Nucleic Acids Research

Molecular modeling

The Insight-II computer package (13) was used for all the modeling. The Gaussian 94 (14) program was used for quantum mechanical (QM) calculations. Molecular dynamics (MD) and MD-thermodynamic integration (TI) calculations were made using AMBER5.0 (15). All calculations used the ORIGIN-2000 parallel supercomputer of the Centre de Supercomputació de Catalunya, as well as work stations in our laboratory.

Theoretical calculations

Chemicals

Protected phosphoramidites and DNA synthesis reagents were purchased from Perkin Elmer-Applied Biosystems (USA). Nucleosides were purchased from Pharma-Waldorf (Germany). Anhydrous solvents were obtained from SDS (France). Phosphodiesterase and alkaline phosphatase were obtained from Boehringer Mannheim (Germany). The rest of the chemicals were from Fluka (Switzerland) and Aldrich (USA) and were used without further purification. 8-Amino-2[prime]-deoxyadenosine (1) was prepared following previously described protocols (27,28).

8-Amino-2[prime]-deoxy-N2,N8-bis(dimethylaminomethyliden) adenosine (2)

8-Amino-2[prime]-deoxyadenosine (27,28) (1) (3.25 g, 12.2 mmol) was suspended in 200 ml of methanol and 8.2 ml of N,N-dimethyl-formamide dimethylacetal (61 mmol) were added. The mixture was stirred overnight at room temperature. The reaction mixture was concentrated to dryness and the residue was dissolved in 10% ethanol (EtOH) in dichloromethane (DCM) and purified on a silica gel column eluted with 15% EtOH in DCM. Yield 3.52 g (9.35 mmol, 76%). TLC (10% EtOH in DCM) R_f 0.29. UV (H₂O) [lambda]_max 267, 334 nm. ¹H-NMR (DMSO-d₆) [delta]_H: 8.81 (1H, s), 8.66 (1H, s), 8.17 (1H, s), 6.57 (1H, t), 5.55 (1H, m), 5.25 (2H, m), 4.46 (1H, m), 3.86 (1H, m), 3.5-3.6 (2H, m), 3.2-3.05 (13H, 3s and 1m), 2.05 (1H, m). ¹³C-NMR (DMSO-d₆) [delta]_C: 157.6, 157.1, 156.0, 155.6, 151.6, 148.4, 124.7, 87.8, 82.9, 71.7, 62.6, 40.4 (under solvent peak), 37.1, 34.5, 34.4. Analysis of C₁₆H₂₄N₈O₃.½H₂O: predicted, C 49.86, H 6.54, N 29.07; found, C 49.52, H 6.57, N 28.88.

8-Amino-2[prime]-deoxy-5[prime]-O-dimethoxytrityl-N2,N8-bis(dimethyl-aminomethyliden) adenosine-3[prime]-O-(2-cyanoethyl)-N,N-diisopropyl phosphoramidite (4)

Compound 2 (1.0 g, 2.65 mmol) was dissolved in 50 ml of dry pyridine and 1.08 g of dimethoxytrityl chloride (3.18 mmol) were added. The mixture was stirred for 2 h at room temperature. To the reaction mixture 1 ml of methanol was added and the solution was concentrated to dryness. The residue was dissolved in DCM and washed with 5% NaCO₃H and saturated NaCl aqueous solution. The organic phase was dried with anhydrous Na₂SO₄ and concentrated to dryness. The residue was purified by chromato-graphy on silica gel. The column was packed with silica gel using a 1% triethylamine solution in DCM. The product was eluted with a 0-4% methanol gradient in DCM. Compound 3 was obtained as a white foam (1.5 g, 2.21 mmol, 83%). TLC (5% EtOH/DCM)R_f 0.6.¹H-NMR (Cl₃CD) [delta]_H: 8.71 (1H, s), 8.68 (1H, s), 8.14 (1H, s), 7.5-7.1 (9H, m), 6.8-6.6 (5H, m), 4.85 (1H, m), 4.18 (1H, m), 3.69 (6H, s), 3.4 (2H, m), 3.16, 3.07, 3.02, 2.93 (13H, 4 s and 1m), 2.2 (1H, m). ¹³C-NMR (Cl₃CD) [delta]_C: 158.1, 157.5, 156.8, 156.7, 155.3, 152.1, 149.2, 144.8, 136.0, 129.9, 128.0, 127.4, 126.4, 124.8, 112.8, 85.9, 85.3, 82.4, 73.2, 64.4, 55.0, 40.8, 40.6, 36.4, 34.8, 34.6.

Compound 3 (1.16 g, 1.7 mmol) and diisopropylethylamine (0.9 ml, 5.1 mmol) were dissolved in 20 ml of dry acetonitrile. The solution was cooled with an ice bath and 0.57 ml of 2-cyanoethoxy-N,N-diisopropylamino-chlorophosphine (2.55 mmol) were added. After 1 h of magnetic stirring at room temperature, the mixture was concentrated to dryness. The residue was dissolved in DCM and washed with 5% NaCO₃H aqueous solution. The organic phase was dried with anhydrous Na₂SO₄ and concentrated to dryness. The residue was purified on silica gel. The column was packed using 1% triethylamine solution in DCM/hexane 9:1 and the product was eluted with DCM/hexane 9:1. Compound 4 was obtained as a white foam. Yield 1.4 g (1.59 mmol, 93%). TLC (DCM/hexane 9:1) R_f 0.8. ¹H-NMR as described in Kawai et al. (12). ³¹P-NMR (Cl₃CD) [delta]_P: 144.96. Analysis of C₄₆H₅₉N₁₀O₆P: predicted, C 62.86, H 6.77, N 15.93; found, C 62.58, H 6.80, N 15.64.

Deprotection studies

Aliquots of dinucleotide supports containing 8-amino-deoxyadenosine (5[prime]-MT-3[prime]) were treated with concentrated ammonia either at room temperature or at 55°C. At different intervals, the ammonia solutions were filtered, concentrated to dryness and analyzed by HPLC. HPLC conditions are described below. A 30 min linear gradient from 0 to 60% B was used. Elution at 260 nm was followed.

Dimer MT. HPLC: retention time 17.2 min; UV_max 269 nm. Mass spectra found 570.4 (expected for C₂₀H₂₇N₈O₁₀P: 570.4).

Dimer MT containing a N⁶,N⁸-bis-dimethylaminomethyliden (dmf) group. HPLC: retention time 18.8 min. UV_max 270, 305 nm. Mass spectra found 625.3 (expected for C₂₃H₃₂N₉O₁₀P: 625.5).

Dimer MT containing two dmf groups. HPLC: retention time: 20 min. UV_max 266, 335 nm. Mass spectra found 679.3 (expected for C₂₆H₃₇N₁₀O₁₀P: 680.5).

Oligonucleotide synthesis

Oligonucleotides were prepared on an automatic DNA synthesizer using standard 2-cyanoethyl phosphoramidites and the modified phosphoramidite 4. Complementary pentadecamers and undecamers containing natural bases were also prepared using commercially available chemicals and following standard protocols. After the assembly of the sequences, the oligonucleotide supports were treated with 32% aqueous ammonia at 55°C for 16 h. Ammonia solutions were concentrated to dryness and the products were purified by reverse phase HPLC. Dimer A and pentamer B were prepared without the last DMT group (DMT-off protocol) on 1 µmol scale. The remaining oligonucleotides were synthesized on 0.2 µmol scale and with the last DMT group at the 5[prime]-end (DMT-on protocol) to help reverse phase purification. All purified products presented a major peak which was collected and analyzed by snake venom phosphodiesterase and alkaline phosphatase digestion followed by HPLC analysis of the nucleosides (HPLC conditions B). The retention times of nucleosides obtained after enzyme digestion were: dC, 4.6 min; dG, 8.4 min; T, 9.2 min; dA, 17.6 min; 8-amino-2[prime]-deoxyadenosine, 21.2 min. Mass spectra: sequence B, found 1501.8 (expected for C₄₉H₆₃N₂₁O₂₇P₄ 1501.9); sequence C, found 4579 (expected for C₁₄₆H₁₈₅N₅₉O₈₆P₁₄ 4575.5). Yield (OD units at 260 nm after HPLC purification, 0.2 µmol): sequence B, 6.3 OD; sequence C, 10.4 OD; sequence D, 16.8 OD; sequence E, 16.9 OD; sequence F, 13.5 OD; sequence G, 9.6 OD.

HPLC conditions

HPLC solutions were as follows: solvent A, 5% ACN in 100 mM triethylammonium acetate (pH 6.5); solvent B, 70% ACN in 100 mM triethylammonium acetate (pH 6.5). For analytical runs the following conditions were used: column, Nucleosil 120C₁₈, 250 × 4 mm; flow rate, 1 ml/min; condition A, a 40 min linear gradient from 0 to 75% B; condition B, a 20 min linear gradient from 0 to 20% B. For preparative runs the following conditions were used: column, PRP-1 (Hamilton), 250 × 10 mm; flow rate, 3 ml/min; 30 min linear gradient from 10 to 80% B (DMT-on) or a 30 min linear gradient from 0 to 50% B (DMT-off)

Melting experiments

Melting experiments of pentadecamer duplexes were carried out by mixing equimolar amounts of two pentadecamer strands dissolved in a solution containing 0.15 M NaCl, 0.05 N Tris-HCl buffer, pH 7.5. Duplexes were annealed by slow cooling from 80 to 4°C. UV absorption spectra and melting curves (absorbance versus temperature) were recorded in 1 cm pathlength cells using a Varian Cary 13 spectrophotometer having a temperature controller with a programmed temperature increase of 0.5°C/min. Melts were run on duplex concentrations of 4 µM at 260 nm.

Melting experiments with triple helix were performed as follows. Solutions of equimolar amounts of the hairpin oligo-nucleotide (h₂₆) and the 11mer (s₁₁) were mixed in the appropriate buffer. The solutions were heated to 80°C, allowed to cool slowly to room temperature and then samples were kept in the refrigerator overnight. UV absorption spectra and melting experiments (absorbance versus temperature) were recorded in 1 cm pathlength cells using a spectrophotometer having a temperature controller with a programmed temperature increase of 0.5°C/min. Melts were run on duplex concentrations of 4 µM at 270 nm.

RESULTS AND DISCUSSION

MD and MD-TI calculations

Quantum mechanical calculations suggest that the replacement of A by M can lead to the formation of an extra H-bond which stabilizes in vacuo the purine·pyrimidine Hoogsteen H-bond. However, 8-aminoadenine behavior in solution cannot be completely described with this method because water greatly hinders the formation of H-bonds. For a more direct estimate, MD-TI calculations were performed. MD-TI calculations can provide a direct estimate of the influence of the replacement of an A by M on triplex stability, by computing the differences in the reversible work necessary to replace an M by A in a duplex and in a triplex DNA (Fig. 3). Calculations were performed on a standard B-DNA d(A·T)₁₀ duplex and a DNA triplex of sequence d(A·T·T)₁₀.

Figure 3. Schematic representation of the thermodynamic cycle used to compute the stabilization of the triplex due to the replacement of an adenine by 8-aminoadenine. Note that the magnitude in a physical sense is [Delta]G(A) - [Delta]G(B), while the magnitude computed from simulations is [Delta]G(1) - [Delta]G(2).

Change of H8 by an amino group does not lead to any significant difference in duplex or triplex geometry. The Watson-Crick and Hoogsteen H-bonds were preserved during all the simulations. The average M·T (WC) and M·T (Hoogsteen) H-bond distances found in the MD are close to the optimum values determined from gas phase calculations. The amino group is placed in the mM groove of the triplex and seems well hydrated.

Mutations were done in the 8-aminoadenine ([lambda] = 1) to adenine ([lambda] = 0) direction. The free energy profiles for the duplex and triplex were smooth, without apparent discontinuity. When the free energy values were computed with the first 10 ps of each window (equilibration part) or with the last 10 ps (averaging part), almost the same free energy profiles were obtained (to <0.1 kcal/mol), confirming the suitability of the sampling protocol used to compute the free energy profile (Fig. 4). There is always concern in TI or FEP calculations about the dependence of free energy differences on the length of the simulation. The perturbation studied here is moderate and implies only small changes in geometry, so 420 ps of trajectory seems reasonable. Test calculations were performed in which the ‘free energy’ difference after seven windows of M->A mutation in the duplex were determined for shorter (equivalent to 210 ps) and longer (equivalent to 1050 ps) runs. The results obtained were 14.2 and 14.8 kcal/mol, respectively, which compare very well with the estimate of 14.1 kcal/mol for the 420 ps trajectory. It seems then that the free energy values reported here converge reasonably. The numbers obtained in the mutations are large due to the introduction of intra-group contributions. Note that such contributions are the same in the duplex and triplex simulations and, accordingly, they should have little effect on the determination of the triplex-inducing effect of the 8-amino group.

Figure 4. Free energy profile for the mutation from 8-aminoadenine ([lambda] = 1) to adenine ([lambda] = 0) in the triplex and duplex systems.

Comparison of free profiles in the duplex and triplex simulations clearly demonstrates that the change from M is 2.7 kcal/mol more favorable in a duplex DNA than in a triplex DNA (Figs 4 and 5), which indicates that the presence of the M leads to a preferential stabilization of the triplex. In fact, the free energy difference of 2.7 kcal/mol detected here indicates a strong preferential stabilization of the triplex over the duplex as a result of the replacement of a single adenine by M in close accord with previous gas phase calculations. Note that the stabilization due to the amino group at position 8 is smaller in DNA in solution than the values obtained for an isolated base pair in the gas phase, confirming the well-known water interference with H-bond formation. However, when these systems are compared to other H-bonding systems (see for instance ref. 17) the effect of water interference on the M·T Hoogsteen H-bond is a lot less than usual, which suggests that the formation of the M·T Hoogsteen dimer is reasonable. In summary, we believe that the roughly 3 kcal/mol stabilization in the triplex due to the existence of a single 8-aminoadenine molecule is related not only to the existence of an extra H-bond, but also to the ability of the amino group to mimic a water molecule in the mM groove of triplex DNA (10).

Figure 5. Differential (triplex-duplex) free energy profile for the mutation of 8-aminoadenine ([lambda] = 1) into adenine ([lambda] = 0).

Moreover, our molecular dynamic simulation results suggest that the change of a single A residue to an 8-aminoadenine in d(A)₁₀ does not introduce dramatic changes in the structural properties of the helix. The 8-amino group does not induce any change of conformation in the glycosidic bond and the amino group interacts nicely with water and the ribose moiety (O4[prime] and O5[prime]). These results suggest that the A->M mutation is conservative in terms of DNA duplex structure. These results confirm QM calculations noted above, which demonstrate the similar stability of Watson-Crick A·T and M·T pairings.

In a similar manner, the introduction of the 8-amino group into a step of the d(A·T·T)₁₀ triplex does not lead to significant changes in the structure of the triplex. MD simulations during 500 ps allowed us to define an average structure (for details see ref. 10) which is very similar to that obtained for the pure d(A·T·T)₁₀ triplex. As in the duplex, the extra amino group seems reasonably solvated and establishes several H-bonding interactions with the ribose moiety and, to a lesser extent, with the phosphate group. In summary, MD suggests that a single A->M mutation in a poly(A) tract does not lead to dramatic alterations in the structure of the triplex.

Preparation of the phosphoramidite derivative and deprotection studies

The synthesis of the phosphoramidite derivative of M is illustrated in Figure 6. 8-Amino-2[prime]-deoxyadenosine (1) was prepared by bromination of dA followed by azide displacement and catalytic hydrogenation of the resulting 8-azido-deoxyadenosine using previously described protocols (27,28). Reaction of 1 with dimethylaminoformamide dimethylacetal gave protected nucleoside 2 in excellent yield. The dmf nucleoside 2 was stable to silica gel purification. Subsequent dimethoxytritylation and phosphitylation gave the desired 8-aminoadenine building block in good yield. The same building block has been described elsewhere (12), but the route described here is shorter and yields are higher.

Figure 6. Preparation of 8-amino-2[prime]-deoxyadenosine phosphoramidite.

The stability of the dmf groups to ammonia was analyzed on the dimer 5[prime]-MT-3[prime]. The dimer was assembled on controlled pore glass using standard phosphoramidite protocols. Aliquots of dinucleotide supports were treated with concentrated ammonia at room temperature and at 55°C. HPLC analysis of the resulting products followed by mass spectrometry analysis of the products showed complete deprotection at 55°C in <3 h. At room temperature only one dmf group was removed rapidly and the dimer containing a single dmf group was slowly converted to the unprotected dimer. Other minor products due to exchange of the dmf groups to acetyl groups during the capping reaction were also observed. All these intermediate compounds were not detected when deprotection was performed at 55°C. Therefore, for the rest of the sequences containing M, all deprotection reactions were carried out overnight at 55°C.

Oligonucleotide synthesis

Oligonucleotide sequences containing M ranging from 5 to 26 bases (Table 1) were prepared using phosphoramidite chemistry on an automatic DNA synthesizer. Coupling efficiency of 8-aminoadenine phosphoramidite 4 was similar to the commercially available phosphoramidites. After standard deprotection with concentrated ammonia, the products were purified by reverse phase HPLC using the DMT-on and DMT-off protocols. In all cases a major peak was obtained and collected. The purified oligonucleotides were analyzed by mass spectrometry (electrospray and MALDI-TOF) and by enzyme digestion followed by HPLC analysis of the resulting nucleosides. In all cases the purified oligonucleotides were obtained in good yield and had the correct nucleoside composition and the expected mass.

Table 1. Oligonucleotide sequences containing 8-amino-2[prime]-deoxyadenosine (M) prepared in this research

		Sequence (5[prime]->3[prime])
A		MT
B		AGMCT
C		GCAATGGAMCCTCTA
D	h261	GAAGGMGGAGATTTTTCTCCTCCTTC
E	h262	GAAGGMGGMGATTTTTCTCCTCCTTC
F	h263	GAMGGMGGMGATTTTTCTCCTCCTTC
G	h264	GMMGGMGGMGMTTTTTCTCCTCCTTC

Base pairing properties

The hybridization properties of 8-aminoadenine were measured spectrophotometrically on a duplex formed by two complementary pentadecamers in which M is paired with the four natural bases (Table 2). 8-Aminoadenine forms the strongest base pairs with T as expected. This base pair is slightly less stable (1.6°C) than the natural A·T. Compared with adenine, 8-aminoadenine base pairs are between 1.6 and 6.4°C less stable than the A·T base pair, while the same base pairs with A were from 0 to 9.6°C less stable than the A·T base pair. There is a tendency to level the melting temperatures of mismatches that comes from a small decrease in the stability of the M·T (1.6°C compared to A·T) base pair and an increase in the M·C (3°C compared to A·C) and M·A (2.9°C compared to A·A) base pairs. This tendency to similar melting temperatures for the different mismatches may assist in the design of degenerate primers or probes. This possibility was investigated with primers containing multiple 8-aminoadenine mismatches for PCR reactions. Unfortunately, no special properties were found in the oligonucleotides carrying M as against the same primers carrying A (data not shown), so precluding its use as a universal base.

Table 2. Melting temperatures (°C) of duplexes containing 8-amino-2[prime]-deoxyadenosine (M) base pairs (0.15 M NaCl, 0.05 N Tris-HCl buffer, pH 7.5)

Xa	Ya
	M	A	G
A	52.6	49.7	54.2
C	54.5	51.5	60.9
T	57.7	59.3	51.3
G	54.9	54.8	56.8

^a5[prime]-TAGAGGXTCCATTGC-3[prime], 3[prime]-ATCTCCYAGGTAACG-5[prime]

QM calculations suggest that the Watson-Crick pairing of M·T is slightly less favorable than A·T. This and the large desolvation penalty upon binding of 8-aminoadenine justify that duplexes containing M are less stable than those with A. Preliminary NMR and circular dichroism analysis of duplexes containing M·T base pairs show no major changes in the duplex structure on the introduction of 8-aminoadenine (data not shown). In summary, neither experimental nor theoretical results suggest that changes of adenine to M cause any dramatic effect on duplex structure.

Triple helix stabilization properties of M were investigated using a triple helix model formed by a self-complementary hairpin of 26 bases (h₂₆) and an all-pyrimidine single-strand oligonucleotide (s₁₁) described previously (3). Sequential replacement of A by M in triple helix results in a stabilization of 5-8°C per substitution in the range from pH 5.5 to 7.0, except when the number of M substitutions goes from three to five residues (Table 3). In any case, the observation of triple helical structures at neutral pH becomes possible only when the M-modified base is used. The pH dependence of triplex stability is due to the presence of G·C·C⁺ triads. These results corroborate those of Kawai et al. (12) obtained on a three-strand triple helix. The degree of stabilization and its additive character are consistent with the existence of an extra hydrogen bond between the 8-amino group of 8-aminoadenine and the 2-keto group of T (Fig. 1) and with theoretical calculations reported above both in the gas phase and in aqueous solution.

CONCLUSIONS

In summary, we predicted by theoretical calculations that the introduction of an amino group at position 8 of adenine would broadly stabilize triple helix formation, due to the combined effect of the gain in one Hoogsteen purine-pyrimidine H-bond and the ability of the amino group to be integrated into the ‘spine of hydration’ located in the mM groove of the triplex structure. Oligonucleotides carrying M residues were synthesized using protected phosphoramidite 4. As we predicted, these oligonucleotides form very stable triple helices. As a matter of fact, the degree of stabilization is one of the highest reported for a non-natural base analog and it could be further increased by adding other reported stabilizing molecules such as intercalating compounds (1,2). The high degree of stabilization obtained by this simple substitution is especially relevant to the development of new applications based on triple helix formation such as structural studies, DNA-based diagnostic tools and antigene therapy (1,2). However, it should not be overlooked that applications using DNA from natural sources such as control of gene expression (1) may not benefit from the use of 8-aminoadenine. Since the modified purine is on the target strand, the direct applicability of this modification in certain applications may be limited. One possible way to overcome this problem may be the preparation of oligonucleotides containing the purine Watson-Crick strand (with 8-aminoadenine) linked with the pyrimidine Hoogsteen strand. If the polarity of the strands is correct, these oligonucleotides may bind the pyrimidine Watson-Crick strand and leave the natural purine Watson-Crick strand unpaired due to the exceptionally high stability of 8-aminoadenine triplexes. Experiments along these lines are currently being undertaken.

Table 3. Melting temperatures^a (°C) for the triplex h₂₆:s₁₁ containing 8-amino-2[prime]-deoxyadenosine (M)

Sequence	Mb	pH 5.5	pH 6.0	pH 6.5	pH 7.0
h26:s11	0	40	~20	-	-
h261:s11	1	48.3	30.9	24	-
h262:s11	2	56	41.8	32.4	22.2
h263:s11	3	65.2	48.7	39.6	26.9
h264:s11	5	68.3	51.9	43.5	32.2

^a1 M NaCl, 100 mM sodium phosphate/citric acid buffer.
^bNumber of substitutions A->M. The duplex T_m values of h₂₆ occurred between 82 and 75°C.

Our results demonstrate the use of ‘state-of-the-art’ quantum mechanics, molecular dynamics and thermodynamic integration techniques in the analysis and design of polynucleotidic sequences. Overall, our results suggest that introduction of amino groups at position 8 of purines may lead to new products with interesting triple helix stabilization properties. Just recently, oligonucleotides carrying 8-aminoguanine have also been shown to form a very stable triple helix (29), which indicates that, indeed, replacement at position 8 of purines by amino groups is key to triple helix stabilization.

See supplementary material available in NAR Online (40 kb PDF file).

ACKNOWLEDGEMENTS

We thank M. Wiersma for valuable suggestions. We also thank Dr Matthias Mann and his group for mass spectroscopic measurements. This research was partially supported by the Centre de Supercomputació de Catalunya (CESCA, Molecular Recognition Project) and the Spanish Dirección General de Investigación Científica y Técnica (DGICYT, PB96-1005).

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*To whom correspondence should be addressed. Tel: +49 6221 387210; Fax: +49 6221 387306; Email: eritja@embl-heidelberg.de

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