table 1. PNA Analysis by MALDI-TOF and HPLCa name
description
sequence
Mcalcd.
Mobsd.
tR, min
I II III IV V VI VII VIII IX
mono-PNA PNA-5′PAM PNA-SH PNA-3′Chl bis-PNA(4K) bis-PNA(4K)-5′Chl bis-PNA(J) bis-PNA(J)-5′Chl bis-PNA
CTCCTCCTC-O-KK PAM-CTCCTCCTC-O-KK CTCCTCCTC-O-KK-S CTCCTCCTC-O-KK(Chl)-S KK-TCCTCCTCC-OOO-CCTCCTCCT-KK Chl-KK-TCCTCCTCC-OOO-CCTCCTCCT-KK KK-TCCTCCTCC-OOO-JJTJJTJJT-KSG Chl-KK-TCCTCCTCC-OOO-JJTJJTJJT-KSG TCCTCCTCC-OOO-K-CCTCCTCCT
2724.5 2982.9 2848.7 3098.9 5577.69 5847.9 5609.7 5881.7 5193.2
2725.3 2983.5 2850.9 3100.6 5577.16 5847.03 5610.2 5882.4 5193.9
14.8 19.4 15.5 17.2 15.1 16.22 15.1 18.64 18.2
a C, T, J ) cytosine, thymine, and pseudoisocytosine PNA residues; O ) linker; K, S, G ) lysine, cysteine, and glycine amino acid residues; PAM ) phenylacetic acid mustard; Chl ) chlorambucil.
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To remove the Mmt protecting group, the resin was treated with 3% TFA in DCM (2 × 200 µL for 10 min each) and washed with DCM and DMF (2 × 2 mL) between and after each treatment. PFP-Chl (2.6 mg, 6 µmol) was dissolved in 150 µL of DCM and mixed with 150 µL of DMF and 50 µL of Et3N. The resulting mixture was added to the resin and bubbled through with argon for 3 h. The resin was filtered and washed with DMF (2 × 2 mL) and DCM (2 × 2 mL). The PNA was then deprotected with 200 µL of piperidine/DMF (1/4 v/v) for 30 min. The resin was filtered, washed with DMF (2 × 2 mL) and DCM (2 × 2 mL), dried under vacuum for 2 h, and left overnight in a desiccator. The resulting PNA-3′Chl conjugate (IV) was cleaved from the resin with TFA/m-cresol, HPLC purified (tR ) 17.2 min) and lyophilized as described above. MALDI-TOF MS analysis for PNA IV (C127 H177Cl2N49O38S): calculated 3098.9; found 3100.6. Bis-PNA-5′Chl Conjugates (PNAs VI and VIII). The unmodified bis-PNAs (V and VII) were obtained on solid support with the free 5′ (N-terminal) amino group but all other side chain protecting groups in place. They were conjugated to Chl at the 5′ end as described above for PNA II, except that PFPChl (2.6 mg, 6 µmol for each PNA) was used in place of PFPPAM. After synthesis, the conjugates were HPLC purified (PNA VI tR ) 16.22 min; PNA VIII tR ) 18.64 min) and lyophilized. MALDI-TOF MS analysis for PNA VI (C241H339Cl2N97O74): calculated 5847.9; found 5947.03. MALDI-TOF MS analysis for VIII (C240H360Cl2N97O75S): calculated 5881.7; found 5882.4. Electrophoretic Mobility Shift Assay (EMSA) Analysis of DNA/PNA Interaction. PNAs were incubated with a 36-base pair HER-2/neu duplex target sequence end-labeled on the pyrimidine strand. Reaction mixtures including increasing concentrations of the PNA with 0.01 µM target duplex in NaPO4 buffer (10 mM, pH 6.5) were incubated for 24 h. The complexes were separated on a 10% nondenaturing gel at room temperature with 1× TAE (pH 6.5) in both the gel and running buffer and visualized by autoradiography. The rate of PNA binding was analyzed by adding 0.1 µM PNAs and 36-base pair HER-2/ neu duplex target sequence (0.01 µM) end-labeled on the purine strand and stopping the reactions by freezing the samples at -80 °C at various time points from 1 to 18 h. The products were then separated on nondenaturing gels and visualized by autoradiography. Piperidine Cleavage. A 250 base pairs (bp) Pst/XmaI fragment of the pGL3/HNP410 plasmid containing a 410 bp HER-2/neu promoter in the pGL3 basic luciferase plasmid (Promega) was used to create an end-labeled template by Klenow filling. DNA/PNA complex formation was performed by incubating 0.02 µM of labeled promoter fragment with 2 concentrations of PNA-5′PAM and PNA-3′Chl (0.1 and 1.0 µM) for 2, 4, 8, and 18 h in a final volume of 10 µL of 10 mM NaPO4 (pH 6.5) at 37 °C. Reactions without PNAs were performed to show nonspecific cleavage sites of the radiolabled template by piperidine. Piperidine cleavage reactions were separated on a 10% denaturing gel and subjected to autoradiography. Maxam-Gilbert G-reactions were performed to identify alkylated guanine residues, and these reactions were run alongside the piperidine cleavage lanes as indicated in Figures 4 and 5; however, images from different autoradiographs were combined into composite figures in these illustrations in order to optimize the intensity of the exposure between the piperidine cleavage lanes and the G-reactions. Transient Transfection Analysis. HeLa cells were obtained from the American Type Culture Collection and cultured as recommended by the supplier. The pGL3/HNP410 plasmid (2 µg) containing the HER-2/neu promoter was incubated in 10mM NaPO4 (pH 6.5) at 37 °C with 1 and 10 µM of PNAs for 18 h. Transfection mixtures included the pGL3/HNP410 plasmid (with
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or without PNAs), 20 ng of an internal control plasmid (pRL/ SV40, Promega), and 2 µL of Lipofectamine 2000 (Invitrogen). HeLa cells were incubated with the transfection mixture, cell lysates were obtained at 24, 48, and 72 h, and dual luciferase assays (Promega) were performed. All experiments were repeated three times, normalized for transfection efficiency, and are presented as a percentage of luciferase activity from plasmid without PNA treatment.
RESULTS Design of the PNA-PAM Conjugates. Our previous results on targeting a polypurine tract in the HER-2/neu promoter with TFOs demonstrated that unmodified TFOs easily dissociated from the target sequence when transfected into tumor cells, while TFOs modified with an alkylating agent prevented helicase activity and suppressed HER-2/neu driven luciferase activity in cells for up to 72 h (31). We postulated that PNAs similarly modified with an alkylating agent would improve the stability of the PNA/DNA complex and improve the PNAs ability to prevent HER-2/neu expression. To simplify synthesis and product analysis, we decided to start with short mono-PNAs, and designed a mono-PNA (I) to target the HER-2/neu polypurine tract from -220 to -228 (Figure 1). This PNA, CTCCTCCTC, has perfect mirror symmetry and was designed to be able to simultaneously bind to the target sequence by Hoogsteen bonding (triplex formation) in parallel to the purine rich strand and by Watson-Crick bonding (strand invasion) in antiparallel orientation to the purine strand. To increase PNA solubility and strand invasion, two lysine residues were added to the C-terminus of the PNAs through a single 8-amino-2,6dioxaoctanoic acid (O) linker (Table 1). To conjugate the N-terminus of PNA I to the drug, we used the R-amino group of the peptide backbone of the last nucleic base in the PNA I sequence. To modify the C-terminus, we used the -amino group of one of the lysines of PNA III. That -amino group was protected with an acid-labile monomethoxytrityl (Mmt) protecting group. Deprotection of the Mmt group can be done with mild acidic conditions (3% TFA in DCM) while keeping nucleic bases, the other lysine side chain, and the N-terminus protected to create PNA-drug conjugation only at C-terminus (46). PNA III was also designed with a cysteine residue at the C-terminus that separated the target lysine from the solid support for the conjugation reaction and could eventually be used as a site of further conjugation, for example with a carrier peptide. For these studies we selected two commonly used nitrogen mustards, phenyl acetic acid mustard (PAM) and chlorambucil (Chl). To demonstrate the feasibility of the conjugation between PNA and alkylating agent, we started with commercially available chlorambucil, which was easily modified with PFP for conjugation in one step (45). PAM has a longer hydrolysis half-life but is slightly less reactive compared to chlorambucil. In this way we could evaluate the ability of two reactive nitrogen mustards conjugated to the PNA to direct site-specific DNA alkylation and regulate gene expression. Conjugation of the Nitrogen Mustards to PNAs. The conjugation of the nitrogen mustard to the PNAs was done manually on solid support using an acylation reaction with the active ester of the nitrogen mustard and a free amino group of the PNA. Pentafluorophenyl esters are widely used in reactions with free amino groups to give amide bonds, and we synthesized the pentafluorophenyl (PFP) ester of phenylactate mustard and chlorambucil according to previously published methods (45). A mixture of anhydrous solvents was used for the coupling, with DMF as a traditional solvent for coupling in peptide synthesis on solid support, DCM as a more suitable solvent for the highly lipophilic PFP-PAM, and an excess of anhydrous triethylamine as an activating agent. The anhydrous conditions
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Scheme 1. Synthesis of PNA-5′PAM (II)
Scheme 2. Synthesis of PNA-3′Chl (IV)
Figure 2. HPLC monitoring of the PNA-PAM conjugation reaction and MALDI-TOF spectra of conjugate II. The top chromatogram shows unconjugated mono-PNA (I), tR) 14.8 min. The bottom chromatogram shows the reaction mixture with a new peak of the PNA-5′PAM conjugate (II), tR) 19.4 min.
protect the nitrogen mustard from hydrolysis, and coupling requires only a small excess of PFP-PAM. PNA I on solid support had a free amino group at the N-terminus (Scheme 1) and was ready for conjugation unlike PNA III, which needed additional preparation for the coupling (Scheme 2). To put the drug molecule specifically on the C-terminus and avoid side reactions, we applied the strategy of selectively deprotecting a lysine bearing a labile protecting group, specifically an Mmt group on the -amine of the lysine at the C-terminus of PNA III. Mmt is a well-known acid-sensitive, trityl-type protecting group that can be removed with 3% TFA in DCM. The mild conditions allowed us to deprotect only the desired -amino group of the lysine at the C-terminus and proceed with coupling at this position. After the coupling reaction (3 h), the resin was dried and then cleaved using standard conditions. HPLC chromatograms (Figure 2) of the coupling reaction mixtures demonstrated the peak of unmodified PNA (tR 14-15 min) and a smaller peak of conjugated product (tR 17-19 min), the estimated yield was 20-40%, and prolonging the coupling reaction up to 18 h did not improve the yield. The conjugates
were characterized by mass spectral analysis, confirming the expected molecular weights (Table 1). Analysis of PNA-Nitrogen Mustard Binding by EMSA. To evaluate the influence of the alkylating agent on the efficiency, mode, and rate of mono-PNA binding, PNA I (unmodified) was compared to PNA II (5′ PAM conjugate) by EMSA. The PNAs were incubated with a synthetic 36-mer duplex target sequence end-labeled on the pyrimidine strand in order to show displacement of the pyrimidine strand (Figure 3A). To monitor the rate of PNA binding, PNAs were incubated with the synthetic 36-mer duplex target end-labeled on the purine strand at a fixed concentration of the PNA, and reactions were stopped at various time points (Figure 3B). PNA I and PNA II showed similar affinities for the target duplex at pH 6.5, producing a transition of the target duplex to single stranded DNA and slow mobility complexes at 100 nM PNA concentrations, with complete disappearance of the duplex band at 1 µM (Figure 3A). PNAs and particularly covalently bound PNAnitrogen mustard conjugates are not expected to dissociate from DNA in solution, so that equilibrium dissociation constants are not practical. C50 values (PNA concentration leading to 50% duplex DNA binding) can be used to estimate and compare the affinity of PNAs for duplex DNA under given experimental conditions, and C50 values for both PNA I and II would be 50% DNA binding (shift in the duplex band to single stranded DNA and slow mobility PNA:DNA complexes at 100 nM PNA). Conjugation to a nitrogen mustard did not improve the pH dependent binding by the cytosine-rich PNA, and although binding was less efficient for both PNAs at pH 7.0, it was similar for PNA I and PNA II, again slightly favoring PNA I (not shown). Interestingly, when the efficiency of PNA binding was evaluated over time, the presence of the nitrogen mustard on the PNA slowed binding to duplex DNA and appeared to favor binding by PNA(DNA)2 triplex formation (Figure 3B). Both PNA I and II probably first bind by triplex formation with duplex DNA, forming PNA(DNA)2 triplexes (seen as a shift in the duplex to a slower mobility complex at 100 nM PNA in
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Figure 3. PNA binding to a synthetic duplex target sequence by EMSA. Representative native EMSAs comparing an unmodified monoPNA (PNA I) to the same PNA conjugated to PAM at the 5′ terminus (PNA II). (A) Increasing concentrations of PNAs were mixed with duplex DNA radiolabeled on the pyrimidine strand for a fixed incubation time of 18 h at pH 6.5. (B) Fixed concentrations of the PNAs (100 nM) were mixed with duplex DNA radiolabeled on the purine strand for increasing incubation times at pH 6.5.
Figure 3A and 3B, accompanied by partial displacement of the pyrimidine strand in Figure 3A). Higher PNA concentrations (Figure 3A) or longer incubation times (Figure 3B) resulted in a further shift of the duplex to even slower mobility complexes (Figure 3A and 3B), accompanied by complete displacement of the pyrimidine DNA strand (Figure 3A), which is a characteristic of strand invasion and (PNA)2DNA triplex formation with the binding of a second PNA molecule. Piperidine Cleavage Analysis of Site-Directed DNA Alkylation by PNA-Nitrogen Mustard Conjugates. To determine the DNA alkylation sites and deduce the orientation of PNA-alkylator conjugate binding, piperidine cleavage was used. Sites of DNA alkylation can be visualized by converting these sites into single strand breaks with piperidine and heat as previously described (30). A 250 base pair promoter fragment was end-labeled on the purine-rich strand and incubated with 0.1 and 1 µM of 5′ and 3′ mono-PNA-alkylator conjugates (PNAs II and IV) over increasing amounts of time. The cleavage products were resolved on a sequencing gel and aligned with a G reaction to determine the alkylated bases. PNA II (Figure 4A) demonstrated highly specific covalent adduct formation with G-229 with 0.1 µM of the conjugate, consistent with binding in a parallel orientation with Hoogsteen bonds to the duplex to direct the alkylation to the duplex/triplex junction. At higher concentrations and after prolonged incubation, alkylation of several additional guanines was observed, most prominently G-219, consistent with strand invasion and PNA binding in antiparallel orientation to the purine strand during the formation of a triplex invasion complex. Minor alkylation sites at G-241, G-238, and G-231 were also observed, probably due to binding of the PNA to less ideal binding sites in the highly repetitive HER-2/neu polypurine tract; for example, the PNA could bind to a single mismatch target sequence from -232 to -240, placing the alkylator at G-241 and G-231 at the junction of the duplex/triplex junction.
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For PNA IV (Figure 4B), the major alkylation site is G-219, again consistent with the conjugate binding in parallel orientation to dsDNA through Hoogsteen bond formation. Minor alkylation at G-232 is most likely due to strand invasion and the influence of the spacing betweent the last PNA base and the alkylator moiety. In PNA IV the Chl is coupled to the -amino group of the second lysine residue and thus separated from the most 3′ PNA base by the 2,6-dioxaoctanoic acid linker, two amino acid residues, and the lysine side chain, resulting in a flexible, 18 atom linker between PNA and Chl. Alternately, the PNA could potentially bind (with less efficiency) to a single mismatch target sequence from -232 to -240, placing the chlorambucil in the proximity of G-232, but alkylation of a guanine in the PNA: DNA complex (G-232) would not be expected, since the N7 of this guanine would not be accessible to the Chl. Unconjugated PNAs did not demonstrate any cleavage sites (not shown). In both cases, the estimate of the alkylation efficiency at major sites was at least 80%. Figures 4C and 4D illustrate the deduced modes of binding and site-directed DNA alkylation by 5′ and 3′ PNA-alkylator conjugates, based on the major alkylation sites observed by piperidine cleavage. We recently reported that a bis-PNA (PNA IX) composed of two linked 9-mers with (TCC)3 sequences could target the HER2/neu PPT at two adjacent (GGA)3 or (AGG)3 sequences (33). Because of the repetitive nature of the target sequence, this bisPNA could bind in different orientations, depending on which of the PNA arms contributes to Hoogsteen binding versus Watson-Crick binding. If the N-terminal arm was the Hoogsteen strand, the bis-PNA would prefer to target 5′(AGG)3 sequences with the linker oriented 3′ to the purine strand of the target sequence, whereas if the C-terminal arm was the Hoogsteen strand, the bis-PNA would prefer to target 5′(GGA)3 sequences with the linker oriented 5′ to the purine strand (Figure 5A). We therefore synthesized bis-PNA-alkylator conjugates and probed for the orientation of bis-PNA binding by piperidine cleavage. PNA V is a bis-PNA with linked (TCC)3 arms, and PNA VII is an analogous bis-PNA with J-base substitutions in the C-terminal PNA arm [(TCC)3-(TJJ)3] to favor Hoogsteen binding by this arm of the bis-PNA at neutral pH. Compared to the PNA we previously studied (PNA IX), these PNAs were designed with additional lysine residues to enhance solubility and improve DNA binding. Both PNAs were conjugated to chlorambucil at their N-termini (PNAs VI and VIII). PNA V and VI could bind to adjacent (AGG)3 sequences in the HER2/neu PPT from -231 to -239 and from -219 to -227 if the N-terminal PNA arm serves as the Hoogsteen strand of the bisPNA. Piperidine cleavage with PNA VI (Figure 5B) shows major alkylation products at G-228 and G-241, which is consistent with binding in this orientation, placing the alkylator at the guanines immediately adjacent (G-228) or near (G-241) the PNA/DNA junction (Figure 5D). PNAs VII and VIII would prefer to bind to the (GGA)3 sequence from -221 to -229, using the C-terminal PNA arm bearing J-base substitutions as the Hoogsteen strand of this bisPNA. Piperidine cleavage with PNA VIII (Figure 5B) confirms this binding orientation with major alkylation products at G-218 and G-219, consistent with PNA binding to the HER-2/neu PPT from -221 to -229, placing the alkylator near the PNA/DNA junction (Figure 5C). An additional alkylation site at -231 is probably explained by PNA binding to an adjacent, single mismatch (GGA)3 sequence from -233 to -241, placing the alkylator at G-231 near the PNA/DNA junction. With TFO-nitrogen mustard conjugates, we and others have observed that guanine adducts form most efficiently with a guanine on the purine strand immediately adjacent to the triple helix (30, 31, 47). It is interesting to note that some of the PNA-
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Bioconjugate Chem., Vol. 17, No. 1, 2006 219
Figure 4. Representative piperidine cleavage gels demonstrating sites of alkylation for (A) the PNA-5′PAM conjugate (II) and (B) the PNA3′Chl conjugate (IV). A ∼250 bp restriction fragment containing the HER-2/neu promoter was labeled on the 3′ end on the purine strand and incubated with 0.1-1 µM PNA for 2-18 h. Maxim-Gilbert G-reactions with the same template were used to identify the alkylation sites. Major alkylation sites are observed at G-229 for the 5′ alkylator conjugate (PNA II) and G-219 for the 3′ alkylator conjugate (PNA IV). Schematic representations of PNA-alkylator conjugate binding and covalent adduct formation deduced from piperidine cleavage analysis for PNA II (C) and PNA IV (D). The DNA:DNA:PNA triplex forms first and directs the nitrogen mustard to form a covalent adduct with the guanine adjacent to the triple helix, producing the major alkylation sites at G-229 for PNA II or G-219 for PNA IV. With either higher concentrations of the PNAalkylators or prolonged incubation times, the PNA:DNA:PNA strand invasion complex is formed, producing the minor alkylation sites at G-219 for PNA II and G-232 for PNA IV. The linker in PNA IV allows the nitrogen mustard to reach G-232, 4 bp upstream of the strand invasion complex. Black circles represent the nitrogen mustards, and the guanines they bind are circled with open circles.
directed guanine adducts with PNAs IV, VI, and VIII are not immediately adjacent to the PNA/DNA junction. For example, it is surprising that PNA VIII does not form a guanine adduct with G-220, immediately adjacent to the PNA/DNA junction, but prefers G-218 and G-219. These findings are most likely explained by the spacing between the chlorambucil and the last PNA base, separated by two amino acid residues in PNA VI and VIII, and by the longer 18 atom linker in PNA IV. In contrast, the major and minor alkylation sites with PNA II, in which the PAM is conjugated to the R-amino group of the PNA base at the 5’terminus, occur immediately adjacent to the PNA binding site. Inhibition of HER-2/neu Transcription. To determine whether conjugation with a nitrogen mustard can improve the antigene activity of a PNA, transient transfection analysis was performed using a HER-2/neu promoter-luciferase expression plasmid (pGL3/HNP410). In these experiments, PNAs were incubated with the plasmid prior to transfection, and the effect of PNA binding to the HER-2/neu promoter on HER-2/neu transcription was measured by luciferase activity (Figure 6). An indirect measurement of the stability of PNA binding in tumor cells is derived from the relative luciferase activity at various time points after transfection (Figure 6B). The specificity
of PNA binding was also evaluated by treating a control luciferase expression plasmid containing the SV40 promoter (not shown). The mono- and bis-PNAs conjugated to nitrogen mustards were generally much more active than their unmodified counterparts in this assay, and two of the PNA-alkylator conjugates were capable of specifically suppressing HER-2/neu expression in HeLa cells by greater than 80%, depending on the concentration of PNA used to drive complete binding to the plasmid. PNA II and PNA VIII demonstrated over 80% suppression of HER-2/neu transcription when the plasmid was incubated with 10 µM PNA and luciferase activity was analyzed at 24 h (Figure 6A). In comparison, the unmodified mono-PNA (I) had only a small effect on HER-2/neu transcription (13% suppression), and the unmodified bis-PNAs (PNAVII and IX) had a modest (40-55% suppression) impact on HER-2/neu transcription under these conditions. The high concentration of the PNAs (10 µM) was used to ensure rapid saturation of the PNA binding site(s) in the plasmid and thereby improve the efficiency of sitedirected alkylation at the target sequence, since PNA binding must occur within the time frame of the hydrolysis half-life of the nitrogen mustards for alkylation to occur. Incubation of the SV40 control plasmid with this high concentration of the PNA-
220 Bioconjugate Chem., Vol. 17, No. 1, 2006
Figure 5. Representative piperidine cleavage gels demonstrating sites of alkylation for bis-PNA-5′Chl conjugate (VI) and bis-PNA(J)-5′Chl conjugate (VIII). (A) Possible orientations of bis-PNA binding to the purine strand of duplex DNA. (B) A ∼250 bp restriction fragment containing the HER-2/neu promoter was labeled on the 3′ end on the purine strand and incubated with 1 µM PNA for 18 h. Maxim-Gilbert G-reactions with the same template were used to identify the alkylation sites. Schematic representations of PNA-alkylator conjugate binding and covalent adduct formation deduced from piperidine cleavage analysis for PNA VIII (C) and PNA VI (D). Black circles represent the nitrogen mustards, and the guanines they bind are circled with open circles.
alkylator conjugates also lead to a 15-20% decrease in SV40 driven luciferase activity, presumably due to some degree of nontarget binding by the PNA (not shown). Incubation of the HER-2/neu luciferase construct with a lower (1 µM) concentration of PNA II improved the specificity for the target sequence, but resulted in a lower level of suppression of HER-2/neu transcription, 55-60%, with minimal effect on the SV40 control plasmid. The effect of the mono-PNA-PAM conjugate (II) on HER-2/neu transcription at both concentrations remained stable for 72 h after transfection (Figure 6B). For comparison, we used the unmodified mono-PNA (I) as well as the unmodified bisPNA (IX) from our recent studies (33), showing that the small effect observed with PNA I was not observed beyond 48 h and that the level of suppression by PNA IX decreased significantly at 72 h. Collectively, these data comparing unmodified PNAs with their nitrogen mustard conjugates demonstrate that the nitrogen mustard significantly improves the biological activity of the PNA and improves the stability of PNA binding within cells.
DISCUSSION This is the first report of a PNA conjugated to a DNA alkylating agent. The PNA-alkylator conjugates directed the site-specific alkylation of the guanines adjacent to the bound PNA, stabilized the binding of the PNA in tumor cells, and markedly enhanced the ability of the PNA to suppress target gene transcription. Intercalating agents have been shown to enhance helix invasion by homopyrimidine PNAs (18, 19), but none have been shown to enhance the ability of a PNA to downregulate gene
Zhilina et al.
Figure 6. Transient transfection analysis of the HER-2/neu promoterluciferase plasmid incubated with PNAs and transfected into HeLa cells. (A) Luciferase activity was analyzed with various PNAs at 24 h. (B) The stability of PNA binding and inhibition of HER-2/neu promoter activity was analyzed for up to 72 h with selected PNAs, when the plasmid was preincubated with the indicated PNA at 10 µM. The luciferase activity from cells transfected with untreated plasmid was defined as 100% promoter activity at each time point, and the effect of the PNAs on HER-2/neu transcription is presented as a percentage of full promoter activity.
expression. In the present experiments, formation of helix invasion complexes with unmodified mono-PNA or bis-PNAs resulted in minimal to modest suppression of target gene transcription, while PNA binding stabilized by covalent adduct formation with a nitrogen mustard resulted in much higher inhibitory activity. Nitrogen mustard conjugated to oligonucleotides have served as valuable probes for nucleic acid structure, for example in the Holliday junctions formed during DNA recombination events (48). The present PNA-alkylator conjugates also function as probes for studiyng the mechanism of PNA interaction with DNA. We took advantage of the ability of the nitrogen mustard to alkylate at a single site adjacent to the PNA binding site which could subsequently be converted to an easily detectable single strand scission by piperidine treatment. Visualizing the alkylation site allowed us to deduce not only the PNA binding site but also the orientation of PNA binding. The binding of a homopyrimidine PNA to dsDNA usually does not result in a conventional triplex with duplex DNA but rather in a triplex invasion complex as the thermodynamically most stable complex (9, 49, 50). However, as also demonstrated here, the formation of a traditional triple helix does indeed take place (at least for cytosine-rich PNAs) (51, 52), and for the PNA-alkylator conjugates used in the present study, the conventional triplex is most probably responsible for their effect on gene transcription. Oxidative cleavage or photocleavage by bis-PNAs conjugated to an anthraquinone DNA intercalator or to a metalloporphyrin metal chelator have previously been used to deduce the sites and modes of PNA binding (17, 19). The present PNAalkylator conjugates predominantly directed covalent adduct
PNA−Nitrogen Mustard Conjugates for HER-2/neu
formation to the duplex-triplex junction, similar to the sites of cleavage seen with the PNA-metalloporphyrin conjugate but in contrast to the PNA-anthraquinone conjugate, which caused the strongest cleavage sites within the displaced strand, perhaps because the single stranded loop is more prone to photooxidation. Our results are consistent with a mechanism in which the nitrogen mustard extends along the major groove and covalently binds to the exposed N7 of nearby guanines. It is well established that for PNA/DNA binding the antiparallel orientation is preferred for Watson-Crick duplex binding while the parallel orientation is preferred for Hoogsteen triplex binding (52). If triplex (invasion) PNA:DNA:PNA complexes are formed using only one type of PNA (mono-PNA), the overall parallel configuration is preferred (52). Concordingly, the mono-PNAnitrogen mustard conjugate also prefers to bind in the parallel orientation. EMSAs with the PNA-PAM conjugate clearly demonstrate a discrete triplex band as well as additional bands of even lower mobility that are ascribed to helix invasion complexes, which are formed upon prolonged incubation or using higher PNA concentrations. We were able to differentiate triplex formation from strand invasion by monitoring the displacement of the pyrimidine strand. The distance between the oligomer and the DNA interactive drug can significantly impact the reaction site and affinity of the conjugate. In the present work we did not systematically study linker length, but the piperidine cleavage experiments demonstrated that without a linker, PAM attached to the PNA N-terminus is able to effectively alkylate a guanine at the duplex/ triplex junction. At the same time, the presence of a additional amino acid residues or a long 18 atom linker between the last PNA base and chlorambucil allows the nitrogen mustard to alkylate guanines a few base pairs away from the duplex/triplex junction, as demonstrated by a minor cleavage sites four base pairs away from the predicted PNA binding site for the PNA3′chl conjugate. In summary, we have demonstrated the facile synthesis of PNA-nitrogen mustard conjugates and their ability to suppress HER-2/neu transcription in a reporter plasmid model system. Such PNA-alkylator conjugates provide a new class of probes for sequence specific PNA-DNA interactions and warrant further study for gene targeting.
ACKNOWLEDGMENT This work is supported by grants from the NIH (CA85306) and the Flinn Foundation (1580). Mass spectra were acquired in the AZCC/SWEHSC Proteomics Core, supported by grants from the NIH (ES06694 and CA023074).
LITERATURE CITED (1) Milligan, J. F., Matteucci, M. D., and Martin, J. C. (1993) Current Concepts in Antisense Drug Design. J. Med. Chem. 36, 1923-1937. (2) Crooke, S. T. (1992) Therapeutic Applications of Oligonucleotides. Bio-Technology 10, 882-886. (3) Helene, C. (1991) Rational Design of Sequence-Specific Oncogene Inhibitors Based on Antisense and Antigen Oligonucleotides. Eur. J. Cancer 27, 1466-1471. (4) Thuong, N. T., and Helene, C. (1993) Sequence-Specific Recognition and Modification of Double-Helical Dna by Oligonucleotides. Angew. Chem., Int. Ed. Engl. 32, 666-690. (5) Uhlmann, E., and Peyman, A. (1990) Antisense Oligonucleotides - A New Therapeutic Principle. Chem. ReV. 90, 543-584. (6) Gryaznov, S., and Chen, J. K. (1994) Oligodeoxyribonucleotide N3′-]P5′ Phosphoramidates - Synthesis and Hybridization Properties. J. Am. Chem. Soc. 116, 3143-3144. (7) Demesmaeker, A., Waldner, A., Lebreton, J., Hoffmann, P., Fritsch, V., Wolf, R. M., and Freier, S. M. (1994) Amides As A New-Type of Backbone Modification in Oligonucleotides. Angew. Chem., Int. Ed. Engl. 33, 226-229.
Bioconjugate Chem., Vol. 17, No. 1, 2006 221 (8) Jones, R. J., Lin, K. Y., Milligan, J. F., Wadwani, S., and Matteucci, M. D. (1993) Synthesis and Binding-Properties of Pyrimidine Oligodeoxynucleoside Analogues Containing Neutral Phosphodiester Replacements - the Formacetal and 3′-Thioformacetal Internucleoside Linkages. J. Org. Chem. 58, 2983-2991. (9) Nielsen, P. E., Egholm, M., Berg, R. H., and Buchardt, O. (1991) Sequence-Selective Recognition of Dna by Strand Displacement with A Thymine-Substituted Polyamide. Science 254, 1497-1500. (10) Kuhn, H., Demidov, V. V., Coull, J. M., Fiandaca, M. J., Gildea, B. D., and Frank-Kamenetskii, M. D. (2002) Hybridization of DNA and PNA molecular beacons to single-stranded and double-stranded DNA targets. J. Am. Chem. Soc. 124, 1097-1103. (11) Liu, B., Han, Y., Corey, D. R., and Kodadek, T. (2002) Toward synthetic transcription activators: Recruitment of transcription factors to DNA by a PNA-peptide chimera. J. Am. Chem. Soc. 124, 18381839. (12) Demidov, V. V., Potaman, V. N., Frankkamenetskii, M. D., Egholm, M., Buchard, O., Sonnichsen, S. H., and Nielsen, P. E. (1994) Stability of Peptide Nucleic-Acids in Human Serum and Cellular-Extracts. Biochem. Pharmacol. 48, 1310-1313. (13) Borgatti, M., Romanelli, A., Saviano, M., Pedone, C., Lampronti, I., Breda, L., Nastruzzi, C., Bianchi, N., Mischiati, C., and Gambari, R. (2003) Resistance of decoy PNA-DNA chimeras to enzymatic degradation in cellular extracts and serum. Oncol. Res. 13, 279287. (14) McMahon, B. M., Mays, D., Lipsky, J., Stewart, J. A., Fauq, A., and Richelson, E. (2002) Pharmacokinetics and tissue distribution of a peptide nucleic acid after intravenous administration. Antisense Nucleic Acid Drug DeV. 12, 65-70. (15) Okamoto, A., Tanabe, K., and Saito, I. (2001) Synthesis and properties of peptide nucleic acids containing a psoralen unit. Org. Lett. 3, 925-927. (16) Mokhir, A. A., and Kraemer, R. (2003) Conjugates of PNA with naphthalene diimide derivatives having a broad range of DNA affinities. Bioconjugate Chem. 14, 877-883. (17) Armitage, B., Koch, T., Frydenlund, H., Orum, H., Batz, H. G., and Schuster, G. B. (1997) Peptide nucleic acid-anthraquinone conjugates: strand invasion and photoinduced cleavage of duplex DNA. Nucleic Acids Res. 25, 4674-4678. (18) Bentin, T., and Nielsen, P. E. (2003) Superior duplex DNA strand invasion by acridine conjugated peptide nucleic acids. J. Am. Chem. Soc. 125, 6378-6379. (19) Bigey, P., Sonnichsen, S. H., Meunier, B., and Nielsen, P. E. (1997) DNA binding and cleavage by a cationic manganese porphyrin-peptide nucleic acid conjugate. Bioconjugate Chem. 8, 267-270. (20) Ross, G. F., Smith, P. M., McGregor, A., Turnbull, D. M., and Lightowlers, R. N. (2003) Synthesis of trifunctional PNA-benzophenone derivatives for mitochondrial targeting, selective DNA binding, and photo-cross-linking. Bioconjugate Chem. 14, 962-966. (21) Jardines, L., Fowble, B., Schultz, D., Mackie, J., Buzby, G., Torosian, M., Daly, J., Weiss, M., Orel, S., and Rosato, E. (1995) Factors associated with a positive reexcision after excisional biopsy for invasive breast cancer. Surgery 118, 803-809. (22) Kern, J. A., Schwartz, D. A., Nordberg, J. E., Weiner, D. B., Greene, M. I., Torney, L., and Robinson, R. A. (1990) P185Neu Expression in Human Lung Adenocarcinomas Predicts Shortened Survival. Cancer Res. 50, 5184-5191. (23) Yu, D., Wang, S. S., Dulski, K. M., Tsai, C. M., Nicolson, G. L., and Hung, M. C. (1994) c-erbB-2/neu overexpression enhances metastatic potential of human lung cancer cells by induction of metastasis-associated properties, Cancer Res. 54, 3260-3266. (24) Agus, D. B., Bunn, P. A., Jr., Franklin, W., Garcia, M., and Ozols, R. F. (2000) HER-2/neu as a therapeutic target in nonsmall cell lung cancer, prostate cancer, and ovarian cancer. Semin. Oncol. 27, 5363. (25) Meric, F., Hung, M. C., Hortobagyi, G. N., and Hunt, K. K. (2002) HER2/neu in the management of invasive breast cancer. J Am. Coll. Surg. 194, 488-501. (26) Ebbinghaus, S. W., Gee, J. E., Rodu, B., Mayfield, C. A., Sanders, G., and Miller, D. M. (1993) Triplex Formation Inhibits Her-2 Neu Transcription In-Vitro. J. Clin. InVest. 92, 2433-2439. (27) Ebbinghaus, S. W., Fortinberry, H., and Gamper, H. B. (1999) Inhibition of transcription elongation in the HER-2/neu coding
222 Bioconjugate Chem., Vol. 17, No. 1, 2006 sequence by triplex-directed covalent modification of the template strand. Biochemistry 38, 619-628. (28) Noonberg, S. B., Scott, G. K., Hunt, C. A., Hogan, M. E., and Benz, C. C. (1994) Inhibition of Transcription Factor-Binding to the Her2 Promoter by Triplex-Forming Oligodeoxyribonucleotides. Gene 149, 123-126. (29) Porumb, H., Gousset, H., Letellier, R., Salle, V., Briane, D., Vassy, J., AmorGueret, M., Israel, L., and Taillandier, E. (1996) Temporary ex vivo inhibition of the expression of the human oncogene HER2(NEU) by a triple helix-forming oligonucleotide. Cancer Res. 56, 515-522. (30) Ziemba, A., Derosier, L. C., Methvin, R., Song, C. Y., Clary, E., Kahn, W., Milesi, D., Gorn, V., Reed, M., and Ebbinghaus, S. (2001) Repair of triplex-directed DNA alkylation by nucleotide excision repair. Nucleic Acids Res. 29, 4257-4263. (31) Ziemba, A. J., Reed, M. W., Raney, K. D., Byrd, A. B., and Ebbinghaus, S. W. (2003) A bis-alkylating triplex forming oligonucleotide inhibits intracellular reporter gene expression and prevents triplex unwinding due to helicase activity. Biochemistry 42, 50135024. (32) Zhilina, Z. V., Ziemba, A. J., Trent, J. O., Reed, M. W., Gorn, V., Zhou, Q., Duan, W., Hurley, L., and Ebbinghaus, S. W. (2004) Synthesis and evaluation of a triplex-forming oligonucleotidepyrrolobenzodiazepine conjugate. Bioconjugate Chem. 15, 11821192. (33) Ziemba, A. J., Zhilina, Z. V., Krotova-Khan, Y., Stankova, L., and Ebbinghaus, S. W. (2005) Targeting and regulation of the HER2/neu oncogene promoter with bis-peptide nucleic acids. Oligonucleotides 15, 36-50. (34) Mizuguchi, G., Kaneiishii, C., Sawazaki, T., Horikoshi, M., Roeder, R. G., Yamamoto, T., and Ishii, S. (1994) Independent Control of Transcription Initiations from 2 Sites by An InitiatorLike Element and Tata Box in the Human C-Erbb-2 Promoter. FEBS Lett. 348, 80-88. (35) Wendtner, C. M., Eichhorst, B. F., and Hallek, M. J. (2004) Advances in chemotherapy for chronic lymphocytic leukemia. Semin. Hematol. 41, 224-233. (36) Panasci, L., Paiement, J. P., Christodoulopoulos, G., Belenkov, A., Malapetsa, A., and Aloyz, R. (2001) Chlorambucil drug resistance in chronic lymphocytic leukemia: the emerging role of DNA repair. Clin. Cancer Res. 7, 454-461. (37) Pukhalsky, A. L., and Shmarina, G. V. (2001) Stimulatory and protective effects of alkylating agents applied in ultra-low concentrations. Pharmacology 62, 129-132. (38) Povirk, L. F., and Shuker, D. E. (1994) DNA damage and mutagenesis induced by nitrogen mustards. Mutat. Res. 318, 205226. (39) Mattes, W. B., Hartley, J. A., and Kohn, K. W. (1986) DNA sequence selectivity of guanine-N7 alkylation by nitrogen mustards. Nucleic Acids Res. 14, 2971-2987. (40) Kutyavin, I. V., Gamper, H. B., Gall, A. A., and Meyer, R. B. (1993) Efficient, Specific Interstrand Cross-Linking of DoubleStranded DNA by A Chlorambucil-Modified, Triplex-Forming Oligonucleotide. J. Am. Chem. Soc. 115, 9303-9304.
Zhilina et al. (41) Vlassov, V. V., Zarytova, V. F., Kutyavin, I. V., and Mamaev, S. V. (1988) Sequence-Specific Chemical Modification of A Hybrid Bacteriophage-M13 Single-Stranded-Dna by Alkylating Oligonucleotide Derivatives. FEBS Lett. 231, 352-354. (42) Belousov, E. S., Afonina, I. A., Kutyavin, I. V., Gall, A. A., Reed, M. W., Gamper, H. B., Wydro, R. M., and Meyer, R. B. (1998) Triplex targeting of a native gene in permeabilized intact cells: covalent modification of the gene for the chemokine receptor CCR5. Nucleic Acids Res. 26, 1324-1328. (43) Christensen, L., Fitzpatrick, R., Gildea, B., Petersen, K. H., Hansen, H. F., Koch, T., Egholm, M., Buchardt, O., Nielsen, P. E., Coull, J., and. (1995) Solid-phase synthesis of peptide nucleic acids. J. Pept. Sci. 1, 175-183. (44) Dueholm, K. L., Egholm, M., Behrens, C., Christensen, L., Hansen, H. F., Vulpius, T., Petersen, K. H., Berg, R. H., Nielsen, P. E., and Buchardt, O. (1994) Synthesis of Peptide Nucleic-Acid Monomers Containing the 4 Natural Nucleobases - Thymine, Cytosine, Adenine, and Guanine and Their Oligomerization, J. Org. Chem. 59, 5767-5773. (45) Reed, M. W., Lukhtanov, E. A., Gorn, V., Kutyavin, I., Gall, A., Wald, A., and Meyer, R. B. (1998) Synthesis and reactivity of aryl nitrogen mustard oligodeoxyribonucleotide conjugates. Bioconjugate Chem. 9, 64-71. (46) Musumeci, D., Roviello, G. N., Valente, M., Sapio, R., Pedone, C., and Bucci, E. M. (2004) New synthesis of PNA-3′DNA linker monomers, useful building blocks to obtain PNA/DNA chimeras. Biopolymers 76, 535-542. (47) Lampe, J. N., Kutyavin, I. V., Rhinehart, R., Reed, M. W., Meyer, R. B., and Gamper, H. B. (1997) Factors influencing the extent and selectivity of alkylation within triplexes by reactive G/A motif oligonucleotides. Nucleic Acids Res. 25, 4123-4131. (48) Podyminogin, M. A., Meyer, R. B., and Gamper, H. B. (1995) Sequence-Specific Covalent Modification of Dna by Cross-Linking Oligonucleotides - Catalysis by Reca and Implication for the Mechanism of Synaptic Joint Formation. Biochemistry 34, 1309813108. (49) Egholm, M., Behrens, C., Christensen, L., Berg, R. H., Nielsen, P. E., and Buchardt, O. (1993) Peptide Nucleic-Acids Containing Adenine Or Guanine Recognize Thymine and Cytosine in Complementary-Dna Sequences, J. Chem. Soc., Chem. Commun. 800-801. (50) Egholm, M., Nielsen, P. E., Buchardt, O., and Berg, R. H. (1992) Recognition of Guanine and Adenine in Dna by Cytosine and Thymine Containing Peptide Nucleic-Acids (PNA). J. Am. Chem. Soc. 114, 9677-9678. (51) Wittung, P., Nielsen, P., and Norden, B. (1997) Extended DNArecognition repertoire of peptide nucleic acid (PNA): PNA-dsDNA triplex formed with cytosine-rich homopyrimidine PNA. Biochemistry 36, 7973-7979. (52) Egholm, M., Christensen, L., Dueholm, K. L., Buchardt, O., Coull, J., and Nielsen, P. E. (1995) Efficient Ph-Independent SequenceSpecific Dna-Binding by Pseudoisocytosine-Containing Bis-PNA. Nucleic Acids Res. 23, 217-222. BC0502964
