Recognition of left-handed Z-DNA of short unmodified oligonucleotides under physiological ionic strength conditions.

Abstract

The left-handed Z-DNA form of the short unmodified alternating guanine-cytosine oligonucleotides, 5′-(dGdC)₂₄ and 5′-(dGdC)₁₈, was selectively detected under physiological ionic strength and pH conditions using the anionic nickel(II) porphyrin, NiTPPS. No spectroscopic signal was observed for NiTPPS with any right-handed oligonucleotides under identical conditions. The 48-mer 5′-(dGdC)₂₄ Z-form was detected at concentrations as low as 100 nM. The binding of NiTPPS to the B- and Z-oligonucleotides was studied quantitatively by UV-vis absorption and circular dichroism spectroscopies. NiTPPS was found to be a universal DNA binder, with binding affinity and geometry depending on the ionic composition of the solution, rather than on the DNA helical twist. This is the first example of a successful spectroscopic detection of the Z-DNA of short unmodified oligonucleotides under physiological pH and ionic strength conditions.

Introduction

Left-handed Z-DNA is an intriguing DNA structure[1, 2] which can be formed by certain sequences containing alternating purine and pyrimidine bases. The discovery of proteins that bind to Z-DNA with high affinity and specificity suggests that Z-DNA can play a still unknown vital role in vivo and has stimulated much research in this area.[3] The Z-DNA binding domain in proteins (called Zα) was first identified from human ADAR1 (double-stranded RNA adenosine deaminase)[4, 5] and subsequently found in other proteins (DLM1, E3L and PKZ). The Z-DNA-binding domain in vaccinia E3L has been showed to act as a transcription modulator of several apoptosis-related genes.[6] Another Z-DNA-binding protein, DLM1, has been found to act in the innate immune system as a cytosolic receptor for dsDNA, recognizing foreign pathogenic DNA.[7] Z-DNA also may play a role in regulating transcription of genes. While Liu et al. provided evidence that Z-DNA formed near the transcription start site of the human colony stimulating factor-1 (CSF-1) gene favors its transcription,[8] recently, inhibitory effects of a short Z-DNA forming sequence on transcription elongation by T7 RNA polymerase have been reported.[9] Beyond alternating purine-pyrimidine sequences and binding proteins, DNA some mutations can also stabilize Z-DNA. Naturally occurring 5-methylcytosine or the synthetically prepared 5-bromocytosine, 8-methylguanine, and 8-bromoguanine have been shown to stabilize the left-handed Z-DNA form via shifting the equilibrium to the syn conformation at the N-glycoside bond.[10-12] However, detection of Z-DNA represents a significant challenge as ZDNA is in equilibrium with the canonical right-handed form, known as B-DNA. Thus, it is very important to develop a probe to selectively and sensitively detect this unusual DNA form without altering the equilibrium.

Small molecule DNA probes offer a fast and convenient approach for detecting DNA secondary structure. Norden and Tjerneld (Fe(II) complexes),[13] the Barton group (Ru(II) complexes),[14] and the Sugiyama group (P-helicine)[15] pioneered the use of chiral probes for detecting the handedness of DNA helices of polynucleotides or synthetically modified oligonucleotides (ODNs). We have reported a different approach using achiral porphyrin spectroscopic probes (achiral molecules have smaller tendency to interfere with B-Z-DNA equilibrium) to recognize the left-handed Z-DNA form of poly(dG-dC)₂ with an average length of ~900 base pairs via induced exciton coupled circular dichroism.[16, 17] However, long tracts of Z-DNA are unlikely to be found in vivo,[1, 18, 19] making the detection of short Z-DNA sequences better reflective of physiological conditions. We report here that the anionic nickel(II) meso-tetra(4-sulfonatophenyl) porphyrin, NiTPPS (Figure 1), is able to discriminate and spectroscopically distinguish the Z-form of short CG ODNs (3-4 helical twists) from the B-forms of both CG and AT ODNs, individually, under physiological pH and ionic strength conditions.

Figure 1.

Structure of nickel(II) meso-tetrakis(4-sulfonatophenyl)porphine, NiTPPS.

Materials and Methods

Water was obtained from Milli-Q system with a resistivity of 18.2 MΩ^-1. NiTPPS was purchased from Frontier Scientific, sodium cacodylate was purchased from Aldrich, and oligodeoxynucleotides (ODNs) were purchased from AlphaDNA. NiTPPS stock solution and ODN stock solutions were prepared in Na-cacodylate buffer (pH= 7.0, 1 mM) and stored in the dark at +4 °C. Concentration of ODNs are per strand. All solutions were prepared on the day of the experiment.

Circular dichroism (CD) spectroscopy

CD spectra were recorded at 20 °C using a Jasco J-815 spectropolarimeter equipped with a single position Peltier temperature control system. A quartz cuvette with a 1 cm path length was used for all CD experiments. Each CD spectrum was an average of at least three scans. UV-vis absorption spectroscopy: UV-vis absorption spectra were collected at 20 °C using a Jasco V-600 UV-vis double beam spectrophotometer equipped with a single position Peltier temperature control system. A quartz cuvette with a 1 cm path length was used for all UV-vis experiments.

UV-vis binding experiments

Binding constants for the porphyrin-ODN interactions were determined by absoption spectrophotometric titration at 20 °C as previously reported.[20-24] The fixed amount of NiTPPS (c= 2.24 μM) in Na-cacodylate buffer (1 mM, pH= 7.0) was titrated with the stock solution of DNA (c= 1.80 μM). The changes in absorbance of the Soret band were monitored upon addition of ODN. The apparent binding constant (K_app) was calculated by the following equation: $\frac{{\left[DNA\right]}_{total}}{∕e_{app}-e_f∕}=\frac{1}{∕e_b-e_f∕}{\left[DNA\right]}_{total}+\frac{1}{K_{app}∕e_{app}-e_f∕}$ where e_app corresponds to absorbance of a given solution divided by the total ligand concentration; e_f corresponds to molar absorptivity of the free ligand; e_b corresponds to molar absorptivity of the total bound ligand. In the plot of [DNA]/ (e_app – e_f) vs. [DNA], K_app is given by the ratio of the slope to the intercept. The higher slope of Scatchard plots at low [DNA]/[bound ligand] ratios is due to the accumulation of gaps shorter than n residues in length (n= number of consecutive lattice residues required by the binding molecule).[20]

Results and Discussion

The Z-form of 48mer 5′-(dGdC)₂₄ was successfully induced using millimolar concentrations of NiCl₂. The B-to-Z-DNA transition was monitored by circular dichroism (CD) spectroscopy using Z-form spectroscopic markers, i.e. negative CD bands at 290 and 195 nm, and a positive CD band at 270 nm.[25] Nickel(II) has been shown to induce a B-to-Z-DNA conformational transition in CG polynucleotides at low ionic strength by simultaneous coordination to a phosphate group and an N7 nitrogen of a guanine.[26, 27] The complete B-to-Z transition of the 5′-(CG)₂₄ (1.7 μM DNA strand concentration) was achieved with 2 mM NiCl₂ (blue curve Figure 2a and Figure S1). Further additions of NiCl₂ (up to 50 mM) only caused left-handed Z-DNA conformational changes (black curve Figure 2a) without changes in the DNA helical twist.[28] Spermine and Co(NH₃)₆³⁺, both highly efficient Z-DNA inducers in poly(dG-dC)₂,[29] failed despite all our efforts to produce a left-handed form of the 48mer (Figures S4-11).

Figure 2.

(a) CD spectra of the 5′-(CG)₂₄ in B- and Z-conformations. Inset: B-to-Z transition of 5′-(CG)₂₄ as a function of NiCl₂ concentration. (b) Z-DNA recognition with NiTPPS in the Soret region. Inset: Intensity of the induced CD at 408 nm as a function of NiTPPS concentration added to Z-form 5′-(CG)₂₄ (50 mM NiCl₂ = black circles; 2 mM NiCl₂ + 48 mM MgCl₂ = blue triangles). Condition: c_ODN = 1.7 μM DNA strand concentration, 1 mM Na-cacodylate buffer, pH =7.0, 20 °C.

Addition of NiTPPS to the left-handed 48mer 5′-(CG)₂₄ (1.7 μM) formed with 2 mM NiCl₂ did not elicit an induced CD signal (NiTPPS is achiral and does not have a CD signal) in the porphyrin Soret band absorption region (Figure S17). However, the Z-form induced with 50 mM NiCl₂ (ionic strength I= 150 mM, Figure 2a) displayed a very strong and distinctive trisignate CD signal (+/−/+, a positive Cotton effect at 425 nm and two negative Cotton effects at 455 nm and 390 nm) after addition of 4 μM of NiTPPS (Figure 2b, black line). In fact, as little as 0.5 μM of NiTPPS was enough for successful Z-DNA recognition (Figure 2b, Inset). The multisignate induced CD signal originates from interactions of non-degenerate transition dipole moments between two chirally oriented porphyrins with restricted rotation.[30]

Importantly, addition of the NiTPPS to the B-form of the 48mer 5′-(CG)₂₄ or B-form of the AT 48mer 5′-(AT)₂₄, under the same pH and ionic strength conditions (c_ODN = 1.7 μM, I= 150 mM, pH=7.0) did not result in an observed CD signal in the porphyrin Soret band region (Figure 2 and Figures S16-17). The ionic strength of B-form solutions was adjusted with MgCl₂, as Mg²⁺ binds to DNA electrostatically as a mobile cloud without a complexation to guanine nitrogen N7.[26,31] Magnesium(II) at millimolar concentrations used in our experiments, however, did not induce the left-handed DNA conformation. This allowed us to compare the Ni²⁺ induced Z-form with the corresponding B-form at the same ionic strength of bivalent cations. To corroborate the critical effect of ionic strength on the Z-DNA spectroscopic recognition, we adjusted the ionic strength of the 2 mM NiCl₂ solution to 150 mM with 48 mM of MgCl₂. Under these conditions, NiTPPS successfully sensed the Z-DNA twist, giving rise to a trisignate CD signal of the same pattern (-/+/-) but with lower intensity as was observed using 50 mM NiCl₂ (Figure 2b, blue line). We anticipate that at low ionic strength (I= 6 mM), the Z-DNA does not provide a scaffold necessary for efficient porphyrin-porphyrin electronic interaction and observation of induced CD signal.

The affinity of NiTPPS for the left- and right-handed forms of 48mer 5′-(dCdG)₂₄ under different ionic strength conditions was determined by Scatchard plot analysis of the Soret band absorbance upon the addition of ODN to the NiTPPS (Figures S22-25).[21-23] Binding characteristics with B- and Z-forms of the 48mer 5′-(dCdG)₂₄ are summarized in Table 1. The NiTPPS-ODN binding constants are in the order of 10⁵ M^-1 and depend on the ionic composition of the solution rather than on the DNA helical twist or the ionic strength. For example, the binding constant of NiTPPS with Z-DNA in the presence of 2 mM NiCl₂ and 48 mM MgCl₂ was twice as strong as in the presence of 50 mM NiCl₂ (identical ionic strength, I= 150 mM). Yet, the latter gave rise to a much stronger induced CD signal in the porphyrin Soret band region. This hints that the cation type and cation distribution on the ODN backbone also define porphyrinporphyrin orientation on the helix and thus determine the CD spectroscopic outcome.

Table 1.

Binding characteristics of NiTPPS with 48mer 5′-(dCdG)24 in Z-form and B-form.^a

DNAb	NiCl2	MgCl2	Ic	ICDd	Kappe / M−1
Z	2 mM	-	6 mM	no	6.50 × 105
Z	50 mM	-	150 mM	yes	6.83 × 105
Z	2 mM	48 mM	150 mM	yes	13.4 × 105
B	-	50 mM	150 mM	no	12.5 × 105

^aSee Materials and Methods and ESI for experimental procedures.

^bDNA helicity.

^cIonic strength of the DNA solution.

^dInduced circular dichroic signal in porphyrin Soret band region.

^ePorphyrin-DNA apparent binding constant.

To explore the potential of NiTPPS as the Z-DNA probe of short ODNs we examined different concentrations of the 48mer 5′-(CG)₂₄ as well as different ODN lengths (48mer and 36mer).

Both, the ODN length and the ODN concentration (100 nM, 0.43 μM, or 1.7 μM) had a profound effect on the Z-DNA formation and resulting CD spectra. Although for all ODNs the CD spectra were collected at end points in the NiCl₂ titrations, the B-to-Z transition CD data were less conclusive using traditional Z-DNA CD marker at 290 nm (Figures S2-3). Therefore we followed the B-to-Z DNA transition via the far UV negative CD band at 205 nm. The Z-DNA formation was shown to cause a shift with a rise in negative intensity from ~205 nm for all of the right-handed conformations to ~195 nm for the left-handed conformations, providing the clearest concentration independent CD hallmark for the Z-form in polynucleotides.[25] Addition of NiCl₂ has indeed induced a blue shift of a negative CD band from 206 nm to 202 nm, accompanied with a large increase in intensity. The amount of NiCl₂ necessary for the B-to-Z-DNA transition varied little with the ODN concentration. NiTPPS selectively and reproducibly detected the Z-form of the 48mer 5′-(CG)₂₄ at concentrations as low as 100 nM (Figure 3a), as well as of the shorter 36mer 5′-(CG)₁₈ (Figure 3b). No CD signal was observed for any of the corresponding right-handed B-forms, making NiTPPS the most selective spectroscopic probe of short Z-form ODNs.

Figure 3.

(a) Induced CD at 409 nm of NiTPPS with the 100 nM 5′-(dCdG)₂₄. Inset: Intensity of the induced CD signal as a function of NiTPPS concentration. (b) Induce CD of NiTPPS with the 36mer 5′-(dCdG)₁₈ (2.2 μM). Inset: Intensity of the induced CD at 410 nm as a function of NiTPPS concentration. Conditions: 1 mM Na-cacodylate buffer, pH 7.0, 20 °C.

Conclusion

The anionic nickel porphyrin NiTPPS is a very promising chiroptical probe for short Z-form oligonucleotides under physiological ionic strength conditions (150 mM) via an induced CD signal resulting from porphyrin-porphyrin exciton coupling. In fact, NiTPPS can detect the Z-form of 48mer CG ODNs at concentrations as low as 100 nM, while being spectroscopically silent with all CG and AT B-DNAs, thus showing high sensitivity and selectivity. Additionally, we have shown that NiTPPS-ODN binding strength and NiTPPS orientation on the DNA depend on the ionic composition of the solution, rather than on the DNA helical twist.