ARTICLE pubs.acs.org/bc
Stabilizing Effect of Propionic Acid Derivative of AnthraquinonePolyamine Conjugate Incorporated into rβ Chimeric Oligonucleotides on the Alternate-Stranded Triple Helix Tomohisa Moriguchi, A. T. M. Zafrul Azam,† and Kazuo Shinozuka* Department of Chemistry and Chemical Biology, Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, Japan
bS Supporting Information ABSTRACT: Two types of anthraquinone conjugates were synthesized as non-nucleosidic oligonucleotide components. These include an anthraquinone derivative conjugated with 2,2-bis(hydroxymethyl)propionic acid and an anthraquinonepolyamine derivative conjugated with 2,2-bis(hydroxymethyl)propionic acid. The conjugates were successfully incorporated into the “linking-region” of the Rβ chimeric oligonucleotides via phosphoramidite method as non-nucleosidic backbone units. The resultant novel Rβ chimeric oligonucleotides possessed two diastereomers that were generated by the introduction of the anthraquinone conjugate with a stereogenic carbon atom. The isomers were successfully separated by a reversedphase HPLC. UV-melting experiments revealed that both stereoisomers formed a substantially stable alternate-strand triple helix, irrespective of the stereochemistry of the incorporated non-nucleosidic backbone unit. However, the enhancing effect on thermal stability depended on the length of the alkyl linker connecting anthraquinone moiety and the propionic acid moiety. The sequence discrimination ability of the chimeric oligonucleotides toward mismatch target duplex was also examined. The Tm values of the triplexes containing the mismatch target were substantially lower than the Tm values of those containing the full-match target. The thermodynamic parameters (ΔH°, ΔS°, and ΔG°) required for the dissociation of the triplexes into the third strand and target duplex were also measured.
’ INTRODUCTION Synthetic oligonucleotide-directed triple-helix formation presents a promising way to interfere with gene expression through direct interaction with genomic dsDNA and has become an area of intense investigation,14 as demonstrated by many recent in vitro and in vivo experiments.57 Indeed, the oligonucleotides forming a triplex with certain gene promoters can modulate the level of transcription of that gene (antigene strategy).811 The limitation to the application of this approach in therapeutics and biotechnology is related to the relatively low stability of triple helical complexes under physiological conditions; sufficiently stable triplexes can be obtained only by targeting substantially long (at least 1617 bases) tracts of homopurines, which are rarely found in biologically relevant DNA sequences. Considerable effort has been devoted to overcoming such limitations of the antigene strategy. A possible method to extend the range of targets is to utilize a specifically designed triplex forming oligonucleotide (TFO) capable of forming a triplex with adjacent and alternating homopurine strands because such a sequence would be more conceivable. On the basis this logic, Horne and Dervan reported a unique type of TFO consisting of 30 -30 linked polythymidylate.12 In the TFO, two short homopyrimidine strands were coupled by 1,2-dideoxy-D-ribose through a 30 -30 r 2011 American Chemical Society
phosphodiester linkage. Thus, the TFO formed a so-called “alternate-stranded triplex” with an adjacent and alternating homopurine strand in the manner of parallel orientation through Hoogsteen hydrogen bonds. In the TFO, the 1,2-dideoxy-Dribose moiety served as the “linker” portion connecting two independent TFO molecules. Many 30 -30 or 50 -50 linked TFOs capable of forming an alternate-strand triple helix by switching strand at the junction between the oligopurine and the oligopyrimidine domain via a Hoogsteen-hydrogen bonding pattern were reported.1322 However, these TFOs are constructed with a natural β-anomeric polypyrimidine strand in combination with a specifically designed “linker molecule”. To create a new class of TFOs based on the strategy described above, we have developed a unique class of chimeric TFOs composed of a contiguous β-anomeric polypyrimidine strand and an R-anomeric polypyrimidine strand.23 In the TFO, mononucleotide units are connected through natural 30 -50 phosphodiester linkages, although the 30 -half of the linkage exclusively consists of R-anomeric polypyrimidylate, and the Received: October 10, 2010 Revised: March 29, 2011 Published: May 02, 2011 1039
dx.doi.org/10.1021/bc100446w | Bioconjugate Chem. 2011, 22, 1039–1045
Bioconjugate Chemistry
ARTICLE
Figure 1. Sequence and structure of Rβ chimeric TFO. In the sequence, italic letters represent the R-anomeric polypyrimidylate component and Roman letters represent the β-anomeric polypyrimidylate component, respectively. The linker portion of the chimera TFO consist of a β-anomeric 50 -AACC-30 sequence in GK-300, whereas AC of the portion is replaced with a non-nucleotidic anthraquinone bearing unit in modified TFOs.
remaining portion consists of β-anomeric polypyrimidylate. In the TFO, the 30 -half binds to the purine tract of a target dsDNA in an antiparallel orientation, while the remaining β-DNA portion, except that which serves as a “linker” connecting the R- and β-DNA portions, binds to another purine tract in a parallel orientation. Several approaches have been attempted to stabilize the triple helix, which is relatively less stable than the double helix. One approach is the conjugation of the intercalation moiety to TFO.2328 In this study, we found that the substitution of a normal nucleoside unit in the linker region of the original chimeric TFO29 with a multiconjugate of the non-nucleosidic unit, 2,2-bis(hydroxymethyl)propionic acid, and an intercalatorpolyamine complex substantially improves the thermal stability of the resulting alternate-strand triplex. This may be due to the intercalation of the complex to the target dsDNA.30,31 In this paper, we will describe the results of our detailed study on the triplex-forming ability of the modified complex including the triplex-stabilizing effect of the polyamine moiety connected to the intercalator and the length of the alkyl chain linking the nonnucleosidic unit and the complex, as well as the sequencediscriminating ability of the modified chimeric TFO.
’ RESULTS AND DISCUSSION Synthesis and Purification of Modified rβ Chimeric TFOs with an Anthraquinone Conjugate as a Non-Nucleosidic Oligonucleotide Component. Two types of anthraqui-
none conjugates were utilized as non-nucleosidic oligonucleotide components. These included an anthraquinone derivative conjugated with 2,2-bis(hydroxymethyl)propionic acid and an anthraquinonepolyamine derivative conjugated with 2,2-bis(hydroxymethyl)propionic acid.32 Synthesis of the modified chimeric chimeric triplex forming oligonucleotides (TFOs), GK-354, GK-355, GK-358, GK-359, GK-366, and GK-367 along with the parental unmodified chimeric TFO (GK-300) (Figure 1) were carried out by an automated DNA synthesizer starting from CPG-bound R-thymidine, which was prepared
Figure 2. Structure of the phosphoramidite derivatives 1 and 2 bearing conjugated anthraquinone.
according to a previously described procedure.32 The phosphoramidite derivatives bearing conjugated-anthraquinone (1a,b,c and 2a,b,c, Figure 2) were incorporated into the middle of the “linker” region of the chimera TFOs instead of a normal nucleotide unit. It should be noted that, in the chimera TFOs, the central 50 -AC-30 portion of the linker region of the parental GK-300 was substituted with the conjugate as shown in Figure 1. The coupling reaction of the phosphoramidite reagents of anthraquinone conjugate was carried out for 360 s with a double coupling procedure using 0.3 M CH3CN solution instead of normally used 0.1 M solution. Under the conditions, the coupling yields of 1a,b,c and 2a,b,c estimated from the conventional trityl assay were 93%, 86%, 88%, 93%, 96%, and 90%, respectively. The incorporation of other unnatural R-nucleoside 1040
dx.doi.org/10.1021/bc100446w |Bioconjugate Chem. 2011, 22, 1039–1045
Bioconjugate Chemistry
ARTICLE
units into the chimera TFO was also accomplished within the extended coupling period (360 s). The obtained CPG-bound oligomers were treated with concentrated ammonia at 55 °C for 12 h. The obtained oligomers were purified by reversed-phase HPLC. As stated previously, the DMTr-off form of the Rβ chimeric oligonucleotides bearing the anthraquinone conjugates gave two peaks with almost equal intensity because of the existence of diastereomers. Thus, the faster elutes (GK-354A, GK-355A, GK-358A, GK-359A, GK-366A, and GK-367A) and the slower elutes (GK-354B, GK-355B, GK-358B, GK-359B, GK-366B, and GK-367B) were separately isolated. The oligomers were further purified by ethanol precipitation followed by Sephadex G-25 gel filtration. The modified TFO, GK-368, with a 3-nucleotide (nt) linker (50 -AAC-30 , Figure 3) was also
synthesized as the reference oligomer according to standard solid-phase DNA synthesis. The yield and characterization data of the oligomers are listed in Supporting Information Table S1. UV-Melting Experiment of Chimera TFOs. The formation and thermal stability of the triplex were examined by UV-melting experiments under slightly acidic and neutral conditions (pH 6.1, 6.5, and 7.0) in appropriate buffer systems. The melting temperature (Tm) was calculated from the first derivative of the melting curve obtained (Supporting Information Figures S1S3) for all the system studied, and the results are summarized in Table 1. GK-368 with the 3-nt linker stabilizes the triplex more efficiently than GK-300 with the 4-nt linker (ΔTm > 2.5 °C) at all pH values. Thus, the use of 3-nt linker instead of the original 4-nt linker in chimera TFOs is more feasible for the triplex formation. In addition, all modified TFOs with the anthraquinone moiety formed a much more stable triplex than the unmodified GK-300 and GK-368. The magnitude of the stabilizing effect on modified TFOs for the triple helix, however, varied considerably. TFOs with a longer linker arm between the anthraquinone and 2,2-bis(hydroxymethyl)propionic acid moieties tend to form a more stable triplex than those with a shorter linker arm. As shown in Table 1, the Tm values of the triplex consisting of modified TFO and full-matched target (ODN-2:ODN-3) are considerably higher (ΔTm > 1017 °C) than that of the triplex containing unmodified GK-300. The triplex-stabilizing effect, however, depends on the length of the alkyl linker connecting the anthraquinone and propionic acid moieties in the incorporated anthraquinone conjugate. Thus, the TFO bearing a longer alkyl linker (GK-354 and GK-355) exhibited higher Tm values compared to the TFO bearing a shorter alkyl linker (GK-358, -359, -366, -367). On the other hand, no apparent difference in thermal stability was observed between the TFOs consisting of two carbon alkyl linkers (GK-358 and GK-359) and three carbon alkyl linkers (GK-366 and GK-367) at pH 6.1 (Table 1).
Figure 3. Sequence and structure of Rβ chimeric TFO (GK-300 and GK-368). In the sequence, italic letters represent the R-anomeric polypyrimidylate component and Roman letters represent the βanomeric polypyrimidylate component, respectively. The linker portion of the chimera TFO consist of a β-anomeric 50 -AACC-30 sequence in GK-300 and β-anomeric 50 -AAC-30 sequence in GK-368.
Table 1. Melting Temperature (Tm) of the Triplex and Full-Match Duplex (ODN-2:ODN-3) at Different pH Levels Tmb (full-match) pH 6.1 ΔTmc
pH 6.5 ΔTmd
ΔTmc
pH 7.0 ΔTmd
ΔTmc
na
triplex
GK-300
-
40.0
-
35.8
-
23.6
-
GK-368 GK-355A
6
43.2 55.7
3.2 15.7
38.3 49.6
2.5 13.8
26.4 38.5
2.8 14.9
TFO
GK-355B
6
56.9
16.9
GK-359A
2
51.2
11.2
GK-359B
2
51.5
11.5
GK-367A
3
51.5
11.5
GK-367B
3
51.6
11.6
GK-354A
6
56.2
16.2
GK-354B GK-358A
6 2
57.5 53.0
17.5 13.0
GK-358B
2
52.8
12.8
GK-366A
3
53.1
13.1
GK-366B
3
53.2
13.2
1.2
triplex
51.4
15.6
45.6
9.8
45.3
9.5
-
-
-
-
50.3
14.5
1.3
51.2 46.4
15.4 10.6
0.2
46.6 -
0.3 0.1
0.1
1.8
triplex
ΔTmd
0.2
38.3
14.7
32.9
9.3
0.3
33.4
9.8
0.5
-
-
-
-
-
-
-
-
40.4
16.8
0.9
40.2 35.5
16.6 11.9
0.2
10.8
0.2
36.4
12.8
0.9
-
-
-
-
-
-
-
-
-
-
a
n indicates the amount of carbon in the alkyl linker that connects anthraquinone moiety with the propionic acid moiety. b Tm values (°C) were determined by computer fitting of the first derivative of the absorbance with respect to 1/T. Each Tm value is the average of three separate experiments. c ΔTm indicates the deviation from the Tm value of the corresponding triplex consisting of GK-300 and the duplex. d ΔTm indicates the Tm differences between diastereomeric counterparts. 1041
dx.doi.org/10.1021/bc100446w |Bioconjugate Chem. 2011, 22, 1039–1045
Bioconjugate Chemistry
ARTICLE
The Tm values tend to depend on the pH of the media. The Tm values showed a greater decrease at high pH values than at low pH values. For example, the Tm of GK-354A decreased from 56.2 to 40.4 °C when the pH changes from 6.1 to 7.0. This may be due to inadequate protonation of cytidine residues in the TFOs. However, even at pH 7.0, the Tm values of GK-354 and GK-355 exceeded the physiological temperature. Interestingly, the data in Table 1 also reveal that the difference in the Tm values between the two diastereomers A and B was very low in all TFOs at all pH values (16 °C with a single modification of the parental chimera TFO. Sequence Discrimination Ability of the Modified Chimera TFOs. Generally, the thermal stability of a triplex decreases substantially (>10 °C) in the case of a mismatch between the TFO and the target duplex.33 To estimate the specificity of modified TFOs in triplex formation, a single mismatch target duplex ODN-4:ODN-5 was prepared as shown in Figure 4. The Tm values of the triplex consisting of the modified chimera TFO and the mismatch target duplex were measured using the thermal denaturation study, as described in the previous section.
Figure 4. Alternate-stranded triplex consisting of modified chimera TFOs and single mismatch target duplex (ODN-4:ODN-5). X represents the non-nucleosidic anthraquinone conjugate.
The Tm values of the triplexes consisting of the modified TFOs and the mismatched target (ODN-4:ODN-5) decreased significantly as shown in Table 2. The magnitude of Tm decrement for the tested TFOs was nearly the same as that of the parental unmodified GK-300 at all pH values (6.1, 6.5, and 7.0). The results clearly indicate that the modified TFO retains at least the same degree of sequence discrimination ability as the unmodified TFO. Thus, the introduction of the intercalatorpropionic acid conjugate to the chimeric TFO will not abolish its ability to discriminate between sequences, despite apparent enhancement of the thermal stability with the full-matched target. The magnitude of Tm decrement with respect to the corresponding diastereomer is also quite small, as shown in Table 2. Thus, the results clearly indicate that the sequence discrimination ability of the modified TFOs is not influenced by the existence of the stereogenic carbon in them. Effect of Polyamine Moiety on the Thermal Stability of the Alternate-Strand Triplex. To check the effect of polyamine moiety on the thermal stability of the alternate-strand triplex, Tm differences among the sets of corresponding TFOs with or without the polyamine moiety on the anthraquinone moiety were analyzed. As shown in Table 3, Tm values increased in the presence of polyamine in the TFO, but only by a small margin (0.23.0 °C). Such smaller Tm increments might be due to the apparent distance of the polyamine moiety from the interacting target duplex. In the case of the short alkyl linker between anthraquinone and 2,2-bis(hydroxymethyl)propionic acid (GK-359 and GK-358), the Tm increment was slightly higher (2.03.0 °C) than that in the case of the long alkyl linker (GK355 and GK-354), because of the presence of the polyamine moiety on the anthraquinone moiety. In this case, the polyamine moiety could interact with the target duplex more easily than in the above case. This notion can be supported by the observation of similar effects of the polyamine moiety on the thermal stability of the triplex consisting of modified TFOs and single mismatch target duplex (Table 3, mismatch). Temperature-Dependent UV-Absorption Changes of the Anthraquinone Moiety. To confirm the interaction of the anthraquinone moiety in the modified TFOs with the
Table 2. Melting Temperature (Tm) of the Triplex and the Full-Match Duplex (ODN-2:ODN-3) and Mismatch Duplex (ODN-4: ODN-5) at Different pH Levels Tmb (mismatch) pH 6.1 TFO
na
triplex
ΔTmc
pH 6.5
ΔTmd
ΔTme
triplex
ΔTmc
pH 7.0
ΔTmd
ΔTme
triplex
ΔTmc
ΔTmd
ΔTme
GK-300
-
29.2
-
10.8
26.2
-
9.6
16.3
-
GK-355A
6
45.4
16.2
10.3
40.4
14.2
9.2
31.9
15.6
GK-355B
6
45.7
16.5
11.2
40.9
14.7
10.5
31.6
15.3
GK-359A
2
41.4
12.2
9.8
34.8
8.6
10.8
26.2
9.9
GK-359B GK-354A
2 6
41.2 45.6
12.0 16.4
0.2
10.3 10.6
35.3 42.4
9.1 16.2
0.5
10.0 7.9
26.4 33.3
10.1 17.0
0.2
7.0 7.1
GK-354B
6
46.4
17.2
0.8
11.1
42.2
16.0
0.2
9.0
33.6
17.3
0.3
6.6
GK-358A
2
43.3
14.1
9.7
39.1
12.9
7.3
28.8
12.5
GK-358B
2
43.4
14.2
9.4
39.4
13.2
7.2
30.1
13.8
0.3
0.1
0.5
0.3
7.3 6.6 0.4
6.7 6.7
6.7 1.3
6.3
a
n indicates the amount of carbon in the alkyl linker that connects the anthraquinone moiety with the propionic acid moiety. b Tm values (°C) were determined by computer fitting of the first derivative of the absorbance with respect to 1/T. Each Tm value is the average of three separate experiments. c ΔTm indicates the deviation from the Tm value of the corresponding triplex consisting of GK-300 and the duplex. d ΔTm indicates the Tm differences between the diastereomeric counterparts. e ΔTm indicates the deviation from the Tm value of the corresponding full-match triplex. 1042
dx.doi.org/10.1021/bc100446w |Bioconjugate Chem. 2011, 22, 1039–1045
Bioconjugate Chemistry
ARTICLE
Table 3. Comparison of Triplex Melting Temperature (Tm) of the TFOs with or without Polyamine Moiety at Different pH Levels full-match pH 6.1 TFO
na
Rb
GK-355A
6
H
55.7
GK-354A
6
polyamine
56.2
GK-355B
6
H
56.9
GK-354B
6
polyamine
57.5
GK-359A
2
H
51.2
GK-358A
2
polyamine
53.0
GK-359B
2
H
51.5
GK-358B
2
polyamine
52.8
Tm
ΔTmc
pH 6.5 Tm
50.3
Tm
þ0.7
40.4
51.2 46.4
0.2
46.6
40.2
þ0.8
35.5 36.4
45.6
þ1.9
46.4
Tm
þ0.2
42.4
43.3 43.4
ΔTmc
Tm
þ2.0
33.3
þ0.7
42.2 39.1
þ1.3
39.4
33.6
þ2.0
26.2 þ4.3
35.3 þ2.2
þ1.4
31.6
34.8 þ1.9
ΔTmc
31.9
40.9
41.2 þ3.0
pH 7.0
40.4
41.4 þ2.6
pH 6.5
ΔTmc
45.7
33.4 þ1.3
Tm 45.4
þ1.9
32.9
45.3 þ1.3
ΔTmc
38.3
45.6 þ1.8
pH 6.1
38.5
51.4 þ0.6
pH 7.0
ΔTmc
49.6 þ0.5
mismatch
28.8
þ2.6
26.4 þ4.1
30.1
þ3.7
a
n indicates the amount of carbon in the alkyl linker that connects the anthraquinone moiety with the propionic acid moiety. b R indicates the C-5 substituent of the anthraquinone moiety. c ΔTm (°C) indicates the deviation from the Tm value of corresponding TFOs without polyamine in the C-5 position of the anthraquinone moiety.
Table 4. Thermodynamic Parameters for Dissociation of the Third Strand from the Target Duplex (ODN-2:ODN-3) at pH 6.5 TFO
ΔH° (kcal mol1)
ΔΔH°a (kcal mol1)
ΔS° (cal mol1 K1)
ΔΔS°a(cal mol1K1)
ΔG°37(kcal mol1)
ΔΔG°37a(kcal mol1)
GK-300 GK-354A
112.9 159.0
46.1
340.3 462.8
122.5
7.3 15.4
8.1
GK-354B
164.2
51.3
478.7
138.4
15.7
8.4
GK-358A
148.3
35.4
436.6
96.3
12.9
5.5
GK-358B
151.7
38.8
446.4
106.0
13.2
5.9
ΔΔH°, ΔΔS°, and ΔΔG°37 values indicate the deviation from the corresponding value of the triplex consisting of the unmodified parental GK-300 and the duplex. a
double-stranded portion of the target dsDNA, experiments monitoring changes in the UV-absorption of the anthraquinone moiety with increasing temperature were carried out. The complex formed between GK-354A or GK-355A and the dsDNA (ODN-2:ODN-3) was heated at an elevated temperature from 20 to 80 °C, and the UV-absorbance was monitored in the range 400700 nm. In this experiment, the same buffer system as that for the UV-melting experiment (pH 6.1) was used, although the concentration of oligomers was increased to 28.0 μM for clear monitoring of the anthraquinone-based absorbance changes. As shown in Supporting Information Figure S7(A), anthraquinone-based absorbance in GK-354A bearing the anthraquinonepolyamine multiconjugate exhibited a marked shift to the shorter wavelength (blue shift, 551.0543.5 nm) according to the elevation of the temperature.3436 At the same time, the absorption maximum also increased (hyperchromicity, 12.4%). A combination of these characteristic behaviors in UVspectroscopy strongly suggests that the anthraquinone moiety actually intercalates to the target dsDNA when the modified TFO binds to form the triplex. Similarly, GK-355A bearing the anthraquinone conjugate without the polyamine moiety showed a blue shift (536.0525.5 nm) and hyperchromicity (12.4%) in UV spectroscopy (Supporting Information Figure S8(A)). Similar patterns of blue shift and hyperchromicity were observed in the case of other TFOs also (data not shown). Subsequently, the UV-absorbances of GK-354A at 550 nm and GK-355A at 535 nm were plotted against temperature and the results are shown in Supporting Information Figures S7(B) and S8(B), respectively. The plots show a prominent increment in the absorbance between 50 and 60 °C. Essentially identical
results were obtained from the corresponding experiment for the other modified TFOs. The obtained plots would reflect the association and dissociation of the anthraquinone conjugate from the target dsDNA. The results indicate that the anthraquinone conjugate in the modified TFO strongly intercalates with dsDNA. Thermodynamic Analysis of the Triplex. The thermodynamic values of the triplex containing TFOs (GK-300, GK354A, GK-354B, GK-358A, and GK-358B) and the full-match duplex ODN-2:ODN-3 were measured at pH 6.5 by using the same buffer system as the UV-melting experiment at different oligomer concentrations (2.5 μM, 1.25 μM, 0.625 μM, and 0.313 μM). Supporting Information Figure S3 showed the van’t Hoff plot of 1/Tm vs ln Ct for the transition of the triplex to the third strand and corresponding target duplex. The plots obtained for GK-354A and GK-354B were almost identical, confirming almost identical stability between the two diastereomers. A similar observation was made in the case of the diastereomers GK-358A and GK-358B. The thermodynamic values, calculated from the slope and intercept of the van’t Hoff plot by using the appropriate equations,3741 are summarized in Table 4. Inspection of these data revealed that the triplex consisting of the anthraquinonepolyamine conjugate was thermally more stable than the triplex consisting of the unmodified parent. In terms of enthalpy, GK-354A and GK-354B were more stable than those with a shorter alkyl linker (GK-358A and GK-358B). The significant difference in enthalpy data revealed that the modified triplex is more favorable than the unmodified parental TFO (GK-300). On the other hand, oligomers with longer alkyl linkers showed a 1043
dx.doi.org/10.1021/bc100446w |Bioconjugate Chem. 2011, 22, 1039–1045
Bioconjugate Chemistry significant loss of entropy than the oligomers with shorter alkyl linkers (ΔΔS° = 30 cal mol1 K1) and GK-300. Comparing the ΔG°37 values of the triplex, GK-354B formed the most stable triplex (15.7 kcal mol1). The ΔG°37 value of GK-300 (7.3 kcal mol1) was significantly smaller than those of the other triplex consisting of modified TFOs, indicating that it has relatively low stability. As a total effect, modified TFOs with longer alkyl linkers result in more stable triplex compared to those with shorter linkers. For instance, GK-354A (ΔG°37 = 15.4 kcal mol1) had greater stability than GK-358A (ΔG°37 = 12.9 kcal mol1). The thermodynamic values of the two diastereomers A and B in both GK-354 and GK-358 are essentially identical; as mentioned above, this indicates the structural flexibility of the two isomers.
’ CONCLUSION Chimera TFO bearing non-nucleosidic anthraquinone conjugates with or without a polyamine moiety on anthraquinone moiety exhibited more significant thermal stability than the unmodified parental TFO GK-300 because of the interaction of the anthraquinone moiety with the target dsDNA. The enhancing effect depended on the length of the alkyl linker between the anthraquinone and propionic acid moieties. However, no apparent differences were observed in the thermal stability of the two diastereomeric counterparts A and B in any case. This indicates that the extent of the intercalation of the anthraquinone moiety to the dsDNA is almost independent of the stereochemistry of the conjugate. It also indicates that the environment around the linker region of the modified TFOs is very flexible. On the other hand, TFOs bearing the polyamine moiety on the anthraquinone ring showed higher stability than those without the polyamine moiety, but with a relatively small margin. The polyamine moiety was found to be more effective in stabilizing the triplex in the case of TFOs bearing shorter alkyl linkers. This may be because the polyamine moiety with a short alkyl linker would be located near the dsDNA, and therefore would interact more efficiently than its counterpart with a longer alkyl linker. Even in this case, however, the thermal stability of the two diastereomeric counterparts was observed to be almost the same. This result indicates that the polyamine moiety introduced into DNA did not distinguish both diastereoisomers. The mismatch studies revealed that Tm values of triplexes consisting of modified TFOs and mismatch target duplex are significantly reduced (about 10 °C). The extent of Tm decrement is almost the same as that of the unmodified parental GK300 which indicates that the modified TFOs retain, at least, the same degree of sequence discrimination ability without abolishing the sequence specificity as the unmodified GK-300. The thermodynamic values also revealed that the alternatestranded triplex consisting of modified TFO and target dsDNA is thermodynamically more stable than the parental GK-300, whereas the TFOs having a longer alkyl linker result in a more stable triplex compared to that of short linker. The effect is brought about by the favorable enthalpic factor. All the above-mentioned properties would make the modified TFOs a unique class of alternate-stranded triplex-forming oligonucleotide feasible for practical use as a gene-regulating agent. ’ ASSOCIATED CONTENT
bS
Supporting Information. Experimental procedures and characterization data for phosphoramidite derivatives 1a, b,c, and
ARTICLE
2a,b,c, and the UV melting curves of the triplexes. This material is available free of charge via the Internet at http://pubs.acs.org/.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected]; Fax: 81-277-301321; Tel: 81-277-30-1320. Present Addresses †
Present address: Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Dhaka, Dhaka-1000, Bangladesh.
’ ACKNOWLEDGMENT The work was partially supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan. K. S. also thanks the Ministry for supporting A. Z. A. as a scholar of the Monbukagakusho Scholarship for Foreign Graduate Students. ’ REFERENCES (1) 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. (2) Doronina, S. O., and Behr, J. P. (1997) Towards a general triple helix mediated DNA recognition scheme. Chem. Soc. Rev. 26, 63–71. (3) Giovannangeli, C., and Helene, C. (1997) Progress in developments of triplex-based strategies. Antisense Nucleic Acid Drug Dev. 7, 413–421. (4) Helene, C. (1999) The antigene strategy: Progress and perspectives in selective gene silencing. Triple Helix Forming Oligonucleotides (Malvy, C., Harel-Bellam, A., and Pritchard, L. L., Eds.) pp 316, Chapter 1, Kluwer Academic Publishers, Norwell, MA, USA. (5) Upegui-Gonzalez, L. C., Francois, J. C., Ly, A., and Trojan, J. (2000) The approach of triple helix formation in control of gene expression and the treatment of tumors expressing IGF-I. Adv. Exp. Med. Biol. 465, 319–332. (6) Barre, F. X., Ait-Si-Ali, S., Giovannangeli, C., Luis, R., Robin, P., Pritchard, L. L., Helene, C., and Harel-Bellen, A. (2000) Unambiguous demonstration of triple-helix-directed gene modification. Proc. Natl. Acad. Sci. U.S.A. 97, 3084–3088. (7) Praseuth, D., Guieysse, L., and Helene, C. (1999) Triple helix formation and the antigene strategy for sequence-specific control of gene expression. Biochem. Biophys. Acta 1489, 181–206. (8) Helene, C. (1991) The anti-gene strategy: Control of gene expression by triplex-forming-oligonucleotides. Anti-Cancer Drug Des. 6, 569–584. (9) Luyten, I., and Herdewijn, P. (1998) Hybridization properties of base-modified oligonucleotides within the double and triple helix motif. Eur. J. Med. Chem. 33, 515–576. (10) Gowers, D. M., and Fox, K. R. (1999) Towards mixed sequence recognition by triple helix formation. Nucleic Acids Res. 27, 1569–1577. (11) Grigoriev, M., Praseuth, D., Guieysse, A. L., Robin, P., Thuong, N. T., Helene, C., and Harel-Bellan, A. (1993) Inhibition of gene expression by triple helix-directed DNA cross-linking at specific sites. Proc. Natl. Acad. Sci. U.S.A. 90, 3501–3505. (12) Horne, D. A., and Dervan, P. B. (1990) Recognition of mixedsequence duplex DNA by alternate-strand triple-helix formation. J. Am. Chem. Soc. 112, 2435–2437. (13) Griffin, L. C., and Dervan, P. B. (1989) Recognition of thymine adenine base pairs by guanine in a pyrimidine triple helix motif. Science 245, 967–971. (14) Fossella, J. A., Kim, Y.-J., Shih, H., Richard, E. G., and Fresco, J. R. (1993) Relative specificities in binding of Watson - Crick base pairs 1044
dx.doi.org/10.1021/bc100446w |Bioconjugate Chem. 2011, 22, 1039–1045
Bioconjugate Chemistry by third strand residues in a DNA pyrimidine triplex motif. Nucleic Acids Res. 21, 4511–4515. (15) Mergny, J.-L., Sun, J.-S., Rougee, M., Garestier, T., Barcelo, F., Chomilier, J., and Helene, C. (1991) Sequence specificity in triple helix formation: Experimental and theoretical studies of the effect of mismatches on triplex stability. Biochemistry 30, 9791–9798. (16) Ono, A., Chen, C.-N., and Kan, L.-S. (1991) DNA triplex formation of oligonucleotide analogs consisting of linker groups and octamer segments that have opposite sugar-phosphate backbone polarities. Biochemistry 30, 9914–9921. (17) Best, G. C., and Dervan, P. B. (1995) Energetics of formation of sixteen triple helical complexes which vary at a single position within a pyrimidine motif. J. Am. Chem. Soc. 117, 1187–1193. (18) Greenberg, W. A., and Dervan, P. B. (1995) Energetics of formation of sixteen triple helical complexes which vary at a single position within a purine motif. J. Am. Chem. Soc. 117, 5016–5022. (19) Gowers, D. M., and Fox, K. R. (1997) DNA triple helix formation at oligopurine sites containing multiple contiguous pyrimidines. Nucleic Acids Res. 25, 3787–3794. (20) Ueno, Y., Mikawa, M., Hoshika, S., and Matsuda, A. (2001) Nucleosides and Nucleotides. 208. Alternate-strand triple-helix formation by the 30 -30 -linked oligodeoxynucleotides with the anthraquinonyl group at the junction point. Bioconjugate Chem. 12, 635–642. (21) Hishika, S., Ueno, Y., and Matsuda, A. (2003) Nucleosides and nucleotides. 218. Alternate-strand triple-helix formation by the 30 -30 linked oligodeoxynucleotides using a purine motif. Bioconjugate Chem. 14, 607–613. (22) Hoshika, S., Ueno, Y., Kamiya, H., and Matsuda, A. (2004) Nucleosides and nucleotides. Part 226: Alternate-strand triple-helix formation by 30 -30 -linked oligodeoxynucleotides composed of asymmetrical sequences. Bioorg. Med. Chem. Lett. 14, 3333–3336. (23) Silver, G. C., Sun, J.-S., Nguyen, C. H., Boutorine, A. S., Bisagni, E., and Helene, C. (1997) Stable triple-helical DNA complexes formed by benzopyridoindole- and benzopyridoquinoxaline-oligonucleotide conjugates. J. Am. Chem. Soc. 119, 263–268. (24) Silver, G. C., Nguyen, C. H., Boutorine, A. S., Bisagni, E., Garestier, T., and Helene, C. (1997) Conjugates of oligonucleotides with triplex-specific intercalating agents. Stabilization of triple-helical DNA in the promoter region of the gene for the a-subunit of interleukin 2 (IL-2Ra). Bioconjugate Chem. 8, 15–22. (25) Garbesi, A., Bonazzi, S., Zanella, S., Capobianco, M. L., Giannini, G., and Arcamone, F. (1997) Synthesis and binding properties of conjugates between oligodeoxynucleotides and daunorubicin derivatives. Nucleic Acid Res. 25, 2121–2128. (26) Belousov, E. S., Afonina, I. A., Kutyavin, I. V., Gall, A. A., Reed, M. W., Gamperm, 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 Acid Res. 26, 1324–1328. (27) Capobianco, M. L., De Champdore, M., Francini, L., Lena, S., Garbesi, A., and Arcamone, F. (2001) New TFO conjugates containing a carminomycinone-derived chromophore. Bioconjuagte Chem. 12, 523– 528. (28) Carbone, G. M., McGuffie, E., Napoli, S., Flanagan, C. E., Dembech, C., Negri, U., Arcamone, F., Capobianco, M. L., and Catapano, C. V. (2004) DNA binding and antigene activity of a daunomycin-conjugated triplex-forming oligonucleotide targeting the P2 promoter of the human c-myc gene. Nucleic Acid Res. 32, 2396– 2410. (29) Shinozuka, K., Matsumoto, N., Suzuki, H., Moriguchi, T., and Sawai, H. (2002) Alternate stranded triplex formation of chimeric DNA composed of tandem R- and β-anomeric strands. Chem. Commun. 2712–2713. (30) Azam, A. T. M. Z., Hasegawa, M., Moriguchi, T., and Shinozuka, K. (2004) Rβ Chimeric oligo-DNA bearing intercalatorconjugated nucleobase inside the linker sequence remarkably improves thermal stability of an alternate-stranded triple helix. Bioorg. Med. Chem. Lett. 14, 5747–5750.
ARTICLE
(31) Azam, A. T. M. Z., Moriguchi, T., and Shinozuka, K. (2006) Modified Rβ chimeric oligoDNA bearing a multi-conjugate of 2,2bis(hydroxymethyl)propionic acidanthraquinonepolyamine exhibited improved and stereo-nonspecific triplex-forming ability. Chem. Commun. 335–337. (32) Gough, G. R., Brunden, M. J., and Gilham, P. T. (1981) Recovery and recycling of synthetic units in the construction of oligodeoxyribonucleotides on solid supports. Tetrahedron Lett. 22, 4177–4180. (33) Mergny, J. -L., Sun, J. -S., Rougee, M., Montenay-Garestier, T., Barcelo, F., Chomilier, J., and Helene, C. (1991) Sequence specificity in triple helix formation: Experimental and theoretical studies of the effect of mismatches on triplex stability. Biochemistry 30, 9791–9798. (34) Ly, D., Sanii, L., and Schuster, G. B. (1999) Mechanism of charge transport in DNA: Internally-linked anthraquinone conjugates support phonon-assisted polaron hopping. J. Am. Chem. Soc. 121, 9400–10. (35) Yamana, K., Nishijima, Y., Ikeda, T., Gokota, T., Ozaki, H., Nakano, H., Sangen, O., and Shimidzu, T. (1990) Synthesis and interactive properties of an oligonucleotide with anthraquinone at the sugar fragment. Bioconjugate Chem. 1, 319–324. (36) Deshmukh, H., Joglekar, S. P., and Broom, A. D. (1995) Selfcomplementary oligodeoxyribonucleotides containing 20 -O-(anthraquinone2-methyl)adenosine. Bioconjugate Chem. 6, 578–586. (37) Puglisi, J. D., and Tinoco, I., Jr. (1989) Absorbance melting curves of RNA. Methods Enzymol. 180, 304–325. (38) Marky, L. A., and Breslauer, K. J. (1987) Calculating thermodynamic data for transitions of any molecularity from equilibrium melting curves. Biopolymers 26, 1601–1620. (39) Fresco, J. R., and Lavelle, L. (1995) UV spectroscopic identification and thermodynamic analysis of protonated third strand deoxycytidine residues at neutrality in the triplex d(Cþ-T)6:[d(A-G)6d (C-T)6]; evidence for a proton switch. Nucleic Acids Res. 23, 2692– 2705. (40) Sugimoto, N., Nakano, S. I., Katoh, M., Matsumura, A., Nakamuta, H., Ohmichi, T., Yoneyama, M., and Sasaki, M. (1995) Thermodynamic parameters to predict stability of RNA/DNA hybrid duplexes. Biochemistry 34, 11211–11216. (41) Giancola, C., Petraccone, L., Pieri, M., and Barone, G. (2001) Thermodynamic and computational studies of DNA triple helices containing a nucleotide or a non-nucleotide linker in the third strand. Biophys. Chem. 94, 23–31.
1045
dx.doi.org/10.1021/bc100446w |Bioconjugate Chem. 2011, 22, 1039–1045
