Bioconjugate Chem. 2001, 12, 635−642
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Nucleosides and Nucleotides. 208. Alternate-Strand Triple-Helix Formation by the 3′-3′-Linked Oligodeoxynucleotides with the Anthraquinonyl Group at the Junction Point† Yoshihito Ueno, Mai Mikawa, Shuichi Hoshika, and Akira Matsuda* Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan. Received February 20, 2001; Revised Manuscript Received April 17, 2001
The synthesis of 3′-3′-linked oligodeoxynucleotides (ODNs) with the anthraquinonyl group at the junction point is described. The ODNs were synthesized on a DNA synthesizer using a controlled pore glass (CPG) carrying pentaerythritol that has an intercalator at one of the four hydroxymethyl groups. Stability of the triplexes with the target duplexes was studied by thermal denaturation. The 3′-3′-linked ODNs with the anthraquinonyl group enhanced the thermal stability of the triplexes when compared with those without the intercalator and the unmodified nonamer 10. It was found that the ODNs 12 and 13 carrying the anthraquinonyl groups can form thermally stable triplexes by skipping two or three extra base pairs between two binding domains of the target duplexes. The ability of the 3′-3′-linked ODNs to inhibit cleavage of the target DNA 22 by the restriction enzyme Hind III was tested. It was found that the 3′-3′-linked ODN 16 with the anthraquinonyl group at the junction point inhibited the cleavage by the enzyme more effectively than the nonamer 14 and the 3′-3′-linked ODN 15 without the intercalator.
INTRODUCTION
In the antigene strategy proposed to control gene transcription, an oligopyrimidine‚oligopurine sequence of double-helical DNA is recognized by a third-strand oligodeoxynucleotide (ODN) that binds to the major groove of the DNA and forms a local triple helix (triplex) (1). Depending on the orientation of the third strand, two major classes of triplexes are identified. When the third strand consists mainly of pyrimidines, Hoogsteen-type Py‚PuPy base triplets (T‚AT and C+‚GC) are formed in which the third strand is parallel to the purine strand of the target duplex (2, 3). When the third strand is predominantly purines, reverse Hoogsteen-type Pu‚PuPy base triplets (G‚GC and A‚AT) are formed in which the third strand is antiparallel to the purine strand of the target duplex (4, 5). However, target sequences in the antigene strategy are very restricted. Since the thermal stability of the triplexes is generally lower than that of the duplexes under physiological conditions, an oligopurine cluster with fairly long chain lengths is required for stable triplex formation. In addition, formation of the pyrimidine motif triplex needs conditions of low pH (pH < 6.0), because unmodified cytosine residues, if present in the third strand, must be protonated to bind with guanine of the G:C duplex (2, 6). Several approaches have been described to overcome this limitation. If the target duplex is composed of two adjacent and alternating oligopurine-oligopyrimidine tracts, the duplex can form a triplex with a single-strand ODN which pairs with oligopurine sequences on alternate strands by crossover in the major groove (7-24). This socalled “alternate-strand triplex” or “switched triplex” can * To whom reprint requests should be addressed.Phone: +81-11-706-3228. Fax: +81-11-706-4980. E-mail: matuda@ pharm.hokudai.ac.jp. † For Part 207 in this series, see: Kojima, N., Minakawa, N., and Matsuda,A. (2000) Tetrahedron 56, 7909-7914.
be formed in two distinct ways. By combining both Hoogsteen and reverse Hoogsteen motifs (7-14), the natural ODNs with oligopurine-oligopyrimidine sequences such as 5′-oligopurine-oligopyrimidine-3′ or 5′oligopyrimidine-oligopurine-3′ can be used as the third strand. In the second approach, only one set of motifs (Hoogsteen or reverse Hoogsteen motif) (15-24) is employed, namely, the 3′- and 3′- or 5′- and 5′-ends of two ODN fragments have to be connected using a suitable linker. The different possibilities for “alternate-strand triplex” formation in the latter case are shown in Figure 1. When the oligopyrimidine sequence follows the oligopurine sequence, the 3′-3′-linked and the 5′-5′-linked ODNs can form the parallel (a) and antiparallel (b) triplexes with the target duplex by Hoogsteen and reverse Hoogsteen hydrogen bonds, respectively. On the other hand, when the oligopurine sequence follows the oligopyrimidine sequence, the 5′-5′-linked and the 3′-3′-linked ODNs can form the parallel (c) and antiparallel (d) triplexes with the target duplex by Hoogsteen and reverse Hoogsteen hydrogen bonds, respectively. On the other hand, it is known that ODNs carrying intercalators, such as acridine (25-29), oxazolopyridocarbazole (30), benzopyridoindole (31, 32), and perylene (33) enhance the thermal stability of the triplexes. We recently reported the synthesis of branched ODNs with a pentaerythritol at the branch points (34). The ODNs formed thermally stable parallel and antiparallel triplexes with the single strand ODNs. On the basis of this information, we planned a synthesis of a 3′-3′-linked ODN with an intercalator at the junction point. We reasoned that the intercalator would stabilize the triplex by intercalating between the base pairs of the target duplex as shown in Figure 2. Since pentaerythritol has four equivalent hydroxymethyl groups, we envisaged that if we could synthesize the controlled pore glass (CPG) with pentaerythritol carrying an intercalator at one of the four hydroxymethyl groups, we would be able to conveniently
10.1021/bc010023i CCC: $20.00 © 2001 American Chemical Society Published on Web 06/30/2001
636 Bioconjugate Chem., Vol. 12, No. 4, 2001
Ueno et al.
Figure 1. Schematic presentation of “alternate-strand triplex” formation by the 3′-3′- or 5′-5′-linked ODNs.
linked ODNs to inhibit cleavage of a target DNA by a restriction enzyme, are also studied. RESULTS AND DISCUSSION
Figure 2. Schematic presentation of “alternate-strand triplex” formation by the 3′-3′-linked ODN with an intercalater at the junction point. Scheme 1
synthesize the 3′-3′-linked ODN with the intercalator at the junction point as shown in Scheme 1. In this paper, we report the synthesis of the 3′-3′-linked ODN with the anthraquinonyl group at the junction point. The thermal stability of the triplexes between these ODNs and the target DNAs, and the ability of the 3′-3′-
Synthesis. The CPG with pentaerythritol that has the anthraquinonyl group was synthesized as shown in Scheme 2. First, 2-[(tert-butyldiphenylsilanyl)oxymethyl]2-(hydroxymethyl)-1,3-propanediol (1) (33) was treated with 2 equiv of 4,4′-dimethoxytrityl chloride (DMTrCl) in pyridine to give the O-bis(DMTr) derivative 2 in 87% yield. Compound 2 was converted into a carbonylimidazolide, which was reacted with ethylenediamine or 1,4diaminobutane to produce 3a or 3b in 81 or 76% yield, respectively. Protection of the primary amino group of 3b with a trifluoroacetyl group followed by desilylation by tetrabutylammonium fluoride (TBAF) gave 5. Compound 5 was modified to the corresponding 3′-succinate 6, which was reacted with CPG to give a solid support containing 6 (23 µmol/g). At the same time, 3a or 3b was treated with anthraquinone-2-carboxylic acid in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSCI) in DMF to afford 7a or 7b in 51 or 83% yield, respectively. After removal of the TBDPS groups, 8a and 8b were modified to the corresponding 3′-succinates 9a and 9b, which were reacted with CPG to give solid supports containing 9a (20 µmol/ g) and 9b (30 µmol/g), respectively. The 3′-3′-linked ODNs 11-13, 15, 16, which consist of thymidine (T) and 5-methyl-2′-deoxycytidine (M), were synthesized on a DNA synthesizer (Figure 3a). The fully protected ODNs (each 1 µmol) linked to the solid support were treated with concentrated NH4OH at 55 °C for 16 h. Purification by C-18 HPLC gave the ODNs 11, 12, 13, 15, and 16 in 52, 41, 44, 31, and 33 OD260 units, respectively. These ODNs were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS), and the observed molecular weights supported their structures. Studies of Triplex Formation by Thermal Denaturation. The stability of the triplexes was studied by thermal denaturation in a buffer of 0.01 M sodium phosphate (both at pH 6.9 and 6.0) containing 0.5 M NaCl
3′-3′-Linked Oligodeoxynucleotides
Bioconjugate Chem., Vol. 12, No. 4, 2001 637
Scheme 2a
a (a) DMTrCl, pyridine, room temperature; (b) (1) N,N′-carbonyldiimidazole, DMAP, DMF, room temperature; (2) H N(CH ) NH 2 2 n 2 (n ) 2 or 4), DMF, room temperature; (c) EtOCOCF3, Et3N, DMF, room temperature; (d) TBAF, THF, room temperature; (e) succinic anhydride, DMAP, pyridine, room temperature; (f) anthraquinone-2-carboxylic acid, WSCI, DMF, room remperature.
Figure 4. Melting profile of the triplexes between the ODN 13 and the DNA 20 at pH 6.9 (curve I) and at pH 6.0 (curve II), and between the ODN 10 and the DNA 17 at pH 6.9 (curve III), respectively. Table 1. Hybridization Dataa ODN 10
Figure 3. Sequences of ODNs and the target duplexes.
and 0.02 M MgCl2. Sequences of target duplexes used in this study are shown in Figure 3b. The DNAs 17-21 have oligopurine sequences, one on each strand of the duplexes, that can be recognized by the ODNs 10-13. The DNAs 18-21 also have extra base pairs between the two binding domains that do not participate in Hoogsteen base pair interactions with the third strands and will be spanned by the linker groups. The sequences of the binding domains are symmetrical. One equivalent of the 3′-3′-linked ODNs or two equivalents of the control ODN 10 to the target DNAs was used for the thermal denaturation. Figure 4 shows melting profiles of the triplexes between the ODN 13 and the DNA 20 at pH 6.9 (I) and at pH 6.0 (II), and between the ODN 10 and the DNA 17 at pH 6.9 (III), respectively. The Tms are listed in Table 1. The Tm for the control triplex between the ODN 10 and the DNA 17 was not detected at pH 6.9 and was 18.2 °C at pH 6.0. All 3′-3′-linked ODNs with and without the anthraquinonyl group enhanced the thermal stability of the triplexes at both pH 6.9 and 6.0 as compared with the control ODN 10. The Tm values of the triplexes at pH 6.0 were greater than those at pH 6.9. The ODNs 12 and 13 with intercalators at the junction points more
ODN 11
ODN 12
ODN 13
target DNA
pH 6.9
pH 6.0
pH 6.9
pH 6.0
pH 6.9
pH 6.0
pH 6.9
pH 6.0
DNA 17 DNA 18 DNA 19 DNA 20 DNA 21
ND -
18.2 -
