Oligonucleotides with conjugated dihydropyrroloindole tripeptides: base composition and backbone effects on hybridization
Oligonucleotides with conjugated dihydropyrroloindole tripeptides: base composition and backbone effects on hybridizationIgor V. Kutyavin*, Eugeny A. Lukhtanov, Howard B. Gamper and Rich B. Meyer
Epoch Pharmaceuticals Inc., 1725 220th Street SE, 104, Bothell, WA 98021, USA
Received April 16, 1997; Revised and Accepted July 23, 1997
ABSTRACT
The ability of conjugated minor groove binding (MGB) residues to stabilize nucleic acid duplexes was investigated by synthesis of oligonucleotides bearing a tethered dihydropyrroloindole tripeptide (CDPI3). Duplexes bearing one or more of these conjugated MGBs were varied by base composition (AT- or GC-rich oligonucleotides), backbone modifications (phosphodiester DNA, 2'-O-methyl phosphodiester RNA or phosphorothioate DNA) and site of attachment of the MGB moiety (5'- or 3'-end of either duplex strand). Melting temperatures of the duplexes were determined. The conjugated CDPI3 residue enhanced the stability of virtually all duplexes studied. The extent of stabilization was backbone and sequence dependent and reached a maximum value of 40-49oC for d(pT)8[middot]d(pA)8. Duplexes with a phosphorothioate DNA backbone responded similarly on CDPI3 conjugation, although they were less stable than analogous phosphodiesters. Modest stabilization was obtained for duplexes with a 2'-O-methyl RNA backbone. The conjugated CDPI3 residue stabilized GC-rich DNA duplexes, albeit to a lesser extent than for AT-rich duplexes of the same length.
INTRODUCTION
Most antibiotics and small molecules that target DNA can be divided into two major families by their site of interaction with double-stranded DNA: intercalators and minor groove binders (MGBs) (1 ). Intercalators are flat aromatic molecules whose interaction with base pairs of nucleic acid duplexes is driven mainly by stacking forces. In contrast, MGBs are relatively long, crescent-shaped molecules which bind isohelically to the minor groove of DNA through van der Waals contacts, hydrophobic and electrostatic interactions (for a review see 2 and references cited therein). Some of them, like netropsin and distamycin A, form highly oriented hydrogen bonds with adenine N-3 and thymidine O-2 atoms on the cleft of the minor groove (2 ). The binding of MGBs in the minor groove of the duplex can be very strong, especially with AT-rich B-form DNA and association constants reach values of 107-109/M (3 -5 ). These affinities are much greater than those exhibited by simple intercalating agents, which bind to DNA with association constants of 104-106/M (6 -8 ). However, most naturally occurring MGBs are quite sensitive to the structure of the duplexes and bind poorly to GC-rich DNA and to A-form nucleic acid duplexes (2 ,4 ). In contrast, synthetic MGBs from the lexitropsin family were shown to decrease the strict AT preference (9 ,10 ), exhibiting in some cases solely GC site binding (11 ). Covalent coupling of two lexitropsin-like MGBs which bind antiparallel in a single DNA site helped to improve both efficiency and specificity of DNA site recognition (12 -14 ). A strategy (15 ) for design of pyrrole imidazole polyamide MGBs to target any predetermined DNA sequence of 5-13 bp (16 -18 ) resulted from this approach.
Addition of a pendant intercalating moiety has proven to be an effective way to improve oligonucleotide (ODN) affinity for a complementary strand (19 -22 ). Covalently appended MGBs can also significantly stabilize duplexes formed by AT-rich ODNs, as we recently reported (23 -25 ). Synthetic analogs of the natural oligopeptide antibiotics netropsin (or distamycin A) and CC-1065 were synthesized and conjugated to ODNs. These oligopeptides consisted respectively of repeating subunits of N-methylpyrrole carboxamide (MPC) (23 ) and 1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate (CDPI) which contained an N-3 terminal carbamoyl group (24 ). The extent of stabilization was judged by an increase in Tmax, the point at which dA/dT is greatest. For AT-rich duplexes stabilization was shown to depend on the length of the peptides. For (dTp)8 conjugated to MPC5 or CDPI3 and hybridized to poly(dA) Tmax increased by the unprecedented value of 38-44oC. In contrast, unconjugated CDPI3 increased the Tmax of the unmodified duplex by only 2oC. It was also shown that the MPC-type conjugates stabilized only AT-rich DNA, whereas the CDPI3 conjugates also stabilized AT-rich DNA+RNA duplexes. We report here the effect of structure on hybridization strength of octanucleotide duplexes with a conjugated CDPI3 residue.
MATERIALS AND METHODS
Synthesis of oligonucleotides
All ODNs were prepared from 1 [mu]mol appropriate CPG support on an ABI 394 synthesizer using the protocol supplied by the manufacturer. Protected [beta]-cyanoethyl phosphoramidites of 2'-deoxyribo and 2'-O-methylribonucleotides, CPG supports, deblocking solutions, cap reagents, oxidizing solutions and tetrazole solutions were purchased from Glen Research. 5'-Aminohexyl modifications were introduced using an N-(4-monomethoxytrityl)-6-amino-1-hexanol phosphoramidite linker (Glen Research). 3'-Aminohexyl and 3'-hexanol modifications were introduced using the CPG prepared as previously described (26 ). All other general methods employed for preparative HPLC purification, detritylation and butanol precipitation were carried out as described (27 ). All purified octanucleotides were analyzed by C-18 HPLC (column 250 * 4.6 mm) in a gradient of 0-30% acetonitrile in 0.1 M triethylamine acetate buffer, pH 7.0, over 20 min at a flow rate of 2 ml/min. Pump control and data processing were performed using a Rainin Dynamax chromatographic software package on a Macintosh computer. ODN purity was further confirmed by capillary gel electrophoresis (CGE) with a P/ACEtm 2000 Series equipped with an eCAPtm cartridge (Beckman, Fullerton, CA). The octanucleotides were >95% pure by C-18 HPLC and showed one major peak on CGE. Thermal denaturation studies were performed as described (24 ,25 ). The melting temperatures (Tmax values) of the hybrids were determined from the first derivative maxima and are shown in Tables 1 -1 .
Synthesis of CDPI3-tailed ODN conjugates
The methods used in this study to conjugate 3-carbamoyl-1,2- dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate trimer (CDPI3) to ODNs have been published (24 ). All CDPI3-tailed octanucleotides were isolated from reaction mixtures and, if necessary, repurified on an analytical (4.6 * 250 mm) PLRP-S column (Polymer Labs) using a gradient of acetonitrile (0-60% for mono- and 0-80% for bis-CDPI3-tailed ODNs) in 0.1 M triethylammonium acetate, pH 7.5. The column was incubated at 70-75oC. All ODN-CDPI3 conjugates prepared were analyzed and characterized as described (24 ).
Molar extinction coefficients of octanucleotides and their derivatives
The concentrations of the octanucleotides and their derivatives were measured spectrophotometrically. The molar extinction coefficients ([epsilon]260) of unmodified octadeoxyribonucleotides were determined by measuring the absorption of the ODNs before and after complete hydrolysis by snake venom nuclease (28 ). With this value, 32P-labeled CDPI3-tailed conjugates with known specific activities were prepared and their [epsilon]260 values determined as described (22 ). Molar extinction coefficients of all octadeoxyribonucleotides and their CDPI3 derivatives used in this study are, in order of unmodified ODN, mono-CDPI3 and di-CDPI3 derivative: d(pT)8p, 65.8, 110.1, 178.1/mM/cm; d(pA)8p, 81.9, 150.0, 218.0/mM/cm; d(pApGpCpGpGpApTpGp), 74.0, 162.9, 230.9/mM/cm; d(pCpApTpCpCpGpCpTp), 65.0, 136.9, 204.9/mM/cm. These extinction coefficients were used to determine the concentration of all other backbone-modified octanucleotides and their CDPI3 conjugates. To calculate [epsilon]260 values for deoxyinosine-containing ODNs the value of 4.6/mM/cm multiplied by the number of hypoxanthine bases was subtracted from the extinction coefficients of the corresponding dG-containing ODNs.
RESULTS AND DISCUSION
CDPI3 residue conjugated to AT-rich duplexes
The antibiotic CC-1065 (29 ) and its numerous synthetic derivatives (30 ), including CDPI3, have a strong affinity for AT-rich sites of double-stranded DNA, as do most of the MGBs. CC-1065 also binds to and alkylates AT-rich sequences in RNA-DNA hybrids (31 ). We expected the binding properties of ODN-conjugated CDPI3 to be similar to that observed for free CDPI3. We reported earlier on binding of oligothymidylates carrying a CDPIn moiety to polyadenylic acids (24 ,25 ). Here we use the short d(pT)8[middot]d(pA)8 duplex as a model system for a more comprehensive investigation. NMR analysis of a CC-1065-DNA complex (32 ) and non-competitive binding of CDPI3 and ethidium bromide to AT-rich DNA duplexes (33 ) have shown that these compounds span 5-6 bp in the DNA minor groove. An octamer duplex, therefore, is a length of double-stranded DNA sufficient to accommodate the conjugated CDPI3 residue and the linkers used in the present study.
A variety of octaadenylate and octathymidylate derivatives with DNA, 2'-O-methyl RNA or DNA phosphorothioate backbones and carrying CDPI3 residues at either or both termini were prepared. The structures of CDPI3 and linkers for 5'- and 3'-tailed ODN conjugates are shown in Figure 1 . Complementary duplexes constructed from these sequences were melted and the Tmax data are shown in Table 1 . Although the complex strands were taken in equimolar ratio and only single melting transitions were observed in all cases, the possibility of higher order structure formation, other than duplex, cannot be completely excluded. Our previous study conducted on d(pT)8[middot]poly(dA)/poly(rA) did not reveal a tendency of CDPI3-tailed ODNs to form triplex structures (24 ).
Melting temperatures (+-1oC) of duplexes formed by octathymidylate and octaadenylate with different backbone modifications and CDPI3 residues attached to different ends
Octaadenylate derivatives
Octathymidylate derivatives
Type of backbone
3'- and 5'-tailsa
DNA
2'-O-Methyl RNA
Phosphorothioate DNA
3'-Hex
5'-Hex-NH2
3'-CDPI3
5'-CDPI3
5',3'-di-CDPI3
3'-Hex-OH
3'-CDPI3
3'-Hex-NH2
3'-CDPI33
DNA
3'-Hex-NH2
14
12
58
58
55
12
30
<0
41
5'-Hex-NH2
13
13
58
56
44
10
28
<0
42
3'-CDPI3
61
61
71
67
64
19
50
49
61
5'-CDPI3
53
53
63
74
61
17
49
41
56
5',3'-di-CDPI3
60
57
69
68
71
-
-
-
-
2'-O-Methyl RNA
3'-Hex-OH
<0
<0
~0
19
-
20
29
NDb
NDb
3'-CDPI3
22
21
54
58
-
24
41
<0
NDb
Phosphorothioate DNA
3'-Hex-NH2
8
9
55
55
-
34
48
<0
35
3'-CDPI3
55
56
70
73
-
43
65
40
57
aThe oligonucleotides with this modification have a terminal phosphate linked to the hydroxy group of 1,6-hexanediol (Hex-OH) or 6-amino-1-hexanol (Hex-NH2) residues. The structure of the CDPI3 residue and linkers for 3'- and 5'-oligonucleotide conjugates are shown in Figure 1.
bNo melting transition detected.
Melting temperatures (+-1oC) of GC-rich octanucleotide duplexes with CDPI3 residues attached to the ends
d(CpApTpCpCpGpCpT) (strand A)
ApGpCpGpGpApTpG (strand B)
Type of backbone
3'- and 5'-tailsa
DNA
2'-O-Methyl RNA
Phosphorothioate DNA
3'-Hex-NH2
5'-CDPI3
3'-CDPI3
5',3'-di-CDPI3 3'-Hex-OH
3'-Hex-OH
5'-CDPI3
3'-CDPI3
Unmodifiedb
5'-CDPI3
3'-CDPI3
DNA
3'-Hex-NH2
41
45
52
50
46
48
47
33
27
40
5'-CDPI3
58
76
79
76
52
71
80
49
70
75
3'-CDPI3
57
78
81
77
51
68
72
50
73
77
3',5'-di-CDPI3
60
BTd
72
65
-
-
-
-
-
-
2'-O-Methyl RNA
3'-Hex-OH
37
20
29
-
66
67
67
28
NDc
NDc
5'-CDPI3 and
3'-Hex-OH
44
69
70
-
68
~95
87
41
58
64
3'-CDPI3
44
71
BTd
-
72
90
82
37
58
40
Phosphorothioate DNA
Unmodifiedb
32
32
43
-
38
43
39
24
16
28
5'-CDPI3
38
67
69
-
38
64
66
28
62
63
3'-CDPI3
45
71
74
-
44
75
53
36
64
69
aStructures of the CDPI3 residue and linkers for 3'- and 5'-oligonucleotide conjugates are shown in Figure 1.
bThese ODNs have no tails.
cNo melting transition was detected.
dMelting transition was too broad for Tmax to be accurately determined.
Effect of addition of an MGB residue to a GC-rich octanucleotide duplex
It is well recognized that A/T preference dominates the binding specificity of most MGBs, including CDPI oligomers. This preference is likely due to the hydrophobicity, depth and narrow width of the groove. These together provide a perfect isohelical and van der Waals fit of the crescent-shaped molecules in the minor groove. Free CDPI3 was shown to bind not only to poly(dA)[middot]poly(dT) but also to poly(dG)[middot]poly(dC), although with a lower strength (33 ). Therefore, it was interesting to investigate the ability of the CDPI3 residue to stabilize short GC-rich and mixed duplexes. Table 3 shows the effect of the same MGB modifications discussed above on GC-rich octanucleotide duplexes, in which the nature of the minor groove is altered. Our test sequence gave a duplex Tmax (with the only modification used in this study, the 3'-hexylamino tail) of 41oC. Addition of a single MGB to either end of strand A increased the Tmax by 16-17oC, while addition to strand B gave a smaller increase in Tmax ([Delta]Tmax = 4-11oC). Strand B showed a preference for the position of CDPI3 conjugation, with its 3'-CDPI3-tailed conjugate forming a more stable duplex (Tmax = 52oC) than its corresponding 5'-CDPI3 derivative (Tmax = 45oC). This effect was seen for all of the other duplexes presented in Table 3 except when strand B has a 2'-O-Me backbone and could be due to the presence of the two AT pairs in the test sequence proximal to the site of conjugation of the MGB.
Addition of the 2'-O-Me modification (Table 3 ) to both strands gave a 25oC increase in Tmax over the 2'-deoxy strands. This has previously been shown to be a stabilizing modification (41 ). A single MGB tethered to strand B did not change the Tmax and addition to strand A gave a modest increase ([Delta]Tmax = 2-6oC). Hybrids between one strand bearing the 2'-O-Me modification and one with a DNA backbone were similar to that of an unmodified DNA duplex (Tmax = 41oC), with duplex stability lower when strand A (Tmax = 37oC) was 2'-O-Me and higher when strand B (Tmax = 46oC) was 2'-O-Me. Interestingly, CDPI3 conjugation destabilized the former of these duplexes. This negative effect was observed for both 3'- (Tmax = 29oC) and 5'-CDPI3-tailed (Tmax = 20oC) derivatives of strand B (the DNA strand). Exacerbating this negative interaction, the phosphorothioate modification of strand B in both these cases gave no detectable melting transition over 0oC, compared with a Tmax of 28oC for the non-conjugated counterpart.
Conversion of both backbones of the DNA octameric duplex to all phosphorothioate linkages reduced the Tmax by 17oC. Addition of a single MGB to strand B gave little change in Tmax (even a decrease of 8oC in the 5'-CDPI3 case) and addition to strand A gave a modest increase of 4-12oC. In general, phosphorothioate analogs of strands A and B demonstrated hybridization properties similar to those observed for phosphodiester ODNs except that all of their complementary complexes have lower Tmax.
The conjugated CDPI3 residue stabilized GC-rich DNA duplexes, with the extent of stabilization being about half, in terms of enhancement of Tmax, of that observed for the d(pT)8[middot]d(pA)8 complex. In contrast, another type of conjugated MGB we have already tested, N-methylpyrrole carboxamide oligomers, failed to stabilize the same GC-rich octadeoxyribonucleotide duplex used in this study (23 ). CDPI3 may be less sensitive to the structure of the minor groove of a duplex than the netropsin-type MPC peptides because it does not form any hydrogen bonds with the bases and the interaction is driven by van der Waals contacts or hydrophobic forces. A narrower minor groove promotes better CDPI3 binding and hence greater duplex stabilization.
CDPI3-conjugated duplexes containing deoxyinosine in place of deoxyguanosine
Substitution of deoxyguanosine (dG) by deoxyinosine (dI) in the modified ODN could create a minor groove environment more suitable for CDPI3 binding, as was observed for netropsin (1 ,42 ,43 ) and Hoechst 33258 (44 ). dI analogs of the GC-rich duplex were prepared and studied with respect to MGB-assisted stabilization (Table 4 ). Replacement of the single dG of strand A with a dI residue gave a 10oC decrease in Tmax. Addition of a single MGB to the 3'-end of either strand of the complex raised the Tmax to a value 7-13oC higher than the parent dG-containing duplex. The effect of the tethered MGB on the duplex containing four dI residues in strand B was dramatic. The duplex formed between this modified stand B and either the native or dI-substituted analog of strand A was weak (Tmax = 11oC) or nonexistent. Addition of the MGB to the 3'-end of strand A or B, however, raised the Tmax to 41-48oC, a 37-48oC increase, close to the stability of the analogous dG-containing native strands. This shows that the conjugated CDPI3 residue stabilizes dIdC-rich sequences as well as dAdT.
Duplexes with two and more conjugated CDPI3 residues
The ability of the minor groove of doubled-stranded DNA to bind two MGB residues in a side-by-side antiparallel orientation has been noted by several groups for different classes of MGBs (45 -49 ). We found that side-by-side binding of two MGB moieties in the duplex minor groove affords hyperstabilization in all duplexes studied which carry two CDPI3 residues tethered to opposite strands (Table 1 and 3 ). For example, a d(pT)8[middot]d(pA)8 duplex, already stabilized by 40-49oC by one conjugated CDPI3 residue, was further stabilized by an additional 5-18oC to reach a Tmax of 74oC for d(pT)8[middot]d(pA)8. This is unprecedented for short phosphodiester-based duplexes. The greatest stabilization occurred when either the 5'- or 3'-end of both strands was modified with an MGB; these could bind in the minor groove in an antiparallel mode (Tmax = 71 and 74oC). The parallel orientation was less beneficial (Tmax = 61 and 68oC). This is consistent with literature data for `free' MGBs in which only the antiparallel orientation was experimentally observed (46 -48 ,50 ).
This hyperstabilization did not seem to depend on either sequence or backbone modification. All AT- and GC-rich duplexes that contained two MGB residues, with one conjugated to each of the opposite strands, were substantially stabilized compared with analogous duplexes bearing a single CDPI3 tail (Tables 1 and 3 ). For example, complexes formed by phosphorothioate analogs of d(pT)8 and/or d(pA)8 possessing two CDPI3 residues showed a Tmax in the same range (Tmax = 56-73oC) as their phosphodiester counterparts (Tmax = 63-74oC). Addition of an MGB to both strands of a duplex with 2'-O-Me modifications increased the stability by 21oC. In the case of the GC duplexes the stabilization resulting from conjugation of a second CDPI3 residue to an opposite duplex strand was even greater than that observed for the first CDPI3 incorporation. For instance, attachment of one CDPI3 residue increased stability of the GC DNA duplex by 4-17oC and addition of the second CDPI3 moiety contributed 18-33oC to Tmax.
The data on duplexes with multiple conjugated MGBs in Tables 1 and 3 show the following trends. If the duplex bore two CDPI3 residues tethered to the same strand at the 3'- and 5'-ends almost no advantage in stability versus the corresponding mono-CDPI3-tailed duplex was seen. This implies a strong hydrophobic interaction between two CDPI3 residues occupying the same site in the minor groove of a short duplex. Furthermore, additional binding between the two MGB moieties attached to the opposite duplex strands appears to add significantly to hybrid stability. Similar effects of interaction of pendant hydrophobic groups on duplex and triplex stabilization were seen with ODNs conjugated to cholesterol residues (51 ). Addition of third and fourth CDPI3 conjugated residues normally had no or a slightly negative effect on stability of the GC-rich duplexes studied. As for the AT-rich duplexes, as more hydrophobic MGB groups were appended less cooperative melting transitions were observed (data not shown). For some of the duplexes we could not accurately determine Tmax.
CONCLUSIONS
The key goal of this study was to develop methods to enhance the stability of a duplex hybrid without sacrificing sequence specificity. It is well established that conjugation of an ODN with intercalating moieties enhances the stability of duplexes formed by those ODNs (19 -22 ). For example, coupling of an acridine residue via a pentamethylene linker to the terminal phosphate of ODNs increased the stability of a d(pT)8[middot]d(pA)8 duplex by 8-9oC under the conditions tested and two acridine residues, each one at the opposite ends of the duplex, increased the Tmax by 14-17oC (unpublished results). Similar effects were reported for 2-methoxy- 6-chloro-9-aminoacridine (52 ) conjugated to the ends of the d(pT)8[middot]d(pA)8 duplex via a pentamethylene linker. At a physiologically relevant ionic strength the duplex was stabilized by 8.8 and 10.7oC for one acridine residue tethered to the 5'- or 3'-end of the octathymidylate strand respectively. Compared with the stabilization provided by the tethered MGBs (Table 1 ), one intercalating residue gives only one quarter the stabilizing effect that a single CDPI3-type MGB does for d(pT)8[middot]d(pA)8 ([Delta]Tmax = 43-46oC). As judged by enhancement of hybrid stability, the MGB conjugates are the strongest binding ODN conjugates thus far reported. This is especially true for AT-rich ODN conjugates. Other research groups investigating properties of ODNs conjugated to Hoechst 33258 (53 ,54 ), netropsin and distamycin A (55 ) came to a similar conclusion. Such greatly enhanced target affinity is a promising way to increase the therapeutic efficacy of ODN derivatives in cell culture.
. Melting temperatures (+-1oC) of dG- and dI-containing octanucleotide duplexes carrying a 3'-CDPI3 residue
Strand A
Strand B
3'-Tailsa
d(AGCGGATG)p
d(AICIIATI)p
3'-Hex-NH2
3'-CDPI3
3'-Hex-NH2
3'-CDPI3
d(CATCCGCT)p
3'-Hex-NH2
41
52
11
-
3'-CDPI3
57
81
48
67
d(CATCCICT)p
3'-Hex-NH2
31
48
~0
41
3'-CDPI3
54
79
48
63
aStructures of the CDPI3 residue and linker for the conjugates are shown in Figure 1.
An interesting application of these conjugates is to lessen the hybridization difference between AT- and GC-rich ODNs. More GC base pairs in the duplex elicit less stabilizing effect of the tethered MGB, but basal duplex stability increases (compared with the AT-rich case) because of the higher GC content. The CDPI3-tailed 8mer duplexes melted at almost the same temperatures: 53-61oC for AT and 45-58oC for GC duplexes (Tables 2 and 2 ).
MGB-tailed ODNs have numerous applications in the diagnostic and therapeutic areas. A particularly relevant therapeutic application for CDPI3-tailed ODNs is targeting single-stranded DNA at preselected AT-rich regions. The large amount of extra energy provided by the conjugated CDPI3 residue is distributed within the short 6-8 bp duplex, locking the modified end of the sequence-specific complex. We have shown that such conjugated ODNs can serve as `clamps' for primer extension of M13mp19 single-stranded DNA (56 ). A DNA polymerase was completely blocked when a complementary 16mer with a conjugated 5'-CDPI3 moiety was hybridized to a site downstream of the primer. On the other hand, 5'-CDPI3-tailed ODNs with an unmodified 3'-end particularly can be used as primers for DNA polymerases. We recently reported that this type of modification allows us to shorten the length of primers in PCR to 8-10mers (57 ). According to our own and recently reported data (58 ,59 ) MGBs conjugated with appropriate linkers can also significantly enhance the hybridization properties of triplex-forming ODNs directed to homopurine runs in double-stranded DNA.
ACKNOWLEDGEMENTS
We thank Dr Vladimir Gorn, David Adams and Deborah Lucas for help with oligonucleotide synthesis. Part of this work was funded by grant GM52774 from the National Institutes of Health, USPHS.
