Formation of Poly[d(A-T)2] Specific Z-DNA by a Cationic Porphyrin

Typical CD spectrum of the right-handed poly[d(A-T)2] was reversed when trans-bis(N-methylpyrimidium-4-yl)diphenyl porphyrin (trans-BMPyP) was bound, suggesting that the helicity of the polynucleotide was reversed to the left-handed form. The formation of the left-handed Z-form poly[d(A-T)2] was confirmed by 31P NMR, in which a single 31P peak of B-form poly[d(A-T)2] was split into two peaks, which is similar to the conventional B-Z transition of poly[d(G-C)2] induced by the high ionic strength. The observed B-Z transition is unique for poly[d(A-T)2]. The other polynucleotides, including poly[d(G-C)2], poly(dG)·poly(dC) and poly(dA)·poly(dT) remained as the right-handed form in the presence of the same porphyrin. This observation suggests that the porphyrin array that was formed along the poly[d(A-T)2] provides a template to which left-handed poly[d(A-T)2] is associated with an electrostatic interaction.

]. Judging from the shape of the CD spectrum, no other trans-BMPyP-polynucleotide complex forms the Z-form ( Figure S1). In particular, alternating GC polynucleotide, poly[d(G-C) 2 ], which is a representative polynucleotide to form the Z-form in the presence of a high salt concentration or in the presence of other stimuli, remained in the B-form in the presence of the same concentration of trans-BMPyP. Recently, the [Ru(dip) 2 dppz] 2+ complex was reported to induce a B-Z transition for a range of DNA sequences including non-alternating purine-pyrimidine and AT-rich segments under low salt condition 11 , whereas the result shown in this study suggests that the formation of Z-DNA is specific to alternating AT sequence, poly[d(A-T) 2 ]. The other cationic porphyrin, for example, cis-BMPyP (Fig. 1), did not induce a B-Z transition for poly[d(A-T) 2 ] ( Figure S2). In the presence of cis-BMPyP, the CD spectrum remained as the B-form with its positive band between 260 ~ 280 nm and a negative band between 230 ~ 260 nm.
The appearance of a negative CD band at a long wavelength does not necessarily guarantee the formation of the Z-form. For an example, poly[d(I-C) 2 ], a synthetic polynucleotide, produced a Z-form-like CD spectrum. 31 P NMR spectroscopy provides convincing evidence for confirmation of the Z-form DNA 13,14 . In the B-form DNA case, the environment of the phosphate group is homogeneous, whereas that for Z-form falls into two categories owing to its zigzag conformation. As a result, two 31 P NMR  peaks were observed for the Z-form DNAs. Fig. 3 shows the 31 P NMR spectrum for various combinations of polynucleotide and cationic porphyrins. The B-form poly[d(G-C) 2 ] produced one P 31 NMR peak at −1.264 ppm. The addition of 4 M NaCl resulted in a split in the 31 P NMR peak to 0.371 ppm and −1.016 ppm, reflecting the zigzag conformation of the phosphate groups. This justifies the suitability of 31 P NMR for distinguishing the B-and Z-forms. Poly[d(A-T) 2 ] also exhibited a single 31 P NMR peak at -1.234 ppm. On the other hand, the binding of trans-BMPyP resulted in a split of the peak to 0.231 ppm and -1.313 ppm, similar to poly[d(G-C) 2 ] in a high salt concentration. In addition to inverse CD, which was discussed previously, the 31 P NMR spectrum also indicated the formation of the Z-form for poly[d(A-T) 2 ]. In contrast, the binding of a similar porphyrin, cis-BMPyP, did not alter the appearance of the 31 P NMR spectrum in a recognizable extent, suggesting that it is only trans-BMPyP that can induce the Z-form specifically for poly[d(A-T) 2 ].

Interaction of trans-BMPyP with poly[d(A-T) 2 ].
In general, the binding mode of cationic porphyrin to poly[d(A-T) 2 ] can be classified as monomeric minor groove binding, moderate and extensive stacking with increasing [porphyrin]/[DNA base] ratio [15][16][17][18][19][20] . The characteristic CD spectrum, corresponding to each binding mode, has been reported. Porphyrins that bind at the minor groove of poly[d(A-T) 2 ] in a monomeric manner produced a positive CD band, whereas moderately stacked porphyrins exhibited a bisignate CD spectrum in the Soret absorption region. For example, one of the structurally related meso-tetrakis(N-methylpyridium-4-yl)porphyrin (TMPyP) produced a positive CD signal at the Soret absorption region when bound to poly[d(A-T) 2 ] at a low [porphyrin]/[DNA base] ratio, which was shown to bind across the minor groove, being stabilized by an electrostatic interaction between the DNA phosphate group and TMPyP 20 . As the relative concentration of TMPyP increased, the bisignate CD spectrum with a positive band between 390 ~ 430 nm and negative band between 430 ~ 460 nm was apparent, which was assigned to the moderately stacked porphyrin, involving a few porphyrin molecules. This type of stacking occurs in the major groove of DNA 19 . Similar behavior in the CD spectrum was observed for trans-BMPyP at low  the formation of an extensive array of trans-BMPyP (Fig. 5). Any helical polymer of repeating, closely spaced negative charges to which trans-BMPyP binds has been suggested to be capable of providing the template needed to produce such an array 15

Mechanism of poly[d(A-T) 2 ] specific B-Z transition. As it was mentioned previously, poly[d(G-C) 2 ]
has been well-known to form Z form in the presence of a high salt concentration, while trans-BMPyP induced B-Z transition was specific for poly[d(A-T) 2 ]. Observed specificity can be elucidated by difference in the binding mode of trans-BMPyP to these synthetic polynucleotides. Trans-BMPyP has been   2 ] are compared in Fig. 6. As it was reported 17 , trans-BMPyP complexed with poly[d(G-C) 2 ] exhibits a negative CD signal, which has been considered to be a diagnostics for intercalated cationic porphyrins. Therefore, it is conclusive that the binding mode of trans-BMPyP, that is stacking vs. intercalation causes poly[d(A-T) 2 ] specific B-Z transition.
A large number of porphyrins have been known to form J-type aggregations either in the presence or absence of template 21,22 . Two types of aggregation namely Δ -and Λ-macromolecular structure can be formed depending on the direction of stacking, and causes a large bisignate induced CD in the Soret absorption region. Apparent large bisignate CD spectrum observed for trans-BMPyP complexed with poly[d(A-T) 2 ] implies the aggregation of porphyrins on the polynucleotide template. The intensity of this CD spectrum for cis-BMPyP in the same condition was smaller by more ten times compared to that of the trans-BMPyP-poly[d(A-T) 2 ] complex (Fig. 6). Therefore, stacking of cis-BMPyP is far less effective and, consequently, efficient B-Z transition is prevented.
In conclusion, poly[d(A-T) 2 ] forms a left-handed Z-conformation when in the presence of trans-BMPyP. The B-Z transition is associated with the formation of an array of stacked porphyrin and is specific to polynucleotide with alternating AT sequence. Polynucleotides with other sequences, including alternating and non-alternating GC and non-alternating AT, do not form the Z-conformation.

Methods
Preparation and reagents. The porphyrins were purchased from Frontier Scientific, Inc.(Utah, USA) and used as received. Polynucleotides were purchased from Sigma-Aldrich. The synthetic polynucleotides investigated in this study, poly[d(G-C) 2 ], poly[d(A-T) 2 ], poly(dA)·poly(dT) and poly(dG)·poly(dC) were dissolved in 5 mM cacodylate buffer, pH 7.0, containing 100 mM NaCl and 1 mM EDTA by exhaustive shaking at 4 °C followed by several dialyses against 5 mM cacodylate buffer, pH 7.0. The latter buffer solution was used throughout this study. The concentrations of the porphyrins were measured spectrophotometrically using the following extinction coefficients: ε 419 nm = 2. Measurements. The absorption spectra were recorded on a Cary 100 Bio (Australia) spectrophotometer and CD on a Jasco J810 (Tokyo, Japan) spectropolarimeter. The polynucleotide concentration was fixed to 100 μM in the base or phosphate (or 50 μM in base pair), and aliquots of porphyrins were added to the polynucelotide solution to obtain the desired [porphyrin]/[DNA base] ratio. The change in volume was corrected. The pathlength for all CD measurement was 0.5 cm. All measurements were carried out at 25 °C. The 31 P NMR (500 MHz) spectra were recorded on a Bruker AVANCE III 500 NMR spectrometer using 5 mm Broad Band Observe (BBFO: for 19 F as well) probe and the chemical shifts were recorded