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Z-DNA

Z-DNA is a left-handed double-helical form of the DNA double helix, distinguished by its elongated, zigzag-shaped phosphate backbone and alternating syn and anti glycosidic bond conformations of the bases, which results in 12 base pairs per helical turn, a helical pitch of approximately 44.6 Å, and a diameter of about 18 Å. Unlike the predominant right-handed B-DNA, which features a smoother backbone and major and minor grooves, Z-DNA lacks a major groove and possesses a single, deep minor groove, making it a higher-energy conformation typically stabilized under non-physiological conditions such as high salt concentrations (e.g., 4 M NaCl) or negative supercoiling. Discovered in 1979 by Alexander Rich and colleagues through X-ray crystallography of the self-complementary oligonucleotide d(CG)3 in high-salt conditions, Z-DNA was initially observed in synthetic poly(dG-dC)·poly(dG-dC) and later confirmed to form preferentially in alternating purine-pyrimidine sequences like (CG)n, (CA)n, or (TG)n. In vivo, Z-DNA emerges dynamically during cellular processes that generate torsional stress, such as transcription by , where negative supercoiling promotes its formation in genomic hotspots including promoters, introns, and regions near replication origins. This structural polymorphism influences key biological functions, including —where it can enhance or repress by altering accessibility and recruiting transcription factors—and , primarily through binding to the Zα of ADAR1, which prevents aberrant immune by editing double-stranded to evade sensors like MDA5. Additionally, Z-DNA serves as a signal in innate immunity via proteins like ZBP1 (also known as ), which recognizes it to trigger necroptosis—a pathway—during viral infections, as evidenced in responses to herpes simplex virus 1 (HSV-1). Z-DNA's dysregulation is implicated in human diseases, particularly interferonopathies and inflammatory disorders; for instance, mutations in ADAR1's Zα domain (e.g., P193A) lead to Aicardi-Goutières syndrome by impairing and causing excessive type I production from accumulated Alu-derived double-stranded RNAs. Similarly, Z-DNA formation contributes to age-related through DICER1 deficiency, resulting in Alu RNA accumulation, retinal , and activation. Genome-wide analyses reveal Z-DNA-prone sequences are enriched in genes associated with , cancer, and neurological conditions, underscoring its role in genome stability, epigenetic modulation, and pathways.

History and Discovery

Initial Observations and Naming

The first experimental indications of a left-handed helical form of DNA came from (CD) spectroscopy studies in 1972 by Fritz Pohl and Thomas M. Jovin on the synthetic polynucleotide poly(dG-dC)·poly(dG-dC) under high-salt conditions. These studies revealed an inversion in the CD spectrum, suggesting a transition from the right-handed B-DNA to a left-handed conformation. Further structural evidence emerged from fiber X-ray studies conducted in 1980 by Stephen Arnott and colleagues on the same synthetic polynucleotide under high-salt conditions. These studies revealed diffraction patterns indicative of a novel helical conformation distinct from the right-handed B-DNA, characterized by a left-handed twist and extended backbone geometry. In 1979, and his team at determined the of the self-complementary DNA hexamer d(CpGpCpGpCpG) at atomic resolution, confirming the existence of this left-handed double helix with 12 base pairs per helical turn and antiparallel strands featuring Watson-Crick base pairing. The structure displayed a striking pattern in the sugar-phosphate backbone, which led and colleagues to name this conformation "Z-DNA" to reflect the distinctive zigzag appearance when viewed along the helical axis. Early reports of Z-DNA were met with significant skepticism throughout the , primarily because the conformation required higher than the stable B-form, necessitating non-physiological conditions like high salt concentrations for stabilization, and raising doubts about its potential occurrence or functional relevance . A key confirmation of the Z-DNA structure in poly(dG-dC) sequences came in from Andrew H.-J. Wang and colleagues, who used on crystals of the polymer to demonstrate the left-handed helical architecture, complementing the 1980 data and solidifying the model's applicability beyond short .

Key Experimental Milestones

In 1981, researchers in Alexander Rich's laboratory used polyclonal antibodies specific for left-handed Z-DNA to enable the first direct detection of Z-DNA conformations in vivo within cellular contexts such as polytene chromosomes. These antibodies, generated against poly(dG-dC)·poly(dG-dC) in its Z-form, bound selectively to the Z conformation independent of sequence and were used to visualize Z-DNA regions in fixed Drosophila salivary gland chromosomes, revealing localization at sites of active transcription. Monoclonal antibodies specific for Z-DNA were subsequently developed in 1982. This breakthrough provided immunological evidence for Z-DNA's physiological existence, shifting the focus from in vitro models to biological relevance. During the and , advances in spectroscopic techniques, particularly (CD), solidified Z-DNA detection in solution and under physiological conditions. Early CD studies in the late 1970s confirmed the inverted helical handedness of Z-DNA through characteristic positive bands at ~290 nm and negative bands at ~260 nm, distinguishing it from B-DNA's spectrum. By the , vacuum ultraviolet CD extended this to higher resolution, showing Z-DNA's red-shifted and inverted spectrum relative to B-DNA, which facilitated monitoring B-to-Z transitions in synthetic polymers like poly(dG-dC) under varying salt and supercoiling conditions. In the , CD was routinely combined with other methods like to quantify Z-DNA stability in plasmids, establishing it as a standard non-invasive tool for conformational analysis without sequence-specific probes. In the 1990s, Terumi Kohwi-Shigematsu and colleagues identified specific Z-DNA-forming sequences within negatively supercoiled plasmids, demonstrating that torsional stress promotes Z-DNA at purine-pyrimidine tracts like (GA/TC)n and (CG)n inserts. Using chemical probing with agents such as and haloacetaldehyde, they mapped hypersensitive sites in supercoiled derivatives, revealing that Z-DNA formation at these loci altered adjacent DNA conformations and correlated with changes. This work highlighted how negative supercoiling, mimicking chromosomal , stabilizes Z-DNA in bacterial and eukaryotic plasmids, providing evidence for its role in DNA packaging and regulatory elements. A pivotal 1999 study by Tamar Schwartz and Alexander Rich elucidated the Zα domain in the RNA-editing enzyme ADAR1, demonstrating its high-affinity, specific recognition of Z-DNA through structural and binding analyses. The Zα domain, a conserved , bound Z-DNA with a in the nanomolar range, distinguishing it from B-DNA and promoting B-to-Z transitions and . Crystal structures revealed that Zα engages the phosphate backbone and syn-guanine conformations unique to Z-DNA, confirming a dedicated protein for Z-DNA interaction and underscoring its potential in and immune responses.

Structural Characteristics

Canonical Z-DNA Architecture

Z-DNA represents the left-handed helical form of double-stranded DNA, characterized by a distinctive zigzag arrangement of its sugar-phosphate backbone. Unlike the right-handed B-DNA, the canonical Z-DNA helix winds in a left-handed manner with approximately 12 base pairs per turn and a helical pitch of about 44.6 . The two strands are antiparallel, as in other DNA forms, but the overall structure is elongated and slender, with a diameter of roughly 18 . This architecture was first elucidated through crystallographic studies of the self-complementary hexadeoxynucleotide d(CGCGCG), revealing the atomic details of the left-handed duplex. The pattern of the phosphate backbone arises from the alternating conformations of the nucleotides in sequences such as poly(dG-dC) or d(CG)_n, where purine bases () adopt a syn glycosidic torsion angle of approximately 60° and pyrimidine bases () adopt an anti glycosidic torsion angle of about -150°. This alternation results in dinucleotide repeats, with guanosines featuring C3'-endo sugar puckering and cytidines showing C2'-endo puckering, which contributes to the irregular, sawtooth-like path of the phosphodiester linkages. The base pairs themselves are Watson-Crick paired, but their stacking is displaced, with over pyrimidine in adjacent steps, leading to the pronounced appearance when viewed along the axis. High-resolution structures, such as the 1.0 Å refinement of the d(CGCGCG) duplex, confirm these features and highlight the role of coordinated ions like Mg²⁺ or in stabilizing the structure by neutralizing phosphate repulsions. Hydration plays a critical role in maintaining the canonical Z-DNA architecture, with ordered water molecules forming specific patterns around the helix. In the convex major groove—facing outward due to the left-handed twist—a spine of bridges phosphate groups and base edges, while the narrow minor groove contains fewer waters but features hydrogen-bonded networks involving guanine amino groups. Crystal structures at resolutions up to 0.55 Å of the d(CGCGCG) hexamer reveal conserved first-shell hydration motifs, including longitudinal water chains along the backbone that mimic RNA-like interactions and enhance structural rigidity. These patterns are integral to the Z-form's stability in high-salt conditions, distinguishing it from hydrated B-DNA.

Prediction and Formation Pathways

The prediction of Z-DNA forming regions in genomic sequences relies on computational algorithms that assess the thermodynamic propensity for B-to-Z transitions based on sequence composition and environmental factors such as superhelical density. Seminal tools like Z-Hunt, introduced in , employ a thermodynamic model to scan DNA sequences for motifs prone to Z-DNA formation, particularly alternating purine-pyrimidine tracts such as (CG)_n repeats, by calculating changes associated with the conformational shift under conditions of negative supercoiling. Subsequent enhancements, including Z-Hunt-II, refine this approach by incorporating more detailed energetic parameters for longer sequences and improved handling of sequence variability, enabling genome-wide mapping of potential Z-DNA sites. Modern variants, such as ZSeeker (as of 2025), optimize these predictions for efficiency in large-scale genomic analyses while maintaining the core reliance on sequence periodicity and torsional stress. The formation of Z-DNA from B-DNA proceeds via a cooperative B-to-Z pathway, primarily driven by negative supercoiling that introduces torsional to favor the left-handed . This involves a cooperative, sequential flipping at dinucleotide steps, with bases rotating from to conformation and sugar puckering adjusting to '-endo for purines and '-endo for pyrimidines, culminating in the characteristic arrangement of the backbone. The is highly cooperative, propagating through dinucleotide steps in a zipper-like manner once initiated, with the overall pathway exhibiting a free energy barrier of approximately 4-5 kcal/mol per base pair at the B-Z junction under physiological conditions. Stabilization of the Z-form during this transition is facilitated by cations such as Mg^{2+}, which screen electrostatic repulsions in the backbone, and polyamines like or spermidine, which bind in the minor groove to neutralize charges and lower the energetic cost of the conformational change. Kinetic models describe the B-to-Z flip as a two-step mechanism, beginning with an at pyrimidine-purine dinucleotide steps where initial base unstacking and rotation occur, followed by along the helix. This stepwise ensures efficient transition in sequences with high Z-propensity, such as poly(dG-dC), under negative superhelical densities typical of cellular environments.

Comparisons with Other DNA Forms

Geometrical and Helical Parameters

Z-DNA exhibits distinct geometrical and helical parameters that distinguish it from the right-handed and B-DNA forms, primarily due to its left-handed helical architecture and alternating dinucleotide backbone conformation. These parameters define the overall shape, compactness, and groove accessibility of . Key metrics such as the number of base pairs per helical turn, helical twist angle, roll angle, and axial rise per highlight these differences, as summarized in the table below.
ParameterA-DNAB-DNAZ-DNA
Base pairs per turn1110.512
Helical twist (°)3336-30
Roll (°)126-7
Rise per base pair (Å)2.63.43.8
The values for Z-DNA are derived from crystallographic studies of hexameric sequences like d(CGCGCG), which reveal a more extended rise and negative roll that contributes to the elongated, slender helical axis. In comparison, A-DNA's positive roll and reduced rise produce a shorter, wider helix suited to dehydrated environments, while B-DNA's parameters support a more uniform, hydrated structure prevalent in vivo. A defining feature of Z-DNA's backbone geometry is the flip in the α and γ torsion angles relative to B-DNA. In B-DNA, both α (g⁻ ≈ -80°) and γ (t ≈ 180°) adopt consistent conformations across nucleotides, facilitating a smooth right-handed twist. In Z-DNA, this alternates: pyrimidines exhibit α (t ≈ -160°) and γ (g⁺ ≈ 50°), while purines maintain α (g⁻) and γ (t), resulting in the characteristic zig-zag phosphate backbone that accommodates the left-handed winding. This torsional alternation is essential for the structural transition from B- to Z-DNA in alternating purine-pyrimidine sequences. Z-DNA's groove dimensions are markedly altered by the syn glycosidic bonds (χ ≈ 60° for purines), which position the purine bases toward the minor groove, deepening and narrowing it to approximately 4 Å wide and 12 Å deep, while the major groove becomes shallow and convex. This configuration contrasts with B-DNA's wide major groove (12 Å) and narrow minor groove (6 Å), enhancing potential interactions with proteins that recognize the exposed minor groove edges in Z-DNA. The left-handed helical of Z-DNA, with approximately 12 pairs completing one full turn in the negative direction, promotes staggered stacking patterns optimized for its dinucleotide repeat. pairs exhibit a propeller twist of approximately -7°, which is intermediate between A-DNA's positive values and B-DNA's more negative ones, aiding hydrophobic stacking despite the inverted .

Stability and Energetic Differences

Z-DNA exhibits higher free energy compared to B-DNA, primarily due to the strain imposed by the syn glycosidic conformation of bases, which contrasts with the more favorable conformation in B-DNA. simulations indicate that this free energy difference is approximately 0.9 kcal/ per dinucleotide unit (or about 0.45 kcal/ per ) in typical sequences. For specific repeats like (GC)_n, the cost is around 0.33 kcal/ per , while for (TG)_n it rises to 0.67 kcal/ per , reflecting sequence-dependent variations in backbone flexibility and base stacking. The thermodynamic instability of Z-DNA is offset by environmental factors that lower the barrier for the B-to-Z . Negative supercoiling stabilizes Z-DNA by relieving torsional stress, with formation typically requiring a superhelical σ < -0.06 (equivalent to ΔLk < -0.06 turns per helical turn in relaxed DNA). High also promotes the by screening repulsions, enabling Z-DNA in poly(dG-dC) sequences at NaCl concentrations exceeding 2.5 M. These conditions shift the B-Z equilibrium toward the Z-form, as the left-handed helix absorbs underwinding energy more efficiently than the right-handed B-form. The B-Z equilibrium can be described by the constant K_{BZ} = \frac{[Z]}{[B]} = \exp\left( -\frac{\Delta G}{RT} \right), where ΔG represents the change for the transition. In the absence of facilitators like supercoiling or high , ΔG is positive and approximately 4 kcal/ for short Z-DNA segments (e.g., 6-10 pairs), rendering the equilibrium strongly favoring B-DNA (K_{BZ} << 1). This value encompasses both the per-base-pair penalty and junction formation costs, highlighting the kinetic and thermodynamic hurdles to Z-DNA under physiological conditions. Sequence composition further modulates Z-DNA , with GC-rich alternating purine-pyrimidine tracts (e.g., d(CG)_n) exhibiting lower transition barriers than AT-rich ones (e.g., d(TA)_n). GC-rich sequences require less salt or supercoiling for stabilization due to better accommodation of the syn conformation and enhanced dehydration effects, whereas AT-rich tracts favor B-DNA persistence owing to higher stacking energies in the right-handed . This intrinsic bias influences Z-DNA propensity in genomic contexts, prioritizing regions with high for potential left-handed adoption.

Biological Functions

Roles in Transcription and Gene Regulation

Z-DNA formation in regions upstream of gene promoters serves to relieve torsional stress generated during transcription, thereby facilitating the unwinding of DNA necessary for transcription initiation. This process is particularly evident in genes such as c-myc, where three distinct Z-DNA-forming sites (Z-1, Z-2, and Z-3) are stabilized by negative supercoiling upstream of the promoter, promoting efficient access and enhancing . Studies have demonstrated that Z-DNA formation in negatively supercoiled DNA absorbs negative superhelical turns during transcription, reducing torsional barriers and allowing more efficient open complex formation by . These findings highlight Z-DNA's role as a dynamic that responds to supercoiling to fine-tune transcriptional output. More recent investigations have revealed Z-DNA enrichment within super-enhancers, clusters of regulatory elements that drive high-level, cell-type-specific . Computational analyses from 2024 to 2025 indicate that Z-DNA-forming sequences are overrepresented in super-enhancers across diverse populations, potentially contributing to variations in profiles and influencing population-specific traits. By acting as a topological sink, Z-DNA stabilizes these regions under torsional stress, promoting the assembly of complexes and enhancer-promoter looping essential for robust . This mechanism underscores Z-DNA's integral function in regulation, where it facilitates open complex formation by dissipating superhelical tension independently of activity.

Involvement in DNA Repair and Replication

Z-DNA formation occurs preferentially under conditions of negative supercoiling, which is prevalent at replication during DNA unwinding. While Z-DNA can contribute to fork stalling, it also relaxes torsional stress, potentially aiding in fork progression in some contexts. In particular, Z-DNA structures can mitigate the formation of other secondary DNA conformations, such as slipped-strand structures, that could otherwise exacerbate fork stalling and lead to replication collapse. In DNA repair pathways, Z-DNA-forming sequences, particularly CG-rich repeats, induce double-strand breaks that are often resolved through (HR), though this can lead to genomic instability such as large deletions. This process is relevant for repairing replication-associated lesions in repetitive regions, but Z-DNA often promotes rather than error-free repair. Computational tools like the 2025 ZSeeker algorithm have enhanced the identification of Z-DNA-prone sequences genome-wide, revealing their enrichment at mutation hotspots in repair-deficient cells, such as those lacking mismatch repair proteins. In mismatch repair-deficient backgrounds, Z-DNA structures amplify genomic instability by increasing deletion frequencies at these sites, as evidenced by elevated rates in MSH2-knockout models. Such findings underscore Z-DNA's in exacerbating repair vulnerabilities, linking structural transitions to in compromised cellular environments. Emerging research indicates potential roles for Z-DNA in mitochondrial DNA maintenance under stress, though specific mechanisms remain under investigation.

Z-DNA Binding Proteins

The Zα Domain and ADAR1

The Zα domain was first identified in 1997 as a Z-DNA-binding motif within the N-terminal region of human adenosine deaminase acting on RNA 1 (ADAR1), a double-stranded RNA editing enzyme. This discovery highlighted Zα as a specific recognition element for left-handed Z-DNA, distinguishing it from the enzyme's primary RNA-editing function. Subsequent studies confirmed that Zα is conserved across vertebrate ADAR1 orthologs and is particularly prominent in the interferon-inducible p150 isoform of the protein. The of the Zα domain from ADAR1, determined at 2.1 in complex with Z-DNA, revealed a compact winged--turn- fold. This architecture features a three- bundle flanked by a twisted antiparallel β-sheet, where a prominent β-hairpin extension from the β-sheet and the third α- (α3) insert into successive grooves of the Z-DNA . The β-hairpin clamps the DNA backbone, while positively charged residues in α3 form electrostatic interactions with the groups, enabling sequence-independent recognition of the Z conformation's zigzag backbone. This binding mode exploits the wider and deeper groove of Z-DNA compared to B-DNA, ensuring high specificity. Zα exhibits nanomolar for Z-DNA, with a (K_d) of approximately 10 nM for high-affinity substrates like poly(dG-dC)·poly(dG-dC) in the Z form, whereas binding to B-DNA is markedly weaker, with K_d values exceeding 1 μM. Notably, Zα not only recognizes preformed Z-DNA but also induces the B-to-Z conformational transition in DNA sequences with moderate , such as (dC-dA)_n·(dT-dG)_n or mixed purine-pyrimidine repeats, by stabilizing the energetically unfavorable Z form through favorable protein-DNA contacts. This inductive capability extends to non-CG dinucleotide repeats, broadening the potential genomic targets for Zα-mediated recognition. In the context of ADAR1, the Zα domain stabilizes Z-DNA structures, thereby facilitating the enzyme's recruitment to sites of potential Z-forming sequences and modulating A-to-I activity. This stabilization is thought to prevent aberrant immune activation by promoting editing of endogenous double-stranded RNAs that could otherwise trigger responses, thus averting . Mutations disrupting Zα function, such as those impairing DNA binding, have been linked to dysregulated editing and heightened autoinflammatory phenotypes in cellular models.

Other Key Binding Proteins

ZBP1, also known as DNA-dependent activator of IFN-regulatory factors () or DLM-1, serves as a cytosolic that specifically recognizes Z-DNA to initiate innate immune signaling. Upon binding Z-DNA, typically formed during viral infections or cellular stress, ZBP1 recruits and activates the TBK1-IRF3 pathway, leading to type I production, as well as the pathway for proinflammatory expression. This function positions ZBP1 as a critical mediator of antiviral and antibacterial defenses, with its discovery as a DNA sensor reported in 2007. The Vaccinia virus E3L protein, a viral inhibitor of host immunity, contains an N-terminal Zα domain that binds Z-DNA with high affinity, allowing the virus to counteract host sensors like ZBP1. By sequestering Z-DNA, E3L disrupts immune activation, inhibits interferon-beta induction, and blocks in infected cells, thereby promoting and . Experimental evidence from models in the early 2000s confirmed that abolishing E3L's Z-DNA binding activity severely attenuate viral virulence, underscoring its role in immune evasion. Recent studies as of 2024 have also identified novel Z-DNA binding domains in giant viruses of the Nucleocytoviricota phylum, expanding the known viral strategies for Z-DNA recognition. Z-DNA binding by these proteins often involves distinct modes, such as the groove-specific insertion of basic residues into the major groove by Zα-like domains, which confers sequence-dependent specificity, versus more general electrostatic interactions with the phosphodiester backbone. affinities typically range from nanomolar to micromolar, with stronger interactions favored by sequences like (CG)_n repeats that stabilize the Z conformation under physiological conditions. Structural analyses, including crystal structures of ZBP1's Zα domain complexed with Z-DNA, reveal how these modes enable precise recognition while accommodating conformational flexibility.

Genomic Occurrence and Evolution

Sequences and Factors Promoting Z-DNA

Z-DNA formation is preferentially adopted by DNA sequences featuring alternating purines and pyrimidines, which facilitate the structural transition from the right-handed B-form to the left-handed Z-form due to their ability to accommodate the zigzag backbone geometry. The canonical example is poly(dG-dC)·poly(dG-dC), or (CG)_n repeats, where the syn conformation of and anti conformation of stabilize the . Other sequences, such as (CA/TG)_n or (TA)_n tracts, can also form Z-DNA, though with varying efficiency depending on the base composition; for instance, GC-rich alternations are more prone than AT-rich ones because of stronger base stacking interactions in the Z conformation. These potential Z-DNA-forming sequences are abundant in eukaryotic genomes, occurring approximately once every 14,000 base pairs in the (corresponding to ~226,000 motifs), representing a significant fraction capable of adopting the Z-form under appropriate conditions. Several physicochemical factors promote Z-DNA adoption by reducing the energetic barrier of the B-to-Z transition, which is endergonic under standard physiological conditions. Negative supercoiling, quantified by superhelical density σ < -0.06, provides the torsional stress necessary to unwind and rewind the helix in the opposite direction, stabilizing Z-DNA even at low salt concentrations. High ionic strength, typically exceeding 1 M NaCl or MgCl_2, screens electrostatic repulsions in the phosphate backbone, favoring the more compact Z-form; this was first demonstrated with poly(dG-dC) transitioning to Z-DNA in high-salt solutions. Dehydration, achieved through low water activity (e.g., via addition of alcohols like ethanol), similarly compacts the helix by minimizing hydration shells around the bases, promoting Z-conformation in vitro. Trivalent cations, such as La^{3+}, further enhance stability by bridging phosphates more effectively than monovalent ions, inducing B-to-Z transitions at lower concentrations than divalent counterparts.90124-6)00301-8) Base modifications, particularly (5mC) in CpG dinucleotides, significantly bolster Z-DNA stability by introducing hydrophobic interactions between the methyl group and adjacent rings, which compensate for the loss of hydrogen bonding in the Z-form. This enhancement is estimated at 2-3 fold in terms of thermal stability for methylated versus unmethylated sequences, making Z-DNA more accessible in CpG islands prevalent in eukaryotic promoters. , these factors are complemented by dynamic torsional stress generated during transcription, where progression creates negative supercoils ahead and positive behind the fork, favoring Z-formation in susceptible sequences; activity modulates this stress, indirectly promoting transient Z-DNA extrusion.

Distribution, Density, and Evolutionary Aspects

In the , potential Z-DNA-forming sequences cover approximately 0.2% of the total length, with around 226,000 motifs spanning over 6.5 million base pairs, as identified using advanced prediction algorithms. These sites exhibit non-random , showing enrichment in promoter regions—up to 3.3-fold near transcription start sites—and intronic sequences, which comprise a significant portion of the predicted motifs, while being underrepresented in intergenic areas. The Z-Hunt , a foundational method for such predictions based on thermodynamic parameters, has been instrumental in mapping these distributions and highlighting their bias toward genic elements. A 2024 study across 154 genomes revealed higher Z-DNA density in birds compared to humans, ranging from 0.15 to 0.25 motifs per kilobase, with species exhibiting the highest levels. This elevated density correlates with phenotypic diversity, including variations in body mass, developmental timing, and ecological adaptations such as flight capability, suggesting Z-DNA contributes to evolutionary flexibility in among avian lineages. In contrast to mammals, avian genomes' compact structure and microchromosomes amplify this effect, linking Z-DNA prevalence to broader trait evolution. Z-DNA motifs demonstrate strong evolutionary within , where they are frequently preserved across like humans and mice, particularly in regulatory regions, but show reduced prevalence and stability in . Recent 2025 analyses indicate that Z-DNA formation extends to (mtDNA), influencing replication-transcription coupling and immune responses, with motifs integrated into conserved mtDNA elements across vertebrate lineages. This underscores Z-DNA's role in maintaining genomic stability and adaptive in complex . The development of the ZSeeker algorithm in 2025 has enhanced detection of Z-DNA in long-read sequencing data, offering improved accuracy and efficiency over predecessors like Z-Hunt by incorporating for motif prediction in large-scale genomic datasets. This tool facilitates precise mapping of Z-DNA densities across diverse species, enabling deeper insights into their evolutionary patterns without relying solely on short-read limitations.

Pathological Implications and Therapeutic Potential

Associations with Diseases

Z-DNA forming sequences are significantly enriched within super-enhancers across various cancer cell lines, including those from , colon, , and cancers, suggesting a role in driving aberrant programs. This enrichment is particularly notable in enhancer elements of super-enhancers, with up to 3.72-fold higher occurrence compared to genomic background, and mutations in these Z-DNA regions can disrupt transcription factor binding sites for SP/KLF family proteins, thereby influencing activation. For instance, in lymphomas, Z-DNA structures within super-enhancers associated with the may contribute to its amplification and overexpression, promoting tumor progression as evidenced by computational analyses of cancer genomes. Dysregulation of Z-DNA binding by ADAR1 has been implicated in autoimmune disorders, particularly Aicardi-Goutières syndrome (AGS), a monogenic interferonopathy. Mutations in the ADAR1 gene impair its Zα domain's ability to bind and resolve Z-DNA or Z-RNA structures, leading to accumulation of unedited self-nucleic acids that activate ZBP1. This triggers excessive type I production and , manifesting as neurological symptoms, skin lesions, and in AGS patients. Studies confirm that AGS-associated ADAR1 mutations result in a prominent interferon signature in patient cells, underscoring the imbalance in Z-nucleic acid sensing as a key pathological mechanism. In viral infections, Z-DNA serves as a danger signal detected by host sensors, but pathogens like poxviruses employ evasion strategies involving Z-DNA binding. The vaccinia virus E3L protein, through its N-terminal Zα domain, binds Z-DNA and Z-RNA to compete with ZBP1, thereby inhibiting necroptosis and responses that would otherwise restrict . This mechanism allows poxviruses to dampen innate immunity during . Conversely, ZBP1-mediated sensing of viral Z-nucleic acids, such as those generated by replication, activates RIPK3-dependent necroptosis in infected cells, limiting viral spread and contributing to antiviral defense. Neurological diseases like amyotrophic lateral sclerosis () involve protein aggregates of FUS, an /DNA-binding protein, leading to through disrupted nuclear functions and cytoplasmic inclusions. In the 2020s, research has highlighted how FUS mutations cause mislocalization and aggregation, impairing and in motor neurons, which exacerbates neurodegeneration. While direct Z-DNA interactions remain under , FUS aggregates in stress granules co-localize with Z-DNA-binding proteins, potentially linking Z-nucleic acid dysregulation to .

Emerging Therapeutic Applications

Recent research has explored inhibitors targeting ZBP1 to mitigate excessive inflammation in autoimmune diseases, where ZBP1 sensing of Z-DNA contributes to pathological immune activation. ZBP1 is implicated in conditions such as systemic lupus erythematosus (SLE) and , driving sterile inflammation through PANoptosis pathways. Small molecules, such as the covalent PROTAC (C-PROTAC) utilizing a DNA aptamer to bind ZBP1 and direct its ubiquitin-proteasome degradation, disrupt Z-DNA sensing by suppressing ZBP1-mediated necroptosis and inflammatory signaling. This approach shows promise in preclinical models for reducing infection-induced and autoinflammatory responses, with potential extension to . Additionally, the MSB binds the RHIM of ZBP1 with high affinity (K_D = 725 nM), preventing PANoptosome assembly and Z-DNA-triggered , as demonstrated in models of ischemia-reperfusion injury that share inflammatory mechanisms with . In cancer therapy, Z-DNA mimetics and stabilizers offer a novel strategy to exploit topological stress and immune activation against tumors. The small molecule CBL0137, from the curaxin family, promotes endogenous Z-DNA formation by stabilizing left-handed Z-conformations, bypassing ADAR1 suppression and activating ZBP1 to induce necroptosis in inflammatory cancer-associated fibroblasts (iCAFs). This process turns "cold" tumors immunogenic, enhancing the efficacy of checkpoint inhibitors like anti-PD-1 in immunotherapy-resistant melanomas and other solid tumors. By targeting Z-prone sequences (flipons) in promoters, these agents leverage DNA to disrupt support networks, reducing tumor progression independently of specific mutations and addressing resistance in preclinical models. CBL0137 has advanced to Phase I safety trials, highlighting its clinical potential when combined with epigenetic therapies. Antiviral strategies focus on disrupting the E3L-Z-DNA interaction in poxviruses to restore host immune sensing. The E3L protein, expressed by and other poxviruses, binds Z-DNA via its N-terminal Zα domain, inhibiting ZBP1-dependent necroptosis and responses to evade innate immunity. Blocking this interaction unmasks proinflammatory signaling, enhancing antiviral activation in infected cells like . Preclinical assays, including one-hybrid screens, support the development of small-molecule inhibitors that reduce E3L Z-DNA affinity, limiting and replication in poxvirus models. Such compounds could potentiate immune clearance in infections like or , with implications for broader threats.