Metal Coordination to Ligand-Modified Peptide Nucleic Acid Triplexes

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Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX

Metal Coordination to Ligand-Modified Peptide Nucleic Acid Triplexes Dilhara R. Jayarathna, Heather D. Stout, and Catalina Achim* Department of Chemistry, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, United States S Supporting Information *

ABSTRACT: A challenging goal in nanotechnology is the precise and programmable arrangement of specific elements in nanosystems in the three-dimensional space. The use of ligand-modified nucleic acids represents an accurate and selective tool to achieve this goal when it comes to metal ion organization. The synthesis of peptide nucleic acid (PNA) monomers that contain ligands instead of nucleobases makes possible the creation of metal-mediated alternative base pairs and triplets at specific locations in PNA duplexes and triplexes, respectively. We report the formation of fourand six-coordinate metal complexes between PNA triplexes modified with 2,2′bipyridine (Bpy) or 8-hydroxyquinoline (Q) ligands and 3d metal ions. These metal complexes function as alternative base triplets or pairs in that they increase the thermal stability of the triplexes if the stability constants of the metal complexes are relatively high. The increase in the triplex melting temperature correlates with the stability constants of the metal complexes with ligand-containing PNA determined by UV−vis titrations. The metal complexes coordinate two or three ligands although three bidentate ligands are in close proximity of each other within a triplex. Metal coordination to ligand-modified PNA triplexes was further studied by electron paramagnetic resonance (EPR) spectroscopy and circular dichrosim (CD) spectroscopy. EPR spectroscopy indicated the formation of a square planar [CuQ2] complex between Cu2+ and Q-containing PNA triplex. Taken together, the spectroscopic results indicate that in the presence of 1 equiv of Fe2+ or Ni2+ the majority, but not all, of the Bpycontaining PNA triplexes contain [MBpy3] complexes, with a minority of them being metal free. We attribute this behavior to a supramolecular chelate effect exerted by the triplex, which favors the formation of tris-ligand complexes, that is balanced by the steric interactions between the metal complex and the adjacent nucleobase triplets, which decrease the stability of the complex and triplex. In contrast, the very high stability of square planar [MQ2] complexes of Cu2+ and Ni2+ leads to formation of bisligand complexes instead of tris-ligand complexes with Q3-containing PNA triplexes. The metal-containing PNA triplexes have a terminal L-lysine and adopt a left-handed chiral structure in solution. The handedness of the PNA triplex determines that of the metal complexes formed with the Bpy-containing PNA triplexes.

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INTRODUCTION

Peptide nucleic acid (PNA) is a synthetic analogue of DNA that typically has a neutral, achiral, and acyclic pseudopeptide backbone based on N-(2-aminoethyl)glycine (Aeg), which is in contrast to DNA that has a negatively charged, chiral, and cyclic sugar phosphate backbone (Figure 1).1−5 PNA has the same nucleobases as DNA; the nucleobases are attached to the pseudopeptide backbone by methylene carbonyl linkages. PNA can form very stable duplexes with DNA, RNA, and PNA by Watson−Crick base pairing, which has been attributed to the lack of electrostatic repulsion between the strands and to the relative flexible nature of the PNA backbone. PNA has attracted attention due to its physical and chemical properties that underlie its potential to be useful in scientific and technological applications. For example, significant research is exploring the use of PNAs to build antisense and antigene probes.6−9 Conversely, the incorporation of metal complexes in PNA duplexes and triplexes can broaden its potential applications to nanotechnology and molecular electronics. © XXXX American Chemical Society

Figure 1. Chemical structures of (a) DNA, (b) PNA, and (c) a T:AT nucleobase triplet in a parallel triplex. The bases shown in black form Watson−Crick hydrogen bonds; the pyrimidine base in red forms Hoogsteen hydrogen bonds with the purine base.

The selective self-assembly of ligand-modified nucleic acids allows the programmable arrangement of functional elements in Received: February 18, 2018

A

DOI: 10.1021/acs.inorgchem.8b00442 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry nanoscale systems.10,11 The synthesis of PNA monomers that contain ligands instead of nucleobases makes possible the creation of metal-mediated, alternative base pairs and triplets at specific locations in nucleic acid duplexes and triplexes, respectively.12 In the last 15 years, several research groups have developed numerous nucleic acid duplexes that contain a variety of transition-metal-based, alternative base pairs.13,14 Given the planar geometry of natural nucleobase pairs as well as the contribution of π-stacking interactions to the stabilization of nucleic acid duplexes, square planar metal complexes with aromatic ligands have been the first, and are the preponderant, type of inorganic alternative base pairs that have been incorporated in nucleic acid duplexes. The substitution of two complementary bases with two aromatic ligands makes possible the formation of planar complexes that are structurally similar to nucleobase pairs. The π-stacking interactions can manifest between the ligands within metal complexes and the adjacent nucleobases. However, the substitution of two complementary bases with ligands limits the coordination number and geometry that a metal complex can adopt inside a duplex. From this point of view, nucleobase substitution with ligands in triplex and quadruplex nucleic acid−based structures can lead to complexes with larger coordination number and more diverse coordination geometries, which need to be balanced with the degree of steric compatibility between the metal complex and nucleic acid structure. To date, there have been two reports of metal binding to a DNA triplex in which nucleobases have been replaced with ligands. In 2002, Shionya and collaborators reported the study of a DNA triplex based on 21-base adenine and 21-base thymine strands, each containing a central pyridine ligand.15 The triplex was formed from these strands both in the absence and presence of Ag+, as demonstrated by Job plots in which the absorbance at 260 nm was monitored. Both melting temperatures of the triplex, the lower one corresponding to breaking the Hoogsteen hydrogen bonds and the higher one to breaking the Watson−Crick bonds, were higher in the presence of 4-fold excess Ag+ than in the absence of Ag+. On the basis of this evidence, the authors concluded that Ag+ coordinates the three pyridine ligands in the triplex. This report was followed in 2009 by a paper from the same group on the di-, tri-, and tetraoligomers based on monomers with the same backbone as DNA but with 3-hydroxy-4-pyridone (H) instead of a nucleobase.16 Mass spectrometry and UV−vis spectroscopy studies showed that, in the presence of Fe3+, the oligomers formed triplexes with the chemical formula [Fen(5′-Hn-3′)3] (n = 2−4) in which each Fe3+ coordinated three H ligands. The triplexes adopted a chiral structure due to the presence of chiral centers in the backbone. Ag+ binding to CGC+ nucleobase triplets in DNA triplexes has been reported in several studies.17−21 Just like DNA,22 polypurine and polypyrimidine PNA strands can form triplexes.23 The metallo-regulation of the formation of a ligand-modified PNA/DNA bimolecular triplex was studied in 2013.24 Here we report the study of 3d metal ion binding to PNA triplexes that contain triplets of 8-hydroxyquinoline (Q, Figure 2a) or 2,2′-bipyridine (Bpy, Figure 2b) ligands, as a natural extension to the successful incorporation in PNA duplexes of [ML2] metal complexes using ligands such as Q, Bpy, pyridine, terpyridine, or 1,2-hydroxypyridone and 3d transition metal ions.12,25−29

Figure 2. Chemical structures of (a) 8-hydroxyquinoline (Q) and (b) 2,2′-bipyridine (Bpy) PNA monomers.

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RESULTS A nucleic acid triplex formally consists of a pyrimidine triplexforming oligonucleotide (TFO) bound in the major groove to the purine strand of a nucleic acid duplex to form, for example in a parallel triplex, T:AT (Figure 1c) or C+:GC base triplets. For DNA, the melting of the triplex occurs in two steps, one corresponding to the breaking of Hoogsteen hydrogen bonds and a higher temperature step corresponding to the Watson− Crick hydrogen bonds.30 In contrast, the melting of the three strands of PNA triplexes is a one step, cooperative process.23 The TFO can have a parallel or an antiparallel orientation with respect to the purine strand; DNA triplexes with a parallel structure are typically more stable than triplexes with an antiparallel structure. To avoid consideration of the relative strand orientation in PNA triplexes, we incorporated a ligand in the middle of the polypurine and polypyrimidine strands (Chart 1). Specifically, we have synthesized 11-mer adenine Chart 1. Ligand-Modified Bpy3 PNA and Q3 PNA Triplexesa

a

Italics C- and N- identify the C and N-end of each strand.

and thymine strands in which the central unit contained either a Bpy or a Q ligand (Chart 1). The triplexes are termed in this paper as Bpy3 PNA and Q3 PNA, respectively. Thermal Stability of Triplexes. UV-melting curves were measured for the Bpy3 PNA and Q3 PNA triplexes in the absence and presence of Fe2+, Ni2+, Cu2+, or Zn2+ (Figure 3). The changes in absorbance of the triplex were monitored at 260 nm, the wavelength corresponding to the absorbance of the nucleobases, at 300−320 nm, and at 388−460 nm, the wavelengths corresponding to the π−π* transitions of the coordinated Bpy and Q ligands, respectively. Table 1 shows the melting temperatures of the Bpy3 and Q3 PNA triplexes in the absence and presence of the metal ions. The unmodified triplex that contains 11 AT2 nucleobase triplets has a melting temperature of Tm = 73 °C. The substitution of a central triplet with a triplet of three L ligands to form the L3 PNA triplexes in which L = Bpy or Q causes a large destabilization of the triplex (Tm = 51−53 °C). The melting temperature of the Bpy3 PNA triplex increases significantly in the presence of Fe2+ (ΔTm = 13 °C) or Ni2+ (ΔTm = 15 °C) but is not affected by Cu2+ or Zn2+ (Figure 3a and Table 1). These observations indicate that Ni2+ and Fe2+ coordinate to Bpy ligands in Bpy3 PNA but that Cu2+ and Zn2+ bind weakly if at all to Bpy3 PNA. The melting temperature of the Q3 PNA triplex increased significantly in the presence of B

DOI: 10.1021/acs.inorgchem.8b00442 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry

metal complex having a worse steric fit in the triplex (even if the complex has a planar structure, it may still not be a good geometric fit to the nucleic acid, as our previous studies have shown).26 The absorbance at the wavelengths corresponding to the π−π* transitions of the coordinated Bpy and of the metal-toligand charge transfer transitions of complexes with Q decreases as the temperature increases (Figure 3b and d), indicating that the complexes formed between the Bpy or Q ligands and the metal ions completely dissociate by 90 °C. The data also indicate that the complexes dissociate at a temperature slightly higher than the temperature at which the triplexes melt. UV−Vis and EPR Titrations. Spectrophotometric titrations with Fe2+ or Ni2+ were carried out for the free Bpy ligand and the Bpy3 PNA. The UV−vis spectra of the Bpy ligand and the Bpy3 PNA triplex in the presence of increasing amounts of Fe2+ or Ni2 and the corresponding titration curves are shown in Figure 4 and Figures S1 and S2. The absorbance band at 260

Figure 3. Melting curves monitored at 260 nm/λML for 5 μM Bpy3 (a, b) or Q3 (c, d) PNA solutions in the absence () and presence of 1 equiv of Fe2+ (---), Ni2+ (···), Cu2+ (- − - −), or Zn2+ (··--··--). λML for each sample is given in the footnote to Table 1.

Table 1. Melting Temperature Tm (°C)a for Bpy3 and Q3 PNA Triplexes in the Absence and Presence of 1 equiv of Metal Ion per Triplex Bpy3 PNA 260 nm 2+

no M +Fe2+ +Ni2+ +Cu2+ +Zn2+

51 64 66 52 52

Q3 PNA λML

b

260 nm

λMLb

c

53 54 65 74 58

NCc NCc 65 71 61

NC 65 71 55 55

Tm values are known within 1 °C. bλML for the complexes of Bpy with Fe2+, Ni2+, Cu2+, and Zn2+ were 304, 315, 316, and 315 nm, respectively, and those for the complexes of Q with Ni2+, Cu2+, and Zn2+ were 460, 408, and 388 nm, respectively. cNC stands for “no change observed in absorbance at this wavelength”. a

Figure 4. Spectrophotometric titrations of Bpy (□) and Bpy3 PNA (■) with Ni2+ (a and b) and Fe2+ (c and d), respectively. The lines through the data in parts b and d represent simulations of the titration curves for Bpy (---) and Bpy3 PNA () using HypSpec with the stability constants given in Table 2. The titration curves with Ni2+ were monitored at 296 nm for Bpy and 313 nm for Bpy3 PNA, and with Fe2+ at 525 nm for Bpy and at 304 nm for Bpy3 PNA.

Cu2+ (ΔTm = 21 °C) and Ni2+ (ΔTm = 12 °C), suggesting that Cu2+ and Ni2+ form complexes with the Q ligands (Figure 3c and Table 1), but showed only small changes in the presence of Fe2+ and Zn2+. On the basis of these results, we have focused our subsequent UV−vis, EPR, and CD studies on the metal ions that show the strongest interaction with the modified PNAs, namely, Fe2+ and Ni2+ with Bpy3 PNA and Ni2+ and Cu2+ with Q3 PNA. The hyperchromicity at 260 nm is lower in the presence of Fe2+ or Ni2+ for the Bpy3 PNA triplex than in the absence of the metal ions while the hyperchromicity for the Q3 PNA triplex increases in the presence of Cu2+ and Ni2+. This suggests that the metal coordination to the Bpy ligands negatively impacts the π-stacking of the nucleobase triplets in the case of the Bpy3 PNA triplex while it “improves” the nucleobase stacking in the Q3 PNA triplex. The slopes of the melting curves are lower in the presence of the transition metal ions suggesting that the melting is more gradual and less cooperative when the duplex contains metal ions, which may be due to the

nm is caused by π−π* transitions of the nucleobases; its intensity changes upon addition of the metal ions. New absorption bands arising from π−π* transitions of the coordinated Bpy are observed at 300−330 nm upon addition of the metal ions. In the case of Fe2+, new MLCT bands at 485 and 525 nm are observed also. We have analyzed the UV−vis titration curves of the free Bpy ligand and of the Bpy3 PNA triplexes using the HypSpec refinement program31,32 (see Supporting Information for details), and obtained the stability constants listed in Table 2 and Table S1. This analysis shows that Fe2+ and Ni2+ form [M(Bpy)3]2+ complexes with Bpy3 PNA triplexes but that these complexes are at most as stable as the corresponding complexes formed by the metal ions with free Bpy despite the fact that a C

DOI: 10.1021/acs.inorgchem.8b00442 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry

planar [CuQ2] complexes between the Cu2+ and the triplex irrespective of the amount of Cu2+ added to the solution (Figure 6). Analysis of the titration curves of the Q3 PNA with

Table 2. Stability Constants for Metal Complexes Formed with L3 PNA Obtained from UV−Vis Data Using HypSpec Refinement Program31,32 triplex Bpy3 PNA Q3 PNA

M2+

log β2

Ni Fe2+ Ni2+ Cu2+

log β3 15.7 ± 0.03 16.7 ± 0.1

2+

9.9 ± 0.7 13.9 ± 0.1

supramolecular chelate effect could be anticipated. This property could be due to negative steric interactions between the [M(Bpy)3]2+ complexes and the PNA triplex, which can also explain the fact that the stabilization of the triplex by coordination bonds does not make the triplex more stable than the nonmodified triplex (vide supra). The UV−vis spectra of the Q3 PNA triplex in the presence of increasing amounts of Ni2+ and Cu2+ are shown in Figure 5a,c.

Figure 6. EPR spectrum (black line) of a sample of 100 μM Q3 PNA triplex containing 1 equiv of Cu2+ in pH 7.0, 10 mM sodium phosphate buffer with 25% glycerol as a glassing agent. The red line represents a simulation with g values 2.048, 2.056, 2.223 and hyperfine A values 64.98, 94.26, and 608.3 MHz, respectively.

Ni2+ or Cu2+ with Hypspec led to the stability constants shown in Table 2. HypSpec simulation shows that Cu2+ and Ni2+ form [MQ2] complexes with the triplexes although each triplex contains three Q ligands. Hence each Q3 PNA triplex contains a [MQ2] complex, and all triplexes are bridged into dimers of triplexes by [MQ2] complexes. We attribute this unusual observation to the high stability of the [MQ2] complexes and are using this information to pursue the synthesis of extended networks of PNA triplexes bridged by [MQ2] complexes. Circular Dichroism (CD) Spectroscopy. The PNA backbone is achiral. Hence, the PNA triplexes adopt a preferred handedness only when a chiral unit is incorporated in the PNA oligomers.33 The Q3 PNA and Bpy3 PNA triplexes contain an L-lysine, causing them to adopt a left-handed helical structure as revealed by the negative features at 220 and 255 nm in the CD spectra (Figure 7). Additionally, the Bpy3 PNA and Q3 PNA triplexes show a bisignate CD band at ∼530−535 nm in the

Figure 5. Spectrophotometric titrations of Q3 PNA (■) with Cu2+ (a, b) or Ni2+ (c, d). Titrations curves for Cu2+ and Ni2+ were monitored at 408 and 460 nm, respectively. Solid lines in parts b and d are simulations obtained using HypSpec and the stability constants from Table 2.

The intensity of the absorption bands at 246 and 325 nm, which arise from π−π* transitions of the noncoordinated Q ligands, and of the bands at 409 nm, caused by π−π* transitions of the coordinated Q, changes. These spectral changes establish that Ni2+ and Cu2+coordinate to the Q ligands in the Q3 PNA triplex. The titration curves of Q3 PNA with Ni2+ do not show a sharp inflection point, which precludes one from determining directly the stoichiometry of the complex(es) formed by Ni2+ with the triplex (Figure 5d). On the other hand, the titration curves of the Q3 PNA triplex with Cu2+ show an inflection point at a [M2+]/[Q ligand] ratio of 1:2 (Figure 5b). This indicates that Cu2+ forms [MQ2] complexes with Q3 PNA, although each triplex contains three Q ligands. This result is corroborated by EPR spectra of solutions of the Q3 PNA triplex in the presence of 0.5, 1.0, and 1.5 equiv of Cu2+, which are similar to each other and indicate the formation of square

Figure 7. (a) CD spectra for 5 μM solutions of Bpy3 PNA triplex in the absence () and presence of 1 equiv of Fe2+ (---) or Ni2+ (···). (b) CD spectra of the Q3 PNA triplex in the absence () and presence of 1 equiv of Cu2+ (---) or Ni2+ (···). D

DOI: 10.1021/acs.inorgchem.8b00442 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry presence of DiSC2(5) dye molecules, both in the absence and presence of metal ions (Figure S3). This feature is indicative of the PNA triplex adopting a preferred left-handed structure in solution based on its similarity to the bisignate CD feature previously observed for solutions of the DiSC2(5) dye that also contained a left-handed PNA duplex.34 The bisignate CD peaks obtained for triplex solutions are consistent with dimeric units of DiSC2(5) dyes forming H-aggregates in a face-to-face orientation along the minor groove of the triplexes, a situation similar to that observed for the interaction of the DiSC2(5) dye with PNA/DNA, PNA/PNA duplexes, or bisPNA/DNA triplexes.35 The intensity of the 255 and 220 nm CD peaks of Bpy3 PNA decreases in the presence of 1 equiv of Fe2+ or Ni2+ (Figure 7a). This observation further supports the implication based on changes in hyperchromicity at 260 nm (vide supra) that the octahedral geometry of the [M(Bpy)3] metal complexes negatively impacts the π-stacking of the nucleobase triplets in Bpy3 PNA. In contrast, the intensity of the CD peaks of Q3 PNA at 255 and 220 nm increases in the presence of 1 equiv of Cu2+ or Ni2+ (Figure 7b), which supports the conclusion that [MQ2] complexes, which are square planar, efficiently π-stack with adjacent nucleobases of the Q3 PNA. In the presence of Fe2+ and Ni2+, the CD spectra of the Bpy3 PNA triplex show another positive feature at 300−330 nm, which corresponds to the π−π* transitions of the coordinated bipyridine (Figure 7a). This feature indicates that the structural context of [M(Bpy)3]2+ complexes in the left-handed PNA triplex leads to a preference by the metal complex for either Λ or Δ conformation. A similar UV CD feature was observed for [Ni(Bpy)3]2+ that acted as the core of a 3-way DNA junction and was interpreted as indicative of the Λ conformation of the complex.36 Hence we propose that the [M(Bpy)3]2+ complex formed in the PNA triplex also has the Λ conformation. We measured UV−vis and CD spectra of Bpy3 PNA annealed in the presence of different equivalents of Fe2+ or Ni2+ per triplex (Figure 8). The data were used to construct the

Figure 9. CD (■) and UV (□) titration curves monitored at 318 nm in the presence of (a) Fe2+ and at 308 nm in the presence of (b) Ni2+ with 5 μM Bpy3 PNA.

Figure 10. Speciation diagrams for (a) Bpy3 PNA with Ni2+ (black lines) and Fe2+ (blue lines), and (b) Q3 PNA with Cu2+ (red lines) and Ni2+ (black lines) drawn using the stability constants from Table 2. Note that the speciation diagram shown in part a is for the formation of [MBpy3] complexes and that in part b is for [MQ2] complexes: [ML3]2+ (), [ML2]2+ (---), free [L3] (−·−). The vertical solid lines identify the [M2+]/[L] ratio of 0.33 (i.e., 1 equiv M2+ per L3 PNA triplex). The red circles in part b represent the % [CuQ2] complex measured by quantitation of the EPR spectra.

(vertical lines in Figure 10), 76% or 56% of the Bpy3 PNA triplexes contain [MBpy3]2+ complexes. Excess metal ion with respect to the triplex increases the proportion of the triplexes that contain [MBpy3]2+ complexes to reach ∼80% and ∼70% when the M2::triplex ratio is 3:1, for Fe2+ and Ni2+, respectively. Notably, the rest of the triplexes are metal free. Only for the Q3 PNA triplexes does one observe a full occupancy of the metal binding sites at Cu2+/Q3 PNA > 1:1, with all triplexes containing [MQ2] complexes and being bridged by [MQ2] complexes.

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CONCLUSION This study shows that [Ni(Bpy) 3 ] 2+ and [Fe(Bpy) 3 ] 2+ complexes form with PNA triplexes that contain three Bpy ligands situated in complementary positions. It also shows that [CuQ2] and [NiQ2] complexes form with Q3 PNA triplexes although three Q ligands are situated in complementary positions. These complexes act as metal-based, alternative base pairs or triplets and increase the thermal stability of the triplexes. The thermal melting and UV titration results suggest that the stabilization effect arising from the coordination bonds, which are much stronger than hydrogen bonds within the nucleobase triplets, is mitigated by the steric interactions between the octahedral complexes and the triplexes. The handedness of the PNA triplexes is not affected by the metal ions, and in the case of [MBpy3]2+ complexes, a preferred handedness is induced in the metal complexes by the PNA

Figure 8. CD spectra for 5 μM Bpy3 PNA solutions in the presence of (a) 0.2, 0.4, 0.7, 0.9, and 1.0 equiv of Ni2+ and (b) 0, 0.5, 0.7, 0.9 equiv of Fe2+.

titration curves shown in Figure 9. The intensity of the CD and UV-absorption at 300−330 nm increases with the amount of metal ion added to the solution up to 1 equiv of metal per triplex. These curves confirm the formation of [M(Bpy)3]2+ complexes in the triplexes. The stability constants obtained by simulation of UV−vis titration data were used to construct the speciation diagrams for free ligand Bpy and for L3 PNA in the presence of Fe2+, Ni2+, or Cu2+, Figure 10 and Figure S5. Examination of the diagrams shows that in the presence of 1 equiv of Fe2+ or Ni2+ per triplex E

DOI: 10.1021/acs.inorgchem.8b00442 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry triplex. These findings are of value in the design of hybrid inorganic−nucleic acid architectures.

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The absorbance was corrected for the dilution using the formula (nV + V )

Acorr = A measured × iV , where n is the number of metal ion additions, Vi is the volume of the metal ion added during each addition, and V is the initial volume in the cuvette. The change in the absorbance (ΔA) was normalized using the formulas ΔAni=Ani−Ainitial and NormΔA = ΔAni/ΔAfinal, where Ani is the absorbance after each addition ni of metal ion. Spectrophotometric data were simulated using the refinement program Hypspec.31 See Supporting Information for details. Melting Curves. Variable temperature UV−vis experiments were performed in a Varian Cary 300 spectrophotometer equipped with a programmable temperature block in 1 cm optical path, quartz cells. The melting curves were recorded over a temperature range 15−90 °C for both cooling (annealing) and heating (melting) cycles at a rate of 1 °C/min. The samples were kept for 10 min at 90 °C before cooling and at 15 °C before heating. Samples that contained Fe2+ were prepared in the glovebox, and melting experiments were performed in airtight cuvettes under nitrogen. The temperatures that show the maxima of the first derivatives were used as a measure of thermal stability for each of the triplexes. CD Spectroscopy. The CD spectra were measured at 20 °C at a rate of 50 nm/min and 10 scan accumulation on a JASCO J-715 spectropolarimeter equipped with a thermoelectrically controlled, single-cell holder. For the spectra obtained in the presence of DiSC2(5) dye, 10 min before recording the CD spectra, a dye solution prepared in pH 7.00, 10 mM sodium phosphate buffer and 10% methanol was added to the PNA solutions such that the final dye concentration was 5 μM. EPR Spectroscopy. X-band EPR spectra were recorded on a Bruker 300 spectrometer equipped with an Oxford ESR-910 liquid helium cryostat. The microwave frequency was calibrated with a frequency counter and the magnetic field with an NMR gaussmeter. A modulation frequency of 100 kHz was used for all EPR spectra. The quantification of all signals is relative to a [Cu(EDTA] spin standard. The concentration of the standard was derived from an atomic absorption standard (Aldrich). Temperature calibrations were performed using devices from Lake Shore Cryonics. The spectra were recorded at 17 K under nonsaturating power conditions. The EPR simulation software (Spin Count) was written by Professor Michael P. Hendrich.39 Samples containing 100 μM Q3 PNA triplex and 0.5, 1.0, and 1.5 equiv of Cu2+ in pH 7.0 10 mM sodium phosphate buffer with 25% glycerol as a glassing agent were kept at 95 °C for 10 min, and then slowly cooled to room temperature, transferred to an EPR tube, and frozen.

MATERIALS AND METHODS

Solid-Phase PNA Synthesis. The A and T PNA monomers were purchased from Polyorg and used with no further purification. The Q and Bpy PNA monomers were synthesized according to previously published procedures.25,26,29 PNA oligomers were synthesized by the solid-phase BOC-protection peptide synthesis strategy.37 p-Methylbenzhydrylamine resin·HCl (1.03 mequiv/g, Peptides International) was used as the solid support for PNA synthesis. BOC-Lys(2-Cl-Z)OH (41.5 mg, 0.10 mmol, Fluka) was coupled to 1 g of the resin using 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU from Chem-Impex) and N,Ndiisopropylethylamine (DIEA from Sigma-Aldrich) as the coupling reagent and the base, respectively. Free amine groups of the resin were capped using acetic anhydride (Sigma-Aldrich). BOC-PNA monomers and ligand-modified BOC-PNA monomers were coupled using (2(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU from Chem-Impex) as the coupling reagent and methyldicyclohexylamine as the base. Success of each coupling was assessed by a qualitative Kaiser test. PNA oligomers were cleaved form the resin using m-cresol/thioanisole/TFMSA/TFA (1:1:2:6) and precipitated using cold diethyl ether. Purification of the PNA was done by reversed-phase high pressure liquid chromatography (HPLC) equipped with a C18 silica column on a Waters 600 controller and pump. Absorbance was monitored with a Waters 2996 photodiode array detector. PNA oligomers were characterized by matrix assisted laser desorption ionization coupled to time-of-flight (MALDI-ToF) mass spectroscopy on an Applied Biosystems Voyager biospectrometry workstation using α-cyano-4-hydroxycinnamic acid as the matrix (10 mg/mL in water/acetonitrile, 0.1% TFA). MALDI calcd/expt Lys-A5Bpy-A5, 3193.23/3193.48; Lys-T5-Bpy-T5, 3103.90/3103.95; Lys-A5Q-A5, 3182.20/3183.22; Lys-T5-Q-T5, 3093.11/3094.27. See Supporting Information for MALDI-ToF spectra (Figure S6). PNA stock solutions were made in nanopure water. The concentrations of the PNA solutions were determined by UV−vis spectroscopy at 90 °C using 13 700, 8600, 9770, and 2574 cm−1 M−1 as the extinction coefficients at 260 nm for A, T, Bpy, and Q PNA monomers, respectively.38 The 5 μM solutions of the PNA triplexes were prepared in pH 7.00, 10 mM sodium phosphate buffer except for the solutions of the Bpy3 PNA triplex used in the study of interactions with Fe2+, which were prepared in pH 6.50, 10 mM MES buffer. The solutions of PNA triplexes were annealed by cooling from 90 to 15 °C at a rate of 1 °C/min in a Varian Cary 300 spectrophotometer equipped with a programmable temperature block. Spectrophotometric Titrations. The binding of metal ions to ligand-modified PNA triplexes was determined by spectrophotometric titrations performed in a Varian Cary 50 spectrophotometer. Titrations of Bpy3 PNA or Bpy with Ni2+ were done in pH 7.0, 10 mM sodium phosphate buffer while those with Fe2+ were done in pH 6.50, 10 mM MES buffer. The concentrations of Bpy solutions were 70 and 100 μM for the titration with Ni2+ and Fe2+, respectively. Titrations were carried out by addition of aliquots of known volumes and concentrations of metal ion solutions in water to PNA solutions of total strand concentration 15 μM. Metal ion concentrations for the titrations with Bpy were 1000 μM (Ni2+) and 2000 μM (Fe2+), and those for the titrations with Bpy3 PNA were 500 μM (Ni2+) and 1500 μM (Fe2+). The Bpy3 PNA solutions for titrations with Fe2+ were prepared in the glovebox, and the titrations were performed using an airtight titration setup. Bpy3 PNA was titrated with Ni2+ in solutions in which the ionic strength was controlled and in solutions in which it was not controlled. Comparison of these titrations showed that ionic strength had no significant effect on the stability constants. 5 μM solutions of Q3 PNA in pH 7.0 10 mM sodium phosphate buffer were titrated with 400 μM solutions of Cu2+. 25 μM solutions of Q3 PNA in the same buffer were titrated with 500 μM solutions of Ni2+.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.8b00442. PNA strand orientation, CD spectra for triplexes in the presence of DiSC2(5) dye, spectrophotometric titrations of free ligands Bpy and triplexes, and description of HypSpec simulations (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (C.A.). ORCID

Catalina Achim: 0000-0001-5420-4656 Author Contributions

All authors contributed to the research design and analysis of the data. D.R.J. was responsible for the synthesis and the UV− Vis and CD spectroscopy experiments. H.D.S. was responsible for the EPR spectroscopy experiments. The manuscript was F

DOI: 10.1021/acs.inorgchem.8b00442 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry

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written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to the US National Science Foundation (1310441) for financial support of this research. We thank Dr. Michael Hendrich for the EPR simulation software. MALDITOF spectrograms were recorded in the Center for Molecular Analysis at Carnegie Mellon University, which was supported by NSF Grant CHE-9808188.

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DOI: 10.1021/acs.inorgchem.8b00442 Inorg. Chem. XXXX, XXX, XXX−XXX