Topologically constrained formation of stable Z-DNA from normal

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Topologically constrained formation of stable Z-DNA from normal sequence under physiological conditions Yaping Zhang, Yixiao Cui, Ran An, Xingguo Liang, Qi Li, Haiting Wang, Hao Wang, Yiqiao Fan, Ping Dong, Jing Li, Kai Cheng, Weinan Wang, Sai Wang, Guoqing Wang, Changhu Xue, and Makoto Komiyama J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b13855 • Publication Date (Web): 07 Mar 2019 Downloaded from http://pubs.acs.org on March 7, 2019

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Topologically constrained formation of stable Z-DNA from normal sequence under physiological conditions Yaping Zhang,#,† Yixiao Cui,#,† Ran An,*,† Xingguo Liang,*,†,‡ Qi Li,† Haiting Wang,† Hao Wang,† Yiqiao Fan,† Ping Dong,† Jing Li,† Kai Cheng,† Weinan Wang,† Sai Wang,† Guoqing Wang,†,‡ Changhu Xue,†,‡ and Makoto Komiyama† †College

of Food Science and Engineering, Ocean University of China, Qingdao, No. 5 Yushan Road, P. R. China.

‡Laboratory

for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine Science and Technology, Qingdao, No. 1 Wenhai Road, P. R. China. ABSTRACT: Z-DNA, a left-handed duplex, has been shown to form in vivo and regulate expression of the corresponding gene. However, its biological roles have not been satisfactorily understood, mainly because Z-DNA is easily converted to the thermodynamically favorable B-DNA. Here we present a new idea to form stable Z-DNA under normal physiological conditions and achieve detailed analysis on its fundamental features. Simply by mixing two complementary mini circles of single-stranded DNA with no chemical modification, the hybridization spontaneously induces topological constraint which twines a half of the doublestranded DNA into stable Z-DNA. The formation of Z-conformation with high stability has been proved by using circular dichroism spectroscopy, Z-DNA-specific antibody binding assay, nuclease digestion, etc. Even at a concentration of MgCl2 as low as 0.5 mM, Z-DNA was successfully obtained, avoiding the use of high salt conditions, limited sequences, ancillary additives, or chemical modifications, criteria which have hampered Z-DNA research. The resultant Z-DNA has the potential to be used as a canonical standard sample in Z-DNA research. By using this approach, further developments of Z-DNA science and its applications become highly promising.

handed helical B-form which is the most stable DNA conformation. Accordingly, to maintain the linking number (Lk) of the whole system unchanged at zero, the other portion of the dsDNA rings has to be inevitably wound to the reverse direction in a left-handed way. If the complementary bases in the rewound portion form Watson-Crick base pairs, Z-DNA is spontaneously produced (similarly as negative supercoiling21). The B-DNA portion and the Z-DNA portion in the hybrid are connected by two junctions where the complementary bases are not hydrogen-bonding because of steric disturbance.22−25 As evidenced here, stable Z-DNA is formed by using this simple methodology.

INTRODUCTION In nature, DNA almost always takes right-handed helical structure, and “right-handed DNA science” has been believed to govern life on earth. The finding of left-handed DNA (Z-DNA) was a great discovery for biology, medicine, and other areas.1−4 Several pieces of evidence showed that this Z-DNA exists in vivo and is strongly related to various biological functions (e.g., regulation of gene expression and RNA editing).5−15 Meanwhile, B-DNA with alternating purine/pyrimidine sequence was found to convert to Z-DNA by many environmental factors (e.g., high ionic strength,1,2 solvent addition,16 Z-DNA-binding molecules,13,17,18 chemical modification of DNA bases19 and negative supercoiling20,21). Interestingly, B-Z transition has also been shown to be triggered by very small tension from magnetic tweezers.22,23 However, there has been no way to form stable Z-DNA under physiological conditions without significant chemical and/or biological perturbation. Accordingly, research on basic molecular properties, biological functions, as well as practical applications of Z-DNA has been difficult.

RESULTS AND DISCUSSION Formation of stable DNA duplex from two mutually complementary single-stranded DNA mini circles. As we designed, after hybridization of two complementary ssDNA rings, a duplex ring can form, in which about half is left-handed and the other portion is right-handed (Scheme 1A). Accordingly, the sequences were designed to have two parts with different characteristics (Scheme 1B−E). One part involves the sequences with alternating purine/pyrimidine bases that are prone to Z-DNA formation, and the other part contains completely random sequences that are hard to form Z-DNA. As we know, more GC dyads can form Z-DNA more easily. Here, 4 kinds of alternating purine/pyrimidine sequences (blue characters in Scheme 1B−E) with various GC dyads were used.

The solution we have developed here is schematically presented in Scheme 1A. When two complementary mini single-stranded DNA circles are hybridized in aqueous solutions (without using high salt conditions, additives, modifications and other factors), a portion of the resultant cyclic double-stranded DNA (dsDNA) should take a right1


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Scheme 1. Topologically constrained formation of Z-DNA. (A) Schematic illustration of the present strategy. Simply by mixing two complementary ssDNA circles, Z-DNA is formed in the left-half as a chimera hybrid with B-DNA (Z-B chimera). (B) Sequences of mini ssDNA circles used to form 89-bp Z-B chimera hybrid (cc-89). The outside strand is c-F89, and the inner side one is c-R89 (see also Table S1 for the sequences). Similarly, the sequences of mini ssDNA circles to form Z-B chimera hybrids of 92-bp (cc-92), 74-bp (cc-74), and 111-bp (cc-111) are presented in (C), (D), and (E), respectively. The alternating purine/pyrimidine sequences are shown by blue letters.

At first, the 89-bp sequence with 6 consecutive GC dyads was investigated (Scheme 1B). The two single-stranded DNA (ssDNA) mini circles (c-F89 and c-R89; 89 nt), which are complementary to each other, were synthesized and purified as described previously (see Table S1 for the sequences).26 The sequence in the right-half is almost completely random (Scheme 1B). In the left-half, two sets of alternating purine/pyrimidine sequences of 16-bp length are intentionally introduced to promote Z-DNA formation there and differentiate this half from the right half. These two sets of alternating purine/pyrimidine sequences are separated by 10bp random sequence. Note that this left-hand sequence never spontaneously forms Z-DNA even under high salt concentrations, as long as it exists in a linear duplex without topological constraint (vide ante). When the mixture of these two ssDNA circles, c-F89 and c-R89, was annealed in 1:1 ratio from 90°C to 20°C at pH 7.5 (10 mM HEPES, 10 mM MgCl2) and analyzed by electrophoresis, a new band clearly appeared (lane 1 in Figure 1A). This new band is assignable to an 89-bp dsDNA ring (cc-89, Lk = 0) involving a right-handed duplex part and a left-handed one. A unique secondary structure of this hybrid was evident from its considerably smaller mobility, compared with the conventional dsDNA ring of the same sequence (lc89(Lig), lane 2 in Figure 1A) which was prepared by enzymatic ring-closure after hybridization between linear lF89 and circular c-R89. Obviously, lc89(Lig) takes B-type conformation throughout the whole structure. Furthermore,

Figure 1. Confirmation of hybridization between two complementary single-stranded DNA circles. (A) Native PAGE (6%) analysis. Lane L, double-stranded DNA ladder; lane 1, cc-89 (89-bp hybrid formed from c-F89 and c-R89); lane 2, the product of circular B-DNA prepared by hybridization of l-F89 (non-cyclic counterpart of c-F89) and c-R89, followed by ligation using T4 DNA ligase; lane 3, cl-89 (hybrid of c-F89 and l-R89); lane 4, lc-89 (hybrid of l-F89 and c-R89); lane 5, ll-89 (hybrid of l-F89 and l-R89); lane 6, cc-92 (92-bp hybrid from c-F92 and c-R92); lane 7, the ligation product from l-F92/c-R92; lane 8, cl-92; lane 9, lc-92; lane 10, ll92. Conditions: [c- or l-ssDNA] = 0.25 μM, [MgCl2] = 10 mM, pH 7.5 ([HEPES] = 10 mM) and 20°C. (B) Schematic illustration of the hybridization products between two complementary linear or circular single-stranded DNA.

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circles cannot form due to the nick(s), and thus the whole sequence takes B-structure. Only when the alternating purine/pyrimidine region was completely absent (cc-72 in Figure S2), Z-DNA was hardly formed under the conditions employed here, indicating that cc-72 was not a Z-B chimera (Figure 2F).

the new band in lane 1 showed smaller mobility than the Btype hybrids in which either cyclic component is replaced by the corresponding non-cyclic ssDNA (cl-89 hybrid formed from c-F89 and l-R89 in lane 3 and lc-89 hybrid formed from l-F89 and c-R89 in lane 4 in Figure 1A). The c-F92/c-R92 combination (Scheme 1C) also formed cc-92 hybrid as a very clear band (lane 6, Figure 1A). Again, cc-92 showed smaller mobility than lc-92, probably because the left part takes Z-DNA conformation and is longer than B-DNA with the same sequence. Stable circular hybrids of 74 bp and 111 bp (Scheme 1D and 1E) were also obtained from the corresponding complementary ssDNA circles (Figure S1). Note that the 111 bp sequence is a part of human ADAM-12 gene, in which the ZDNA formation has been shown to regulate gene expression.6 Thus, it can be concluded that two complementary circular ssDNAs could form a dsDNA ring (Lk = 0) involving both a lefthanded and a right-handed portion even at low ionic strength. This circular dsDNA (designated as Z-B chimera) was proved later to involve two discrete parts of Z-DNA and B-DNA by CD and specific binding of Z-DNA antibody (vide post). Circular dichroism (CD) spectroscopic evidence of Z-DNA formation in the duplex prepared from two mutually complementary mini circles of single-stranded DNA. One may doubt that the formed circular duplex (the Z-B chimera, as shown in Scheme 1A) cannot form regular Watson-Crick base pairs in the left-handed part, due to very strong topological constraint. However, CD spectroscopy confirmed that the lefthanded DNA in these hybrids took the typical Z-DNA conformation (Figure 2). Here, the concentration of MgCl2 in HEPES buffer was only 10 mM. Notably, the cc-89 chimera hybrid clearly showed a positive Cotton effect at around 270 nm and a negative one at 290 nm (red line in Figure 2A), which is the characteristic and well-recognized signature of Z-DNA structure.27−30 As shown from the difference spectrum (blue line, Figure 2B), in which the contribution from the B-DNA portion in the hybrid was subtracted, the CD profile for Z-DNA was still more explicit.

Figure 2. CD spectra of the Z-B chimera hybrids. (A) Original spectrum of cc-89 (red line). For comparison, the spectra of the partially or completely non-cyclic analogs (cl-89, black; ll-89, purple) are also presented. Only cc-89 shows a distinct CD spectrum different from typical CD of B-form (ll-89). (B) Difference spectrum (cc-89 – 0.47 × cl-89) in which the contribution from the right-handed portion in the hybrid was subtracted from the original spectrum (see supporting materials and methods for details). A typical CD spectrum of Z-DNA was obtained. The spectra for (C) cc-74, (D) cc-92, (E) cc-111, and (F) cc-72 are also shown. Red and black lines show the original spectra for the Z-B chimera hybrid and its non-cyclic analog, respectively. The difference spectra, obtained as described for (B), are presented by blue lines. Conditions: [chimera hybrid] = 4 μM, [MgCl2] = 10 mM, pH 7.5 ([HEPES] = 10 mM) and 20°C.

Similar CD results were obtained for cc-92, indicating that the left-handed part in this chimera hybrid also took Z-DNA conformation (Figure 2D). Note that the left-half of this hybrid is even less favorable for Z-DNA formation compared with cc89, since shorter alternating purine/pyrimidine sequences without continuous GC repeats (12 and 16 bp) are interrupted by a longer (15 bp) random sequence (Scheme 1C). Remarkable effect of topological constraint for Z-DNA formation can be expected. In addition, both cc-74 and cc-111 chimera hybrids also exhibited typical CD spectra for Z-DNA (Figures 2C and 2E). Note that the 12-bp purine/pyrimidine sequence in cc-74 contains no consecutive GC dyads and is highly unfavorable for Z-DNA formation.31

Very importantly, these Z-B chimera hybrids are very stable even at low concentrations of metal salts (Figure 3). As long as [MgCl2] was higher than 0.5 mM (or [NaCl] > 50 mM), all the chimera hybrids were successfully formed. Only when no MgCl2 or NaCl was added to the solutions, chimera hybrids were not formed, and the CD spectra were of typical B-type DNA (black curves in Figures 3A−3F). Z-DNA cannot form spontaneously for these sequences without this strong topological constraint even under an extremely high salt concentration (e.g. 5 M NaCl). The above results show that the topological constraint we used here can induce B-Z transition like strong negative supercoil. At present, it seems that no other left-handed DNA duplex conformation (different from ZDNA) has been formed in our system, although the detailed structure may differ to some extent.

As expected, when non-cyclic analog was used in place of either (or both) of the two ssDNA circles, the Z-DNA signatures were never perceived for the hybrids of all the different sizes (purple and black lines in Figures 2A, 2C, 2D, and 2E). These hybrids showed only a negative Cotton effect at 250 nm and a positive one at 280 nm, which are assignable to the conventional B-DNA. Here, the topological constraint like that induced by the hybridization of two complementary ssDNA 3


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Journal of the American Chemical Society protein) to our Z-B chimera was also evident (Figures S3D, S3E, and S3F). We further used HhaI restriction enzyme to digest the “GCGC” site in the left-part of cc-89 (see the sequence in Scheme 1B). The result clearly showed that the digestion rate was much slower than that for cl-89, indicating that non-BDNA structure was formed there (Figure S4). All these results demonstrated that Z-DNA conformation was present for each Z-B chimera we constructed.

Figure 4. Gel shift assay of selective binding of Z-DNA-specific antibody Z22 to the Z-B chimera hybrids of cc-89 (A) and cc-92 (B). Z22 is an antibody binding to Z-DNA (but not B-DNA). In (A), lane 1, cc-89 hybrid only; lanes 2–6, the molar ratios of cc-89 to Z22 antibody are 1:1, 1:2, 1:3, 1:4 and 1:5, respectively; lane 7, ll-89 only; lane 8, ll-89 with Z22 (the molar ratio 1:5); lane 9, cl-89 only; lane 10, cl-89 with Z22 (the molar ratio 1:5). In (B), lane 1, cc-92 hybrid only; lanes 2–6, the molar ratios of cc-92 to Z22 antibody are 1:1, 1:2, 1:3, 1:4 and 1:5, respectively; lane 7, ll-92 only; lane 8, ll-92 with Z22 (the molar ratio 1:5); lane 9, cl-92 only; lane 10, cl-92 with Z22 (the molar ratio 1:5). Conditions: [cc hybrid, cl hybrid, ll hybrid] = 0.25 μM, [MgCl2] = 10 mM, pH 7.5 ([HEPES] = 10 mM). The mixtures were incubated at 25°C for 2.5 h.

Figure 3. Effects of metal ion concentrations on CD spectra of the Z-B chimera hybrids. (A), (C), and (E): Difference spectra (cc hybrid– 0.47 × cl hybrid) for cc-89, cc-74 and cc-92 in the buffers containing different concentrations of MgCl2. [MgCl2] = 0 mM (black line), 0.5 mM (green line), 2.0 mM (purple line) and 10.0 mM (brown line), pH 7.0 (10 mM HEPES) and 25°C. (B), (D), and (F): Difference spectra for cc-89, cc-74 and cc-92 in the buffers containing different concentrations of NaCl. [NaCl] = 0 mM (black line), 50 mM (red line), 100 mM (blue line) and 200 mM (orange line), pH 7.0 ([HEPES] = 10 mM) and 25°C. [chimera hybrid] = 4 μM.

Selective binding of Z-DNA binding proteins to the Z-DNA portion in the chimera hybrid. The formation of Z-DNA was further supported by gel shift assay using Z22,6 an antibody specifically binding to Z-DNA (Figure 4). With increasing ratio of the antibody to the chimera hybrid, the band of hybrid gradually decreased, and new bands with much smaller mobility concurrently prevailed. Even at the Z22/cc-89 ratio of 1, more than half of the hybrid was bound to the antibody (lane 2 in Figure 4A). When the ratio was further increased, several new bands with still smaller mobility appeared, probably due to the binding of the second antibody molecule (or the third one and more) to the Z-DNA portion in the chimera hybrid. It should be noted that only 1×TBE buffer (89 mM Tris, 89 mM boric acid and 2 mM EDTA) was used during PAGE analysis, indicating again that the binding was very strong under low ion-strength conditions. Similar results were obtained for the cc-92 hybrid (Figure 4B).

Pinning down the positions of B-Z junctions in the cc-89 chimera hybrid. In Figure 5A, the positions of B-Z junctions in the cc-89 chimera hybrid were clarified in details by the digestion with nuclease S1. Two scission bands in lane 4 are assignable to the nicked product (the top) and the linearized one (the bottom). In order to pin down the position of this S1 scission, the linearized product was extracted from the gel, and digested by two restriction enzymes of PvuI and BsaAI (Figure 5B). Significantly, the clear-cut scission bands were formed (lanes 3 and 4 in Figure 5B), showing that the scission of the chimera hybrid by nuclease S1 was never random, and occurred at a predetermined position. Through the analysis of the sizes of restriction fragments (Figure 5C), it was concluded that nuclease S1 selectively cut the cc-89 chimera hybrid at the top of dsDNA ring. Note that 8 alternating purine/pyrimidine dyads terminate near there. It can be deduced that B-Z junction, in which base pairings hardly occur, was formed at the expected position and digested by nuclease S1 as the hotspot.

Consistently, Z22 did not bind, when either of the DNA components was non-cyclic (lanes 8 and 10 in Figures 4A and 4B), because only B-type dsDNA was formed throughout the hybrids. The cc-74 and cc-111 chimera hybrids were also found to bind Z22 (Figures S3A and S3B). Figure S3C shows that another Z-DNA-specific antibody (anti-z DNA antibody) could also bind to all the four Z-B chimeras with a relatively low affinity. The binding of ZBP1 (recombinant Z-DNA-binding

It is interesting that only one of the two junctions was digested, and this can be explained as follows. Once one B-Z junction is cut, the topological constraint in the cc-89 chimera hybrid is removed, and thus Z-DNA should transfer to B-DNA. As a result, the other junction (at the bottom in Figure 5C) 4



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came from the break of circular DNA or the mix of linear ssDNA during purification. For example, the additional melting point of cc-74 was about 79.1°C (Figure S5E), which is approximately equal to the melting point of cl-74 (78.9°C, Figure S5D). In brief, we conclude that the Z-DNA part in all the Z-B chimera hybrids we constructed is almost completely intact up to 37°C, and sufficiently stable even at 60°C (Figures 6 and S5). Extended insight into the properties of Z-DNA could be gained by means of other appropriate analytical techniques.

disappears and cannot be digested further by nuclease S1. Furthermore, another effect of the released topological constraint is that a nick structure should be formed at the digested position, if only one strand is digested. As we know, the phosphodiester bond at the nick can be digested by S1. However, the lc-89 formed by one strand scission of cc-89 was only digested very slowly to ll-89. One possible reason is that this circular duplex is not long enough so that there is a strong stretching of phosphodiester bond at the nick position, causing slow digestion by S1 (lane 4 in Figure 5A).

Figure 6. Thermodynamic analysis of the cc-89 chimera hybrid. (A) Temperature dependence of CD spectra of cc-89 (4 μM). [MgCl2] = 10 mM, pH 7.5 ([HEPES] = 10 mM). (B) Melting curves of cc-89 by HRM (black line) and UV (red line). [MgCl2] = 10 mM, pH 7.5 ([HEPES] = 10 mM).

Figure 5. Analysis of the positions of B-Z junctions in the cc-89 chimera hybrid. (A) PAGE analysis for the digestion of cc-89 with nuclease S1 (lane 4). Lane 1, ll-89; lane 2, cl-89; lane 3, cc-89 without nuclease S1 treatment. (B) PAGE analysis for the restriction enzyme digestion of the linearized cc-89 product in lane 4 (A) which was purified by gel electrophoresis. Lane 1, ll-89; lane 2, the linearized cc-89; lanes 3 and 4 show the digestion products of the linearized cc-89 by PvuI and BsaAI, respectively. (C) Illustration of the plausible position of nuclease S1 scission site in cc-89 (shown as S1), and the expected fragments obtained by the restriction enzyme digestion of the linearized cc-89 (another flipping base pair is shown by red arrows). Conditions: [ll hybrid, cl hybrid, cc hybrid] = 0.25 μM, [MgCl2] = 10 mM, pH 7.5 ([HEPES] = 10 mM).

Even when the concentration of MgCl2 decreased to 0.5 or 1.0 mM, Z-DNA was effectively formed in our Z-B chimera (Figures 3 and S5L). All these results indicate that the present method is directly employable to in vivo applications, since intracellular metal ions are sufficient for the Z-DNA formation, and administration of additional metal ions is unnecessary (in usual cells, [K+] = 140 mM, [Na+] = 10 mM, and [Mg2+] = 0.5 mM). Introduction of the chimera hybrids into cells to regulate the activity of Z-DNA-binding proteins should be one of the most attractive themes. The effects of Z-DNA on gene expression can be also analyzed. Furthermore, many other in vitro applications should be promising (e.g., assay of binding activity of Z-DNA-binding molecules (Figures 4 and S3), and developments of medicines for Z-DNA-related diseases). All these studies have been for a long time hampered by a lack of appropriate specimens of Z-DNA as experimental materials, especially as a standard sample. The present finding should be a straightforward solution to this problem, resulting in still deeper understanding on the roles of “left-handed DNA science” in nature.

Applications of the present method to the analysis of fundamental features of Z-DNA under physiological conditions. One of the most significant features of the present strategy is that intrinsic and detailed properties of Z-DNA (thermodynamic, physicochemical, and others) can be measured by spectroscopic means under physiological conditions. This can help us to better understand Z-DNA in the physicochemical environments of the human body. In Figure 6A, the thermal stability of the cc-89 chimera hybrid was assayed by CD spectroscopy under the conditions of pH 7.5 and 10 mM MgCl2. The temperature dependence of the signature of Z-DNA agreed fairly well with the melting temperature of cc89 (Tm = 69.7°C) which was independently determined by UV spectroscopy in Figure 6B (red line). Almost the same Tm value (68.7°C, black line in Figure 6B) was obtained by using high resolution of melting (HRM),32−37 which is carried out by measuring the temperature-dependent change of the fluorescence from a DNA-binding dye on a Real Time PCR machine. Other Z-B chimera hybrids are also very stable, and the Tms are higher than 65°C even when [MgCl2] is only 10 mM (Figure S5). An extra peak appeared in the derivative of melting curves of the Z-B chimera hybrids (Figure S5E, S5H and S5K). It may be caused by the small amounts of lc/cl hybrid which

CONCLUSION Simply by mixing two complementary mini circles of singlestranded DNA, stable Z-DNA is formed under physiological conditions due to the topological constraint induced by the hybridization of the mini circles. Its Z-conformation has been concretely confirmed by circular dichroism spectra with authentic signature for this non-B-DNA. The Z-conformation is further supported by Z-DNA-specific antibody binding assays, nuclease digestion, and other analyses. To obtain the detailed structure of the Z-B chimera, especially for the two junctions and the left-handed helix with random sequence, further structural analysis using NMR38 or FRET39-42 is required. High salt conditions, limited DNA sequences, ancillary additives, or chemical modifications, which have represented an obstacle for Z-DNA researchers, were shown to not be necessary in our 5


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system. The resultant Z-DNA, which is stable under physiological conditions, can be used as canonical probes to directly reflect in vivo situations. A variety of applications of the present strategy should be promising for either fundamental or practical purposes. ASSOCIATED CONTENT Supporting Information This material is available free of charge via the Internet at http://pubs.acs.org. Experimental details, PAGE analysis, results of antibodies and ZBP1 protein binding assays, digestion of the cc-89 chimera hybrid by restriction enzyme, and thermodynamic features of the Z-B chimera hybrids (PDF).

AUTHOR INFORMATION Corresponding Authors *[email protected] *[email protected]

Author Contributions #Yaping Zhang and Yixiao Cui contributed equally.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENT We thank the late Prof. Alexander Rich for the discussion about our idea and kind comments in 2005 in Albany. We also appreciate Prof. Hiroshi Sugiyama (Kyoto University, Japan), and Prof. Hiroyuki Asanuma (Nagoya University, Japan) for their kind advices. Supports by National Natural Science Foundation of China (31571937 to X.L.), China Postdoctoral Science Foundation (2018M632718 to R.A.), and Fundamental Research Funds for the Central Universities (201713050 to R.A.) are acknowledged.

REFERENCES (1) Pohl, F. M.; Jovin, T. M. Salt-induced co-operative conformational change of a synthetic DNA: equilibrium and kinetic studies with poly(dG-dC). J. Mol. Biol. 1972, 67, 375−396. (2) Wang, A. H.; Quigley, G. J.; Kolpak, F. J.; Crawford, J. L.; van Boom, J. H.; van der Marel, G.; Rich, A. Molecular structure of a left-handed double helical DNA fragment at atomic resolution. Nature 1979, 282, 680−686. (3) Doluca, O.; Withers, J. M.; Filichev, V. V. Molecular engineering of guanine-rich sequences: Z-DNA, DNA triplexes, and G-quadruplexes. Chem. Rev. 2013, 113, 3044−3083. (4) Rich, A.; Zhang, S. Z-DNA: the long road to biological function. Nat. Rev. Genet. 2003, 4, 566−572. (5) Haniford, D. B.; Pulleyblank, D. E. The in-vivo occurrence of Z DNA. J. Biomol. Struct. Dyn. 1983, 1, 593−609. (6) Ray, B. K.; Dhar, S.; Shakya, A.; Ray, A. Z-DNA-forming silencer in the first exon regulates human ADAM-12 gene expression. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 103−108. (7) Oh, D. B.; Kim, Y. G.; Rich, A. Z-DNA-binding proteins can act as potent effectors of gene expression in vivo. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 16666−16671. (8) Schwartz, T.; Rould, M. A.; Lowenhaupt, K.; Herbert, A.; Rich, A. Crystal structure of the Zα domain of the human editing enzyme ADAR1 bound to left-handed Z-DNA. Science 1999, 284, 1841−1845. (9) Herbert, A.; Alfken, J.; Kim, Y. G.; Mian, I. S.; Nishikura, K.; Rich, A. A Z-DNA binding domain present in the human editing enzyme,

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(36) Malentacchi, F.; Forni, G.; Vinci, S.; Orlando, C. Quantitative evaluation of DNA methylation by optimization of a differential-high resolution melt analysis protocol. Nucleic Acids Res. 2009, 37, e86. (37) Wang, J.; Pan, X.; Liang, X. Assessment for melting temperature measurement of nucleic acid by HRM. J. Anal. Methods Chem. 2016, 5318935. (38) Sugiyama, H.; Kawai, K.; Matsunaga, A.; Fujimoto, K.; Saito, I.; Robinson, H.; Wang, A. Synthesis, structure and thermodynamic properties of 8-methylguanine-containing oligonucleotides: Z-DNA under physiological salt conditions. Nucleic Acids Res. 1996, 24, 1272−1278. (39) Yamamoto, S.; Park, S.; Sugiyama H. Development of a visible nanothermometer with a highly emissive 2'-O-methylated guanosine analogue. RSC Adv. 2015, 5, 104601–104605. (40) Han, J. H.; Yamamoto, S.; Park, S.; Sugiyama, H. Development of a vivid FRET system based on a highly emissive dG-dC analogue pair. Chemistry 2017, 23, 7607–7613. (41) Dumat, B.; Larsen, A. F.; Wilhelmsson, L. M. Studying Z-DNA and B- to Z-DNA transitions using a cytosine analogue FRET-pair. Nucleic Acids Res. 2016, 44, e101. (42) Wranne, M. S.; Füchtbauer, A. F.; Dumat, B.; Bood, M.; ElSagheer, A. H.; Brown, T.; Gradén, H.; Grøtli, M.; Wilhelmsson, L. M. Toward complete sequence flexibility of nucleic acid base analogue FRET. J. Am. Chem. Soc. 2017, 139, 9271–9280.

(29) Miyahara, T.; Nakatsuji, H.; Sugiyama, H. Similarities and differences between RNA and DNA double-helical structures in circular dichroism spectroscopy: a SAC-CI study. J. Phys. Chem. A. 2016, 120, 9008−9018. (30) Kawara, K.; Tsuji, G.; Taniguchi, Y.; Sasaki S. Synchronized chiral induction between [5]helicene–spermine ligand and B–Z DNA transition. Chem. Eur. J. 2017, 23, 1763−1769. (31) Peticolas, W. L.; Wang, Y.; Thomas, G. A. Some rules for predicting the base-sequence dependence of DNA conformation. Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 2579−2583. (32) Vossen, R. H.; Aten, E.; Roos, A.; den Dunnen, J. T. Highresolution melting analysis (HRMA): more than just sequence variant screening. Hum. Mutat. 2009, 30, 860−866. (33) Zhou, L.; Wang, L.; Palais, R.; Pryor, R.; Wittwer, C. T. Highresolution DNA melting analysis for simultaneous mutation scanning and genotyping in solution. Clin. Chem. 2005, 51, 1770–1777. (34) Lin, J.; Tseng, C.; Chen, Y.; Lin, C.; Chang, S.; Wu, H.; Cheng, J. Rapid differentiation of influenza A virus subtypes and genetic screening for virus variants by high-resolution melting analysis. J. Clin. Microbiol. 2008, 46, 1090–1097. (35) Croxford, A. E.; Rogers, T.; Caligari, P. D.; Wilkinson, M. J. Highresolution melt analysis to identify and map sequence-tagged site anchor points onto linkage maps: a white lupin (Lupinus albus) map as an exemplar. New Phytol. 2008, 180, 594–607.

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Table of Contents

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Scheme 1. Topologically constrained formation of Z-DNA. (A) Schematic illustration of the present strategy. Simply by mixing two complementary ssDNA circles, Z-DNA is formed in the left-half as a chimera hybrid with B-DNA (Z-B chimera). (B) Sequences of mini ssDNA circles used to form 89-bp Z-B chimera hybrid (cc89). The outside strand is c-F89, and the inner side one is c-R89 (see also Table S1 for the sequences). Similarly, the sequences of mini ssDNA circles to form Z-B chimera hybrids of 92-bp (cc-92), 74-bp (cc-74), and 111-bp (cc-111) are presented in (C), (D), and (E), respectively. The alternating purine/pyrimidine sequences are shown by blue letters. 125x109mm (300 x 300 DPI)


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Figure 1. Confirmation of hybridization between two complementary single-stranded DNA circles. (A) Native PAGE (6%) analysis. Lane L, double-stranded DNA ladder; lane 1, cc-89 (89-bp hybrid formed from c-F89 and c-R89); lane 2, the product of circular B-DNA prepared by hybridization of l-F89 (non-cyclic counterpart of c-F89) and c-R89, followed by ligation using T4 DNA ligase; lane 3, cl-89 (hybrid of c-F89 and l-R89); lane 4, lc-89 (hybrid of l-F89 and c-R89); lane 5, ll-89 (hybrid of l-F89 and l-R89); lane 6, cc-92 (92-bp hybrid from c-F92 and c-R92); lane 7, the ligation product from l-F92/c-R92; lane 8, cl-92; lane 9, lc-92; lane 10, ll-92. Conditions: [c- or l-ssDNA] = 0.25 μM, [MgCl2] = 10 mM, pH 7.5 ([HEPES] = 10 mM) and 20°C. (B) Schematic illustration of the hybridization products between two complementary linear or circular single-stranded DNA. 84x49mm (300 x 300 DPI)



Figure 2. CD spectra of the Z-B chimera hybrids. (A) Original spectrum of cc-89 (red line). For comparison, the spectra of the partially or completely non-cyclic analogs (cl-89, black; ll-89, purple) are also presented. Only cc-89 shows a distinct CD spectrum different from typical CD of B-form (ll-89). (B) Difference spectrum (cc-89 – 0.47 × cl-89) in which the contribution from the right-handed portion in the hybrid was subtracted from the original spectrum (see supporting materials and methods for details). A typical CD spectrum of ZDNA was obtained. The spectra for (C) cc-74, (D) cc-92, (E) cc-111, and (F) cc-72 are also shown. Red and black lines show the original spectra for the Z-B chimera hybrid and its non-cyclic analog, respectively. The difference spectra, obtained as described for (B), are presented by blue lines. Conditions: [chimera hybrid] = 4 μM, [MgCl2]= 10 mM, pH 7.5 ([HEPES] = 10 mM) and 20°C. 84x94mm (300 x 300 DPI)


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Figure 3. Effects of metal ion concentrations on CD spectra of the Z-B chimera hybrids. (A), (C), and (E): Difference spectra (cc hybrid – 0.47 × cl hybrid) for cc-89, cc-74 and cc-92 in the buffers containing different concentrations of MgCl2. [MgCl2] = 0 mM (black line), 0.5 mM (green line), 2.0 mM (purple line) and 10.0 mM (brown line), pH 7.0 (10 mM HEPES) and 25°C. (B), (D), and (F): Difference spectra for cc-89, cc-74 and cc-92 in the buffers containing different concentrations of NaCl. [NaCl] = 0 mM (black line), 50 mM (red line), 100 mM (blue line) and 200 mM (orange line), pH 7.0 ([HEPES] = 10 mM) and 25°C. [chimera hybrid] = 4 μM. 84x94mm (300 x 300 DPI)



Figure 4. Gel shift assay of selective binding of Z-DNA-specific antibody Z22 to the Z-B chimera hybrids of cc-89 (A) and cc-92 (B). Z22 is an antibody binding to Z-DNA (but not B-DNA). In (A), lane 1, cc-89 hybrid only; lanes 2–6, the molar ratios of cc-89 to Z22 antibody are 1:1, 1:2, 1:3, 1:4 and 1:5, respectively; lane 7, ll-89 only; lane 8, ll-89 with Z22 (the molar ratio 1:5); lane 9, cl-89 only; lane 10, cl-89 with Z22 (the molar ratio 1:5). In (B), lane 1, cc-92 hybrid only; lanes 2–6, the molar ratios of cc-92 to Z22 antibody are 1:1, 1:2, 1:3, 1:4 and 1:5, respectively; lane 7, ll-92 only; lane 8, ll-92 with Z22 (the molar ratio 1:5); lane 9, cl-92 only; lane 10, cl-92 with Z22 (the molar ratio 1:5). Conditions: [cc hybrid, cl hybrid, ll hybrid] = 0.25 μM, [MgCl2] = 10 mM, pH 7.5 ([HEPES] = 10 mM). The mixtures were incubated at 25°C for 2.5 h. 81x52mm (300 x 300 DPI)


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Figure 5. Analysis of the positions of B-Z junctions in the cc-89 chimera hybrid. (A) PAGE analysis for the digestion of cc-89 with nuclease S1 (lane 4). Lane 1, ll-89; lane 2, cl-89; lane 3, cc-89 without nuclease S1 treatment. (B) PAGE analysis for the restriction enzyme digestion of the linearized cc-89 product in lane 4 (A) which was purified by gel electrophoresis. Lane 1, ll-89; lane 2, the linearized cc-89; lanes 3 and 4 show the digestion products of the linearized cc-89 by PvuI and BsaAI, respectively. (C) Illustration of the plausible position of nuclease S1 scission site in cc-89 (shown as S1), and the expected fragments obtained by the restriction enzyme digestion of the linearized cc-89 (another flipping base pair is shown by red arrows). Conditions: [ll hybrid, cl hybrid, cc hybrid] = 0.25 μM, [MgCl2] = 10 mM, pH 7.5 ([HEPES] = 10 mM). 84x47mm (300 x 300 DPI)



Figure 6. Thermodynamic analysis of the cc-89 chimera hybrid. (A) Temperature dependence of CD spectra of cc-89 (4 μM). [MgCl2] = 10 mM, pH 7.5 ([HEPES] = 10 mM). (B) Melting curves of cc-89 by HRM (black line) and UV (red line). [MgCl2] = 10 mM, pH 7.5 ([HEPES] = 10 mM). 84x31mm (300 x 300 DPI)


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Table of Contents 82x29mm (300 x 300 DPI)


Topologically constrained formation of stable Z-DNA from normal

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