Structural Basis of the Mispairing of an Artificially Expanded Genetic Information System
The synthetic P-Z genetic system base pairs by a unique arrangement of three hydrogen bonds. Burley and colleagues describe a study that highlights how loss of this synthetic genetic information is driven by Z mispairing with G. They show that G-Z mispairing has two highly pH-dependent pairing regimes whereby Z acts as an anionic mimic of C in a G-C base pair at pH > 7.8.
INTRODUCTION<!>The Bigger Picture<!>Experimental Design<!>Crystal Structural Analysis of DNA Duplexes Containing P-Z and G-Z Base Pairs<!>Analysis of Maintenance of Z-P Base Pairs during PCR<!>DISCUSSION<!>A G-Z Mismatch Leads to Significant Local Distortion within a B-Form Duplex at Neutral pH<!>Stabilization of the Z Anion Is the Source of Mispairing<!>G-Z Mispairing Compromises Orthogonality of P-Z Pairing<!>Conclusion<!>EXPERIMENTAL PROCEDURES<!>CD Analysis<!>Crystallization and Data Collection<!>Structure Solution and Refinement<!>PCR<!>ACCESSION NUMBERS<!>SUPPLEMENTAL INFORMATION
<p>Nucleic acids are the fundamental repository of genetic information found in living organisms. Unique to this family of biomacromolecules is the ability of DNA to act as a template for the replication and storage of genetic information; levels of fidelity approach a single-base substitution every 10 6 nucleotides for proof-reading-deficient DNA polymerases. 1 Maintaining the genetic integrity (replication) and high-fidelity information transfer (transcription and translation) of the primary sequence of nucleic acids is contingent on the discrimination of two hydrogen-bond regimes: pairing A with T via two hydrogen bonds and pairing G with C via three hydrogen bonds 2,3 (Figure 1A). Base pairing that deviates from conventional Watson-Crick profiles is a source of mutation and, subsequently, adaptive evolution.</p><p>The development of an expanded genetic repertoire might have many uses, but it would require the design of synthetic base pairs that pair orthogonally and exclusively with each other and not with naturally occurring nucleotides. [4][5][6][7][8][9][10][11] The Hirao and Romesberg groups have developed synthetic base pairs that rely on complementarity of shape rather than hydrogen bonding to pair preferentially with each other. Both systems enable replication fidelities approaching that of natural Watson-Crick base pairs. 4,5,10,[12][13][14][15][16][17][18][19] A particular mutational hallmark of these hydrophobic base pairs is the gradual conversion of the synthetic base pair into an A-T pair observed both in cellulo and via repeated rounds of PCR, although the structural basis for the loss of synthetic information is at present not known. 16,18 An alternative design approach is an artificially expanded synthetic genetic information system (AEGIS) where synthetic base pairs present a unique arrangement of</p><!><p>Base pairing is a fundamental structural and functional property that allows the storage and propagation of genetic information by the exploitation of two types of pairing rules: A pairs with T, and G pairs with C. Although nature has confined itself to a four-letter code, synthetic base pairs that pair with each other and not with naturally occurring building blocks (i.e., A, G, C, and T) can expand our genetic repertoire. This is important because such designs can be used in a variety of applications ranging from the development of new synthetic biological systems to new diagnostic agents. Contingent on their wider application is the inherent need to understand not only how synthetic genetic systems pair with each other but also how they mispair with naturally occurring nucleotides. This article describes how an established synthetic genetic system can mispair with a naturally occurring nucleotide. hydrogen bonds in relation to Watson-Crick base pairs. [20][21][22] The most recent incarnation of this strategy is the P-Z base pair (Figure 1B). 20,[23][24][25] This design archetype has been used in applications ranging from aptamer development to their development as primers in DNA diagnostics applications. [26][27][28] A recent structural study of an oligodeoxyribonucleotide (ODN) duplex containing four P-Z base pairs revealed a structure that adopts a conventional B-type conformation when in complex with a host protein (N-terminal fragment of Moloney murine leukemia virus reverse transcriptase [MMLV-RT]). However, an ODN duplex containing six consecutive P-Z base pairs formed an A-type duplex in complex with MMLV-RT, 23,25 which suggests a level of structural plasticity, although it is unclear whether this is due to the sequence context of the ODN and/or host protein binding.</p><p>Another unique feature of the P-Z base pair is that the loss of this synthetic genetic information occurs via conversion into a G-C base pair under PCR amplification conditions. 21,22 The putative mechanism for this loss is that the conjugate base of the Z nucleoside (i.e., deprotonation of N1-H of Z, pK a $ 7.8) 21 might be an anionic mimic of C, resulting in the competitive incorporation of dGMP in relation to dPMP opposite a Z-containing DNA template. 20,21 Critical for future applications of synthetic genetic systems is the need for a comprehensive understanding of the molecular determinants of mispairing between synthetic nucleotides and naturally occurring nucleotides when incorporated into duplex DNA. Using a combination of PCR analysis of base-pair fidelity, X-ray crystallography, UV-visible (UV-Vis) thermal melts, and circular dichroism (CD) spectroscopy, we show that G-Z mispairing is remarkably plastic; it pairs via a mixture of canonical (i.e., three hydrogen bonds) and non-canonical (i.e., two hydrogen bonds) regimes that are aligned with the pK a of the Z N1-H (Figure 1C). 22</p><!><p>This work had two principal aims: (1) to determine the structural and thermodynamic differences between a ''matched'' P-Z pair and a ''mismatched'' G-Z pair as a function of the pK a of the Z N1-H and (2) to understand whether the fidelity and amplification of P-Z information is influenced by pH. The experimental design that was used to address our first aim focused on obtaining crystal structures of dodecamer ODN duplexes containing P-Z or G-Z base pairs in the absence of a host protein. This was an important consideration given that host protein binding could alter the pairing profile, particularly with short oligonucleotides, and thus complicate their validity and interpretation. The archetypical Dickerson dodecamer d(CGCGAATTCGCG) 2 (ODN1), for which a crystal structure has previously been determined in the absence of host protein and presence of Ca 2+ (PDB: 463D), 29 was used as a comparison for our structural and spectroscopic studies because this self-complementary sequence forms B-type DNA duplexes with both natural and modified nucleotides. [30][31][32][33][34] The d(CGCPAATTZGCG) 2 sequence (ODN2) was prepared by solid-phase DNA synthesis according to established protocols and consists of two P-Z base pairs in a self-complementary duplex in positions 4 and 9. 22 The dodecamer sequence d(CGCGAATTZGCG) 2 (ODN3), prepared by the same methodology, contained a double G-Z mismatch in the same positions. Finally, the structural and thermodynamic properties of ODN1-ODN3 were compared to a naturally occurring duplex containing a double G-T mismatch in positions 4 and 9 (ODN4 [PDB: 113D]). 35 All four ODNs formed well-defined B-type duplexes. Unlike ODN4, which was solved in the presence of Mg 2+ 22 , ODN2 and ODN3 did not readily crystallize at pH < 7.8 in the presence or absence of that cation. ODN2 and ODN3 were therefore solved in the presence of Ca 2+ at pH 7 (for ODN2), pH 7.8 (for ODN3a), and pH 8.5 (for ODN3b). The previously published ODN1 structure (at pH 7 in the presence of Ca 2+ ) 29 had similar crystal packing (indexed as space group R3 for ODN1 and H3 for ODN2 and ODN3, which are equivalent).</p><!><p>Within the crystal lattices, the duplex structures of ODN2 and ODN3 are asymmetric, leading to non-identical conformations for each of the P-Z (ODN2) or G-Z (ODN3) pairs. An overlay of ODN1-ODN3 (Figure 2A) indicates broad similarity with no substantial distortion away from the B-type conformation as a consequence of the insertion of the artificial bases in both ODN2 and ODN3 (main chain RMSD < 0.6 A ˚). A triple-hydrogen-bonded P-Z pairing was observed in ODN2 with hydrogen-bond lengths of 2.7-2.8 A ˚, which is comparable to G-C hydrogen-bond lengths observed elsewhere in this 2.2 A ˚resolution structure (Figures 2B and 2C). The hydrogen-bonding distances were also equivalent to those reported in an earlier P-Z-containing structure, 23 suggesting that the adjacent bases do not affect the P-Z pairing (Table S4). The inclusion of two P-Z pairs in ODN2 is accompanied by a slight widening of the major groove by approximately 1 A ˚. In ODN2, ODN3a, and ODN3b, the Z NO 2 group is closely planar to the remainder of the aromatic ring system. Consequently, one of the Z NO 2 oxygens is positioned where it could establish an intramolecular hydrogen bond with the exocyclic Z NH 2 (N = O$$$H-N bond length of 2.7 A ˚), thus forming a pseudo-bicyclic ring system. 23,25 Because the Z NO 2 group projects into the major groove, this additional steric bulk might contribute to the slight widening of the groove observed in ODN2 and other Z-P sequences.</p><p>Geometric analysis of ODN3a (pH 7.8) suggests that distortion in relation to ODN1 is greatest around the G-Z mismatch pairs (Table S5). For example, the G-Z pair located at position 4 in ODN3a has propeller twists of approximately 10 greater than those observed in either of the ODN1 or ODN2 structures and, similarly, about 3 increases in buckle angles. This substantially reduces the co-planarity of the base pairs and alters their interconnecting hydrogen bonds. G-Z pairing in ODN3a also exhibits shearing of up to 1.5 A ˚, which is significantly greater than the shearing observed for P-Z pairs (0.3 A ˚in this study) in ODN2, although the analysis is partially limited by the lower resolution (2.5 A ˚) of the G-Z structure at pH 7.8.</p><p>There is a striking difference in the G-Z base-pairing profile between the structures at pH 7.8 (ODN3a, Figure 2C) and pH 8.5 (ODN3b, Figure 2D). The observed electron density indicates that the G-Z in position 4 of ODN3a is present as a slipped or ''wobble-like'' pair containing two hydrogen bonds: (G)O6-N1(Z) at 3.0 A ˚and (G)N1-O2(Z) at 3.1 A ˚(Figure 2D). In this arrangement, both the ring N1 atoms of G and Z must be protonated. This pairing is reminiscent of G-T mispairing observed in ODN4; however, the distortion of the base pair is less pronounced (Table 1). In contrast, as with the G-C and Z-P interactions in ODN1 and ODN2, respectively, the G-Z base pair present in position 9 of ODN3a forms three hydrogen bonds in a pseudo-G-C Watson-Crick arrangement: (G)N1-N1(Z) 3.1 A ˚, (G)O6-N4(Z) 3.1 A ˚, and (G)N2-O2(Z) 3.2 A ˚. It is likely that the anionic form of Z is present, as exemplified by the longer bond lengths than those of the nearest natural base pairs (2.8-3.0 A ˚). Collectively, our crystal structural analysis has shown that G-Z pairing is highly pH dependent.</p><p>Because the ODN3a crystals were grown at a pH corresponding to the pK a of the N1-H of Z (i.e., 7.8), 22 where a mixed population of the neutral and anionic forms of Z is possible, we conclude that Z can pair with G in two different hydrogenbonding arrangements. When crystals of ODN3b were grown at pH 8.5, only a single type of triple-hydrogen-bonded arrangement was observed (Figure 2E), which is consistent with the presence of both Z nucleotides in their deprotonated form. Although these observed structures are consistent with the expected protonation states of Z given that its pKa was measured with the free base, we cannot rule out that its incorporation in B-type DNA causes a shift in the pK a . 36,37 Duplex Stability of G-Z Pairing Is Highly pH Dependent UV-Vis melting and CD experiments were then conducted for further exploration of how the pH dependence of G-Z mispairing affect the thermal stability and structure of ODN duplexes in solution. Whereas ODN1 and ODN4 exhibited virtually no pH dependence by UV-Vis melting, the P-Z-containing ODN2 exhibited slight (2 C) destabilization as the pH was raised from 5.5 to 7.5 (Figure 3A). In contrast, the duplex melt of ODN3 was highly pH dependent and resulted in a 12 C stabilization as the pH was increased from 5.5 to 8.0 (Table S1). No further increase in the duplex melt was observed above 8.0, which indicates that the thermal stability of duplex ODN3 is aligned with the pK a of the N1 of Z. There was also a significant difference in the duplex melts of ODN3 and ODN4, the latter of which contains two G-T mispairs. No pH dependence was observed for ODN4, which is significantly destabilized in relation to ODN1 and ODN3. All four ODNs exhibited a characteristic right-handed B-type duplex according to CD (i.e., negative peak around 245 nm and positive peak between 260 and 280 nm) 38 using a sodium cacodylate buffer at both pH 5.5 and 7.5 (Figure 3B). Collectively, UV-Vis thermal melts and CD show that G-Z mispairing is both highly pH dependent and significantly more stable than a corresponding G-T mispair. These results align with the pairing plasticity seen in the crystal structures of ODN1-ODN3.</p><!><p>We set out to explore the capacity of the Z-P information to be maintained in the absence of the corresponding modified triphosphate (i.e., dZTP when P is present in the DNA template or dPTP when Z is present in the DNA template). Taq polymerase was chosen because it has been shown previously to incorporate dZTP and dPTP. 26 However, extensive PCR analyses using a plasmid template and primers containing Z or P, closely following published conditions, 22 showed that the other nucleotides readily misincorporated during PCR (Figure S35 and S36).</p><p>To test whether the propensity to lose P-Z information during PCR amplification might arise from the pH-dependent ionization state of Z, we analyzed the pH dependency of PCR by using a range of P-Z DNA templates. To maintain consistency with our UV-Vis melting data, we then conducted PCR by using the same cacodylate buffer composition from pH 5.5 to 7.5. Again, there was no evidence that amplification was reduced in the absence of the cognate dPTP or dZTP (Figure 4). To maximize the possibility of observing a pH-dependent effect, we investigated the PCR amplification of a strand containing three consecutive P-Z base pairs. PCR amplification was unaffected by the omission of the complementary dPTP and dZTP (Figure S37). We conclude that AEGIS nucleotides do not allow faithful propagation of a template containing P or Z nucleotides across a pH range used in PCR.</p><!><p>These experiments were designed to determine the structural, spectroscopic, and functional impact of pH on P-Z ''matched'' pairing in relation to that of G-Z ''mismatched'' pairing. Here, we discuss three major conclusions that emerged from our results.</p><!><p>The crystal structures of ODN2 and ODN3 clearly illustrate the ability of Z-containing ODNs to pair with both artificial P and natural G nucleotides while maintaining an overall B-form duplex (Figure 2). An unexpected observation is that the ODN3 structure obtained at pH 7.8 captured both two (Wobble-like) and three (pseudo-Watson-Crick) hydrogen-bond arrangements of the G-Z mispair, highlighting the pairing plasticity of Z mispairing with G at physiologically relevant pH. We infer that our difficulty in obtaining reproducible diffraction-quality crystals of ODN3 at a pH < 7.8 is most likely due to the instability of the double-hydrogen-bonded G-Z slipped pair to promote well-ordered crystallization. This is supported by our UV-Vis thermal melting data, which show a strong stabilization effect under slightly basic conditions (Figure 3A). It is notable that significant accompanying geometric distortions are limited to the G-Z base pairings and their immediately adjacent bases. Further, the ODN3 structure at pH 7.8 implies that a transition between the double-and triple-hydrogen-bonded pairing arrangements can be readily accessed without disruption of the adjacent duplex structure. These observations imply that G-Z mismatches would be readily tolerated in thermal cycling conditions used in PCR amplification. 20,22 One explanation of the results might be that the effective local pH in the active site of the enzyme is closer to the optimal pH ($8.3) for the reaction.</p><!><p>UV-Vis melting experiments confirm that the stability of the G-Z mispair is pH dependent, i.e., contingent on a negative charge forming on the Z nucleobase. The Z NO 2 group and the O2 carbonyl provide extensive conjugation of the resultant anion, which could bias the negative charge either within the interior of the duplex (Z N1 À ) 39 or projected into the major (Z NO 2 À ) or minor (Z O2 À ) groove, as depicted schematically in Figure 5. Furthermore, the Z NH 2 group in position 6 could provide additional stabilization of this anion via an intramolecular hydrogen bond with the Z NO 2 in position 5. A similar intramolecular hydrogen-bonding profile of a negatively charged nucleobase has also been observed with 5-carboxycytosine, where the carboxylate group also projects into the major groove and forms an intramolecular hydrogen bond with the adjacent exocyclic amine. 40 Thus, the combined structural characteristics of a strongly electron-withdrawing Z NO 2 group increasing the acidity of the Z N1-H, extensive stabilization of the anion by resonance, and the possible presence of an intramolecular hydrogen bond between the Z NO 2 and the exocyclic Z NH 2 provide a unique synthetic nucleotide where base-pairing stability can be tuned within a physiologically relevant window. 41</p><!><p>One of the most important applications of synthetic genetic systems is in PCR, where the orthogonal nucleotides might be introduced into selected sites and, after amplification, used as the basis for incorporation of modified versions that would provide site-specific probes for fluorescence or cross-linking in either DNA or RNA transcripts. [42][43][44] For these applications, complete fidelity in practical terms is an essential requirement, given that PCR products or transcripts containing P or Z could not be readily purified in preparative experiments from contaminants containing G or C. We set up a series of stringent tests of the ability of the P-Z pair to be faithfully propagated during PCR. Various P-and Z-containing templates were amplified in the absence of the corresponding modified triphosphate (i.e., dPTP or dZTP). We observed the preparation of full-length PCR amplicons over a pH range below the pK a of Z (i.e., where Z should be in the fully protonated state), and there was no indication of significantly reduced efficiency. We therefore conclude that sufficiently faithful propagation of P-Z information is not possible with the current design.</p><!><p>In summary, our study illustrates the complexities of designing synthetic base pairs directed by subtle differences in hydrogen bonding. The incorporation of the nitro group in the Z nucleotide design was originally introduced in order to reduce the rate of C1 0 epimerization. 21 However, this increases the acidity of the N1-H proton, which contributes to mispairing with G. This subtle interplay highlights the need for new AEGIS designs that are chemically stable while maintaining high pairing fidelity. Furthermore, this study highlights the need for further structural and biochemical studies to be conducted on understanding the mechanisms of the loss of synthetic genetic information. We envisage that this work will assist in the design process of new AEGIS and other synthetic base pairs that have a reduced propensity to mispair with natural nucleotides.</p><!><p>Synthesis and Purification of P-Z-Containing ODN2 and ODN3 ODN2 and ODN3 were synthesized according to standard solid-phase oligonucleotide-synthesis protocols on an ABI 394 synthesizer. 22 Phosphoramidites and controlled pore glass (CPG) supports loaded with standard nucleosides were purchased from LINK Technologies. AEGIS phosphoramidites (dZ and dP) were purchased from Firebird Biomolecular Sciences (Alachua).</p><p>Deprotection Protocol for ODN2 and ODN3 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU, 1 M in MeCN, 3 mL/mmol) was added to Z-containing ODNs immobilized on a CPG (1 mmol) support. The suspension was shaken overnight at room temperature for the removal of the 1-(2-nitrophenyl)ethyl protecting group from the Z nucleobase. The DBU solution was removed, and the CPG support was washed once with MeCN. Ammonia (DNA grade, 3 ml/mmol) was added, and the suspension was shaken for 3 hr at 55 C. The yellow supernatant was removed after 3 hr. The CPG support was then washed with ammonia (1 mL/mmol), and the combined layers were concentrated under reduced pressure to yield crude ODN2 and ODN3 as a yellow solution, which was purified by reverse-phase high-performance liquid chromatography (RP-HPLC) on a Dionex UltiMate 3000 System. ODN1 and ODN4 were purchased from Eurofins and Eurogentec, respectively.</p><p>Analytical Data MALDI-ToF mass spectra were recorded with a Shimadzu Biotech Axima CFR spectrometer with 3-hydroxypicolinic acid as the matrix. All mass spectra were recorded in negative mode (Figures S1-S4). Analytical RP-HPLC was performed on a Dionex UltiMate 3000 system with a Clarity 5 mM Oligo-RP Column (250 3 4.6 mm) in a triethylammonium acetate (TEAA) buffer system (buffer A: 100 mM TEAA [pH 7.5] in water; buffer B: 100 mM TEAA [pH 7.5] in 80% MeCN; flow rate: 1 mL/min; gradient as described in Table S1). For ODN1 and ODN2, spectra were obtained at a column temperature of 80 C for minimizing peaks caused by secondary structures (duplex formation) (Figures S5-S8). Thermal UV-Vis Measurements Cacodylate buffer (10 mM sodium cacodylate, 10 mM KCl, 10 mM MgCl 2 , and 5 mM CaCl 2 ) was used as the buffer system. The different pH values were adjusted with 1 M HCl and 1 M NaOH. 40-50 mM stock solutions of ODN1-ODN4 were diluted to 4 mM in 10 mM cacodylate buffer (10 mM sodium cacodylate, 10 mM KCl, 10 mM MgCl 2 , and 5 mM CaCl 2 ) previously set to the desired pH. UV measurements were obtained with a Shimadzu UV-1800 with an eight-series micro multi-cell, each of which contained a sample volume of 100 mL. The UV spectra were obtained over a range of 20 C-90 C for ODN1 and ODN2 and 20 C-70 C for ODN3 and ODN4 with a ramp speed of 0.5 C/min and measurement intervals of 0.2 C. The measured wavelength was 260 nm. All measurements were performed at least four times to provide an average value (Tables S2 and S3 and Figures S9-S28).</p><!><p>For CD analysis, 40-50 mM stock solutions of ODN1-ODN4 were diluted to 10 mM in 10 mM cacodylate buffer (10 mM sodium cacodylate, 10 mM KCl, 10 mM MgCl 2 , and 5 mM CaCl 2 ) set to either pH 5.5 or 7.5, the same conditions as for the UV spectroscopy. CD spectra were obtained on a Jasco J-810 instrument at 25 C, a rate of 20 nm/min, and a wavelength increment of 0.2 nm (Figure 3B). In addition, ellipticity (mdeg) was recorded for ODN1-ODN3 between 320 and 202 nm in intervals of 5 C over a range of 25 C-85 C at pH 5.5 and 7.5. All spectra were corrected against elliptic readings obtained by the buffer (10 mM sodium cacodylate, 10 mM KCl, 10 mM MgCl 2 , and 5 mM CaCl 2 ) (Figures S29-S34).</p><!><p>Thin, platelike crystals of ODN2 duplex (CGCPAATTZGCG) 2 were obtained through sitting-drop vapor diffusion after 200 nL of 0.5 mM ODN2 in water was mixed with 200 nL precipitant solution, containing 20 mM sodium cacodylate [pH 7.0] and 600 mM calcium chloride. The drop was equilibrated against a reservoir containing 50% hexylene glycol at 298 K. The crystals were flash frozen in liquid nitrogen and diffracted to 2.17 A ˚. These crystals were indexed in space group H3 with unit cell a = b = 41.7 A ˚and c = 99.9 A ˚, a = b = 90 , and g = 120 , containing two strands of DNA. Diffraction data were collected on beamline I04-1 at Diamond Light Source and processed with MOSFLM. 45 Small crystals of duplex ODN3 d(CGCGAATTZGCG) 2 were obtained by the same method, whereby 350 nL of 1 mM ODN3 was mixed in 100 mM NaCl and 20 mM Tris (pH 8.5) with 350 nL precipitant solution containing 0.1 M calcium acetate (pH 7.8) and 32% hexylene glycol. The crystals also belonged to space group H3 with unit cell a = b = 41.5 A ˚and c = 101.7, a = b = 90 , and g = 120 , containing two strands of DNA, and were diffracted to 2.46 A ˚. Diffraction data were collected on beamline I02 at Diamond Light Source and processed with MOSFLM. 45 Crystals of ODN3 were also grown at pH 8.5 by the same method, and data were collected on beamline IO3 and processed as above. The crystals belonged to space group H3 with unit cell a = b = 40.9 A ˚and c = 102.2 A ˚, a = b = 90 , and g = 120 , containing two strands of DNA, and were diffracted to 2.35 A ˚(Table S4).</p><!><p>All three crystal structures were solved with the CCP4 package 46 by molecular replacement using the PHASER program 47 with native DNA oligomer (PDB: 463D 29 ) as the starting model. The structures were refined with iterative cycles of manual rebuilding with the COOT program 48 followed by refinement with Refmac5. The P and Z nucleotides were unambiguously identified in the electron density with the use of positive density for the nitro group specific to this unnatural base. The crystallographic data are summarized in Table 1, and the models and structure factors have been deposited in the Protein Data Bank (PDB: 5LJ4, 5I4S, and 5KTV for ODN2, ODN3a, and ODN3b, respectively). Although not outliers, the Rfree values for two of these structures compare less favorably with averages determined across the entire (mainly protein) PDB. This arises partially from the lack of clear refinement restraints for the artificial base pairs. However, the structures compare very favorably with DNA-only structures averaged across the Nucleic Acid Database (http://ndbserver.rutgers.edu). The final structures were analyzed with 3DNA software, 49 and figures were drawn with the PYMOL program 50 (Table S5).</p><!><p>Initial PCR studies were conducted in MOPS (pH 7.34) or Tris (pH 8.30) with 50 ng of GloC plasmid DNA template, 51 primers containing P and Z nucleotides, and the corresponding reverse primer at 0.5 and 0.3 mM, respectively. In the experiments with primer Z, concentrations of the dNTP were 0.5 mM dA, dG, and dT and 0.6 mM dC and dPTP. In the experiments with primer P, the concentrations of the dNP were 0.1 mM dA, dG, dT, and dC and 0.6 mM dZTP. Each PCR contained 0.25 and 0.3 units of REDTaq polymerase (Sigma) for each amplification using primers Z and P, respectively. Amplification was for the indicated number of cycles. Samples were analyzed by electrophoresis on a 1.5% agarose gel and visualized by staining with ethidium bromide (Figures S35 and S36).</p><p>Further PCR studies (Figures 4 and S37) were done in 10 mM sodium cacodylate (pH adjusted with HCl), 50 mM KCl, 10 mM MgCl 2 , 5 mM CaCl 2 , 0.1 mM DTT, and 0.1 mg/ml BSA. Reactions were done in 20 mL with 40 fmol of template DNA (ODNs 3P-Temp and 3Z-Temp, and Bsp-Z and Bsp-P 20 ) and primers at 0.5 mM. All dNTPs were at 0.25 mM in the experiment shown, but the concentrations and ratios were varied in other experiments. Each PCR contained 0.5 units of REDTaq polymerase (Sigma). Amplification was for 30 cycles. Samples were analyzed by electrophoresis on a 3% agarose gel, staining with ethidium bromide, and quantitative imaging on a Typhoon imager. The control lanes, lacking either template or amplification, were blank (Figures 4 and S37). Sequences for all Z-and P-containing templates and primers are described in Table S6.</p><!><p>The supplemental crystallographic data reported in the paper have been deposited in the RSCB Protein Data Bank under accession numbers PDB: 5LJ4 (ODN2), 5I4S (ODN3a), and 5KTV (ODN3b). These data are freely available via http://www.rcsb. org/pdb/home/home.do.</p><!><p>Supplemental Information includes MALDI-ToF and HPLC analysis, full data from the CD and UV-Vis measurements (including individual melting curves, further comparisons, and thermal CD analysis of ODN1-ODN3), data collection for X-ray crystallographic analysis, PCR amplification of Z-P DNA templates, and all Z-P-containing templates and primers used and can be found with this article online at http://dx. doi.org/10.1016/j.chempr.2016.11.009.</p>