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Genomic alterations are the driving force behind pancreatic cancer (PC) tumorigenesis, but they do not fully account for its diverse phenotypes. Investigating the epigenetic landscapes of PC offers a more comprehensive understanding and could identify targeted therapies that enhance patient survival. In this study, we have developed a new promising methodology of spatial epigenomics that integrates multiplexed molecular imaging with convolutional neural networks. Then, we used it to map epigenetic modification levels in the six most prevalent PC subtypes. We analyzed and semi-quantified the resulting molecular data, revealing significant variability in their epigenomes. DNA and histone modifications, specifically methylation and acetylation, were investigated. Using the same technique, we examined DNA conformational changes to further elucidate the transcriptional regulatory mechanisms involved in PC differentiation. Our results revealed that the foamy-gland and squamous-differentiated subtypes exhibited significantly increased global levels of epigenetic modifications and elevated Z-DNA ratios. Overall, our findings may suggest a potentially reduced efficacy of therapeutics targeting epigenetic regulators for these subtypes. Conversely, the conventional ductal PC subtype has emerged as a promising candidate for treatment with epigenetic modulators. [ABSTRACT FROM AUTHOR]

Abstract The untranslated regions (UTRs) of genes significantly impact various biological processes, including transcription, posttranscriptional control, mRNA stability, localization, and translation efficiency. In functional areas of genomes, non-B DNA structures such as cruciform, curved, triplex, G-quadruplex, and Z-DNA structures are common and have an impact on cellular physiology. Although the role of these structures in cis-regulatory regions such as promoters is well established in eukaryotic genomes, their prevalence within UTRs across different eukaryotic classes has not been extensively documented. Our study investigated the prevalence of various non-B DNA motifs within the 5′ and 3′ UTRs across diverse eukaryotic species. Our comparative analysis encompassed the 5′-UTRs and 3′UTRs of 360 species representing diverse eukaryotic domains of life, including Arthropoda (Diptera, Hemiptera, and Hymenoptera), Chordata (Artiodactyla, Carnivora, Galliformes, Passeriformes, Primates, Rodentia, Squamata, Testudines), Magnoliophyta (Brassicales), Fabales (Poales), and Nematoda (Rhabditida), on the basis of datasets derived from the UTRdb. We observed that species belonging to taxonomic orders such as Rhabditida, Diptera, Brassicales, and Hemiptera present a prevalence of curved DNA motifs in their UTRs, whereas orders such as Testudines, Galliformes, and Rodentia present a preponderance of G-quadruplexes in both UTRs. The distribution of motifs is conserved across different taxonomic classes, although species-specific variations in motif preferences were also observed. Our research unequivocally illuminates the prevalence and potential functional implications of non-B DNA motifs, offering invaluable insights into the evolutionary and biological significance of these structures.

Simple Summary: Various cancer therapies are often ineffective for advanced metastatic liver disease. While immunotherapy can be effective in some cases, it does not provide significant benefits for metastatic cancer in the liver. CBL0137 is an experimental drug that helps form a special type of DNA called Z-DNA, which may help boost the body's immune response against tumors. We investigated how CBL0137 affects metastatic liver tumor models from colon (CT26) and breast (4T1) cancers focusing on how it triggers immune responses. The results showed that CBL0137 hepatic arterial infusion (HAI) enhanced anti-tumor effects by depleting immune-suppressing cells while preserving effector T cells. Combining CBL0137 HAI with conventional immunotherapy improved survival in 4T1 tumors but not in CT26 tumors, highlighting the importance of targeting specific immune cell populations for effective treatment. Background: Surgery, chemotherapy, and radiation often have limited utility for advanced metastatic disease in the liver, and despite its promising activity in select cancers, PD-1 blockade therapy similarly has minimal benefit in this setting. Curaxin, CBL0137, is an experimental anti-cancer drug that disrupts the binding of DNA to histones, destabilizes chromatin, and induces Z-DNA formation which may stimulate anti-tumor immune responses. Methods: Murine cell lines of colon (CT26) and breast (4T1) cancer were interrogated for survival and CBL0137-associated DNA changes in vitro. Immunocompetent models of liver metastases followed by CBL0137 hepatic arterial infusion (HAI) were used to examine in vivo tumor cell DNA alterations, treatment responses, and the immune contexture associated with CBL0137, both alone and in combination with anti-PD-1 therapy. Results: CBL0137 induced immediate changes to favor tumor cell death in vitro and in vivo with an efficient tumor uptake via the HAI route. Toxicity to CBL0137 was minimal and anti-tumor treatment effects were more efficient with HAI compared to intravenous delivery. Immune effects were pronounced with CBL0137 HAI with concurrent depletion of a specific population of myeloid-derived suppressor cells and maintenance of effector T cell populations. Conclusions: Combination of CBL0137 HAI with PD-1 blockade improved survival in 4T1 tumors but not in CT26 tumors, and therapeutic efficacy relies on the finding of simultaneous and targeted depletion of myeloid-derived suppressor cells and skewing of T cell populations to produce synergy with PD-1 blockade therapy. [ABSTRACT FROM AUTHOR]

As with all new fields of discovery, work on the biological role of G-quadruplexes (GQs) has produced a number of results that at first glance are quite baffling, sometimes because they do not fit well together, but mostly because they are different from commonly held expectations. Like other classes of flipons, those that form G-quadruplexes have a repeat sequence motif that enables the fold. The canonical DNA motif (G3N1–7)3G3, where N is any nucleotide and G is guanine, is a feature that is under active selection in avian and mammalian genomes. The involvement of G-flipons in genome maintenance traces back to the invertebrate Caenorhabditis elegans and to ancient DNA repair pathways. The role of GQs in transcription is supported by the observation that yeast Rap1 protein binds both B-DNA, in a sequence-specific manner, and GQs, in a structure-specific manner, through the same helix. Other sequence-specific transcription factors (TFs) also engage both conformations to actuate cellular transactions. Noncoding RNAs can also modulate GQ formation in a sequence-specific manner and engage the same cellular machinery as localized by TFs, linking the ancient RNA world with the modern protein world. The coevolution of noncoding RNAs and sequence-specific proteins is supported by studies of early embryonic development, where the transient formation of G-quadruplexes coordinates the epigenetic specification of cell fate. [ABSTRACT FROM AUTHOR]

African swine fever (ASF) is an infectious disease characterized by hemorrhagic fever, which is highly pathogenic and causes severe mortality in domestic pigs. It is caused by the African swine fever virus (ASFV). ASFV is a large DNA virus and primarily infects porcine monocyte macrophages. The interaction between ASFV and host macrophages is the major reason for gross pathological lesions caused by ASFV. Necroptosis is an inflammatory programmed cell death and plays an important immune role during virus infection. However, whether and how ASFV induces macrophage necroptosis and the eect of necroptosis signaling on host immunity and ASFV infection remains unknown. This study uncovered that ASFV infection activates the necroptosis signaling in vivo and macrophage necroptosis in vitro. Further evidence showed that ASFV infection upregulates the expression of ZBP1 and RIPK3 to consist of the ZBP1-RIPK3-MLKL necrosome and further activates macrophage necroptosis. Subsequently, multiple Z-DNA sequences were predicted to be present in the ASFV genome. The Z-DNA signals were further confirmed to be present and colocalized with ZBP1 in the cytoplasm and nucleus of ASFV-infected cells. Moreover, ZBP1-mediated macrophage necroptosis provoked the extracellular release of proinflammatory cytokines, including TNF-α and IL-1β induced by ASFV infection. Finally, we demonstrated that ZBP1-mediated necroptosis signaling inhibits ASFV replication in host macrophages. Our findings uncovered a novel mechanism by which ASFV induces macrophage necroptosis by facilitating Z-DNA accumulation and ZBP1 necrosome assembly, providing significant insights into the pathogenesis of ASFV infection [ABSTRACT FROM AUTHOR]

Background: Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with an unpredictable course of recurrent exacerbations alternating with more stable disease. SLE is characterized by broad immune activation and autoantibodies against double-stranded DNA and numerous proteins that exist in cells as aggregates with nucleic acids, such as Ro60, MOV10, and the L1 retrotransposon-encoded ORF1p. Results: Here we report that these 3 proteins are co-expressed and co-localized in a subset of SLE granulocytes and are concentrated in cytosolic dots that also contain DNA: RNA heteroduplexes and the DNA sensor ZBP1, but not cGAS. The DNA: RNA heteroduplexes vanished from the neutrophils when they were treated with a selective inhibitor of the L1 reverse transcriptase. We also report that ORF1p granules escape neutrophils during the extrusion of neutrophil extracellular traps (NETs) and, to a lesser degree, from neutrophils dying by pyroptosis, but not apoptosis. Conclusions: These results bring new insights into the composition of ORF1p granules in SLE neutrophils and may explain, in part, why proteins in these granules become targeted by autoantibodies in this disease. [ABSTRACT FROM AUTHOR]

Objective: PANoptosis, a new form of regulated cell death, concomitantly manifests hallmarks for pyroptosis, apoptosis, and necroptosis. It has been usually observed in macrophages, a class of widely distributed innate immune cells in various tissues, upon pathogenic infections. The second-generation curaxin, CBL0137, can trigger necroptosis and apoptosis in cancer-associated fibroblasts. This study aimed to explore whether CBL0137 induces PANoptosis in macrophages in vitro and in mouse tissues in vivo. Methods: Bone marrow-derived macrophages and J774A.1 cells were treated with CBL0137 or its combination with LPS for indicated time periods. Cell death was assayed by propidium iodide staining and immunoblotting. Immunofluorescence microscopy was used to detect cellular protein distribution. Mice were administered with CBL0137 plus LPS and their serum and tissues were collected for biochemical and histopathological analyses, respectively. Results: The results showed that CBL0137 alone or in combination with LPS induced time- and dose-dependent cell death in macrophages, which was inhibited by a combination of multiple forms of cell death inhibitors but not each alone. This cell death was independent of NLRP3 expression. CBL0137 or CBL0137 + LPS-induced cell death was characterized by simultaneously increased hallmarks for pyroptosis, apoptosis and necroptosis, indicating that this is PANoptosis. Induction of PANoptosis was associated with Z-DNA formation in the nucleus and likely assembly of PANoptosome. ZBP1 was critical in mediating CBL0137 + LPS-induced cell death likely by sensing Z-DNA. Moreover, intraperitoneal administration of CBL0137 plus LPS induced systemic inflammatory responses and caused multi-organ (including the liver, kidney and lung) injury in mice due to induction of PANoptosis in these organs. Conclusions: CBL0137 alone or plus inflammatory stimulation induces PANoptosis both in vitro and in vivo, which is associated with systemic inflammatory responses in mice. [ABSTRACT FROM AUTHOR]

Antibodies to DNA are a diverse set of antibodies that bind sites on DNA, a polymeric macromolecule that displays various conformations. In a previous study, we showed that sera of normal healthy subjects (NHS) contain IgG antibodies to Z-DNA, a left-handed helix with a zig-zig backbone. Recent studies have demonstrated the presence of Z-DNA in bacterial biofilms, suggesting a source of this conformation to induce responses. To characterize further antibodies to Z-DNA, we used an ELISA assay with brominated poly(dGdC) as a source of Z-DNA and determined the isotype of these antibodies and their binding properties. Results of these studies indicate that NHS sera contain IgM and IgA as well as IgG anti-Z-DNA antibodies. As shown by the effects of ionic strength in association and dissociation assays, the anti-Z-DNA antibodies bind primarily by electrostatic interactions; this type of binding differs from that of induced anti-Z-DNA antibodies from immunized animals which bind by non-ionic interactions. Furthermore, urea caused dissociation of NHS anti-Z-DNA at molar concentrations much lower than those for the induced antibodies. These studies also showed IgA anti-Z-DNA antibodies in fecal water. Together, these studies demonstrate that antibodies to Z-DNA occur commonly in normal immunity and may arise as a response to Z-DNA of bacterial origin. [ABSTRACT FROM AUTHOR]

Under different conditions, the DNA double helix can take different geometric forms. Of the large number of its conformations, in addition to the "canonical" B form, the A, C, and Z forms are widely known, and the D, Hoogsteen, and X forms are less known. DNA locally takes the A, C, and Z forms in the cell, in complexes with proteins. We compare different methods for detecting non-canonical DNA conformations: X-ray, IR, and Raman spectroscopy, linear and circular dichroism in both the infrared and ultraviolet regions, as well as NMR (measurement of chemical shifts and their anisotropy, scalar and residual dipolar couplings and inter-proton distances from NOESY (nuclear Overhauser effect spectroscopy) data). We discuss the difficulties in applying these methods, the problems of theoretical interpretation of the experimental results, and the prospects for reliable identification of non-canonical DNA conformations. [ABSTRACT FROM AUTHOR]

The dynamic processes operating on genomic DNA, such as gene expression and cellular division, lead inexorably to topological challenges in the form of entanglements, catenanes, knots, "bubbles", R-loops, and other outcomes of supercoiling and helical disruption. The resolution of toxic topological stress is the function attributed to DNA topoisomerases. A prominent example is the negative supercoiling (nsc) trailing processive enzymes such as DNA and RNA polymerases. The multiple equilibrium states that nscDNA can adopt by redistribution of helical twist and writhe include the left-handed double-helical conformation known as Z-DNA. Thirty years ago, one of our labs isolated a protein from Drosophila cells and embryos with a 100-fold greater affinity for Z-DNA than for B-DNA, and identified it as topoisomerase II (gene Top2, orthologous to the human UniProt proteins TOP2A and TOP2B). GTP increased the affinity and selectivity for Z-DNA even further and also led to inhibition of the isomerase enzymatic activity. An allosteric mechanism was proposed, in which topoII acts as a Z-DNA-binding protein (ZBP) to stabilize given states of topological (sub)domains and associated multiprotein complexes. We have now explored this possibility by comprehensive bioinformatic analyses of the available protein sequences of topoII representing organisms covering the whole tree of life. Multiple alignment of these sequences revealed an extremely high level of evolutionary conservation, including a winged-helix protein segment, here denoted as Zτ, constituting the putative structural homolog of Zα, the canonical Z-DNA/Z-RNA binding domain previously identified in the interferon-inducible RNA Adenosine-to-Inosine-editing deaminase, ADAR1p150. In contrast to Zα, which is separate from the protein segment responsible for catalysis, Zτ encompasses the active site tyrosine of topoII; a GTP-binding site and a GxxG sequence motif are in close proximity. Quantitative Zτ-Zα similarity comparisons and molecular docking with interaction scoring further supported the "B-Z-topoII hypothesis" and has led to an expanded mechanism for topoII function incorporating the recognition of Z-DNA segments ("Z-flipons") as an inherent and essential element. We further propose that the two Zτ domains of the topoII homodimer exhibit a single-turnover "conformase" activity on given G(ate) B-DNA segments ("Z-flipins"), inducing their transition to the left-handed Z-conformation. Inasmuch as the topoII-Z-DNA complexes are isomerase inactive, we infer that they fulfill important structural roles in key processes such as mitosis. Topoisomerases are preeminent targets of anti-cancer drug discovery, and we anticipate that detailed elucidation of their structural–functional interactions with Z-DNA and GTP will facilitate the design of novel, more potent and selective anti-cancer chemotherapeutic agents. [ABSTRACT FROM AUTHOR]

Z-DNA refers to the left-handed double-helix DNA that has attracted much attention because of its association with some specific biological functions. However, because of its low content and unstable conformation, Z-DNA is normally difficult to observe or identify. Up to now, there has been a lack of unified or standard analytical methods among diverse techniques for probing Z-DNA and its transformation conveniently. In this work, NaCl, MgCl2, and ethanol were utilized to induce d(GC)8 from B-DNA to Z-DNA in vitro, and Fourier transform infrared (FTIR) spectroscopy was employed to monitor the transformation of Z-DNA under different induction conditions. The structural changes during the transformation process were carefully examined, and the DNA chirality alterations were validated by the circular dichroism (CD) measurements. The Z-DNA characteristic signals in the 1450 cm−1–900 cm−1 region of the d(GC)8 infrared (IR) spectrum were observed, which include the peaks at 1320 cm−1, 1125 cm−1 and 925 cm−1, respectively. The intensity ratios of A1320/A970, A1125/A970, and A925/A970 increased with Z-DNA content in the transition process. Furthermore, compared with the CD spectra, the IR spectra showed higher sensitivity to Z-DNA, providing more information about the molecular structure change of DNA. Therefore, this study has established a more reliable FTIR analytical approach to assess BZ DNA conformational changes in solutions, which may help the understanding of the Z-DNA transition mechanism and promote the study of Z-DNA functions in biological systems. [ABSTRACT FROM AUTHOR]

The classical view of gene regulation draws from prokaryotic models, where responses to environmental changes involve operons regulated by sequence-specific protein interactions with DNA, although it is now known that operons are also modulated by small RNAs. In eukaryotes, pathways based on microRNAs (miR) regulate the readout of genomic information from transcripts, while alternative nucleic acid structures encoded by flipons influence the readout of genetic programs from DNA. Here, we provide evidence that miR- and flipon-based mechanisms are deeply connected. We analyze the connection between flipon conformation and the 211 highly conserved human miR that are shared with other placental and other bilateral species. The direct interaction between conserved miR (c-miR) and flipons is supported by sequence alignments and the engagement of argonaute proteins by experimentally validated flipons as well as their enrichment in promoters of coding transcripts important in multicellular development, cell surface glycosylation and glutamatergic synapse specification with significant enrichments at false discovery rates as low as 10−116. We also identify a second subset of c-miR that targets flipons essential for retrotransposon replication, exploiting that vulnerability to limit their spread. We propose that miR can act in a combinatorial manner to regulate the readout of genetic information by specifying when and where flipons form non-B DNA (NoB) conformations, providing the interactions of the conserved hsa-miR-324-3p with RELA and the conserved hsa-miR-744 with ARHGAP5 genes as examples. [ABSTRACT FROM AUTHOR]

Cytoplasmic mitochondrial DNA (mtDNA) can trigger the interferon response to promote disease progression, but mtDNA sensing mechanisms remain elusive. Lei et al. have shown that Z-DNA binding protein1 (ZBP1) cooperates with cyclic GMP-AMP synthase (cGAS) to sense Z-form mtDNA and transmit mtDNA stress signals to promote diseases such as cardiotoxicity, providing an important piece of the mtDNA stress landscape. [ABSTRACT FROM AUTHOR]

The self‐complementary l‐d(CGCGCG)2 purine/pyrimidine hexanucleotide was crystallized in complex with the polyamine cadaverine and potassium cations. Since the oligonucleotide contained the enantiomeric 2′‐deoxy‐l‐ribose, the Z‐DNA duplex is right‐handed, as confirmed by the ultrahigh‐resolution crystal structure determined at 0.69 Å resolution. Although the X‐ray diffraction data were collected at a very short wavelength (0.7085 Å), where the anomalous signal of the P and K atoms is very weak, the signal was sufficiently outstanding to clearly indicate the wrong hand when the structure was mistakenly solved assuming the presence of 2′‐deoxy‐d‐ribose. The electron density clearly shows the entire cadaverinium dication, which has an occupancy of 0.53 and interacts with one Z‐DNA duplex. The K+ cation, with an occupancy of 0.32, has an irregular coordination sphere that is formed by three OP atoms of two symmetry‐related Z‐DNA duplexes and one O5′ hydroxyl O atom, and is completed by three water sites, one of which is twofold disordered. The K+ site is complemented by a partial water molecule, the hydrogen bonds of which have the same lengths as the K—O bonds. The sugar‐phosphate backbone assumes two conformations, but the base pairs do not show any sign of disorder. [ABSTRACT FROM AUTHOR]

Non-B DNA promotes genomic instability and large-scale rearrangements. In some genomic regions (e.g., promoters and enhancers), G-quadruplex (G4) motifs evolve under purifying selection because of their functionality, yet in some other genomic regions (e.g., coding exons), they might be selected against because of their detrimental effects. Non-B DNA is over-represented in transposable elements (TEs), participates in the life cycle of some of them, and can affect their integration. Many eukaryotic centromeres are specified by non-B DNA. Elevated mutagenicity of non-B DNA may explain some instances of repeated evolution and trans-species polymorphism in fishes and is likely to contribute to phenotypic evolution in other species. In addition to the canonical right-handed double helix, other DNA structures, termed 'non-B DNA', can form in the genomes across the tree of life. Non-B DNA regulates multiple cellular processes, including replication and transcription, yet its presence is associated with elevated mutagenicity and genome instability. These discordant cellular roles fuel the enormous potential of non-B DNA to drive genomic and phenotypic evolution. Here we discuss recent studies establishing non-B DNA structures as novel functional elements subject to natural selection, affecting evolution of transposable elements (TEs), and specifying centromeres. By highlighting the contributions of non-B DNA to repeated evolution and adaptation to changing environments, we conclude that evolutionary analyses should include a perspective of not only DNA sequence, but also its structure. [ABSTRACT FROM AUTHOR]

Despite structural differences between the right-handed conformations of A-RNA and B-DNA, both nucleic acids adopt very similar, left-handed Z-conformations. In contrast to their structural similarities and sequence preferences, RNA and DNA exhibit differences in their ability to adopt the Z-conformation regarding their hydration shells, the chemical modifications that promote the Z-conformation, and the structure of junctions connecting them to right-handed segments. In this review, we highlight the structural and chemical properties of both Z-DNA and Z-RNA and delve into the potential factors that contribute to both their similarities and differences. While Z-DNA has been extensively studied, there is a gap of knowledge when it comes to Z-RNA. Where such information is lacking, we try and extend the principles of Z-DNA stability and formation to Z-RNA, considering the inherent differences of the nucleic acids. [ABSTRACT FROM AUTHOR]

Alternative non‐B‐DNA conformations formed under physiological conditions by sequences called flipons include left‐handed Z‐DNA, three‐stranded triplexes, and four‐stranded i‐motifs and quadruplexes. These conformations accumulate and release energy to enable the local assembly of cellular machines in a context specific manner. In these transactions, nucleosomes store power, serving like rechargeable batteries, while flipons smooth energy flows from source to sink by acting as capacitors or resistors. Here, I review the known biological roles for flipons. I present recent and unequivocal findings showing how innate immune responses are regulated by Z‐flipons that identify endogenous RNAs as self. Evidence is also presented supporting important roles for other flipon classes. In these examples, the dynamic exchange of energy between flipons and nucleosomes enables rapid switching of genetic programs without altering flipon sequence. The increased phenotypic diversity enabled by flipons drives their natural selection, with adaptations evolving faster than is possible by codon mutation alone. [ABSTRACT FROM AUTHOR]

Z-DNA is a left-handed double helix form of DNA that is believed to be involved in various DNA transactions. However, comprehensive investigations aimed at global profiling of Z-DNA landscapes are still missing in both humans and plants. We here report the development of two techniques: anti-Z-DNA antibody-based immunoprecipitation followed by sequencing (ZIP-seq), and cleavage under targets and tagmentation (CUT&TAG) for characterizing Z-DNA in nipponbare rice (Oryza sativa L., Japonica). We found that Z-DNA-IP + (Z-DNA recognized by the antibody) exhibits distinct genomic features as compared to Z-DNA-IP - (Z-DNA not recognized by the antibody). The concomitant presence of G-quadruplexes (G4s) and i-motifs (iMs) may promote Z-DNA formation. DNA modifications such as DNA-6mA/-4acC generally disfavours Z-DNA formation, while modifications like DNA-5mC (CHH) and 8-oxodG promote it, highlighting the distinct roles of DNA base modifications in modulating Z-DNA formation. Importantly, Z-DNA located at transcription start sites (TSSs) enhances gene expression, whereas Z-DNA in genic regions represses it, underscoring its dual roles in regulating the expression of genes involved in fundamental biological functions and responses to salt stress. Furthermore, Z-DNA may play a role in transcriptional initiation and termination rather than in transcriptional elongation. Finally, the presence of Z-DNA in promoters is correlated with the coevolution of overlapping genes, thereby regulating gene domestication. Consequently, our study represents as a pivotal point and a solid foundation for reliably launching genome-wide investigations of Z-DNA, thereby advancing the understanding of Z-DNA biology in both plants and non-plant systems., (© 2025 The Author(s). Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

DNA can fold into noncanonical left-handed Z-DNA conformation beyond the right-handed B-DNA. While its crystal structure was discovered nearly four decades ago, it was predominantly considered a structural curiosity. Recent evidence suggests that Z-DNA formation occurs in nuclear and mitochondrial DNA (mtDNA), with significant biological implications. However, our understanding of its roles remains in its infancy, primarily due to a lack of study tools. In this review we summarize the structure and function of Z-DNA within the genome while addressing the difficulties associated with identifying and investigating its role(s). We then critically evaluate several intracellular factors that can modulate and regulate Z-DNA. Additionally, we discuss the recent technological and methodological advances that may overcome the challenges and enhance our understanding of Z-DNA., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2025 Elsevier Ltd. All rights reserved.)

A complete understanding of DNA double-helical structure discovered by James Watson and Francis Crick in 1953, unveil the importance and significance of DNA. For the last seven decades, this has been a leading light in the course of the development of modern biology and biomedical science. Apart from the predominant B-form, experimental shreds of evidence have revealed the existence of a sequence-dependent structural diversity, unusual non-canonical structures like hairpin, cruciform, Z-DNA, multistranded structures such as DNA triplex, G-quadruplex, i-motif forms, etc. The diversity in the DNA structure depends on various factors such as base sequence, ions, superhelical stress, and ligands. In response to these various factors, the polymorphism of DNA regulates various genes via different processes like replication, transcription, translation, and recombination. However, altered levels of gene expression are associated with many human genetic diseases including neurological disorders and cancer. These non-BDNA structures are expected to play a key role in determining genetic stability, DNA damage and repair etc. The present review is a modest attempt to summarize the available literature, illustrating the occurrence of noncanonical structures at the molecular level in response to the environment and interaction with ligands and proteins. This would provide an insight to understand the biological functions of these unusual DNA structures and their recognition as potential therapeutic targets for diverse genetic diseases. [ABSTRACT FROM AUTHOR]

The transition from right-handed to left-handed DNA is not only acts as the controlling factor for switching gene expression but also has equal importance in designing nanomechanical devices. The (CG) n and (GC) n repeat sequences are well known model molecules to study B-Z transition in the presence of higher concentration of monovalent cations. In this communication, we report a cyclic transition in (CG) 6 DNA using millimolar concentration of trivalent lanthanide salt LaCl 3. The controlled and reversible transition was seen in (CG) 12 , and (GC) 12 DNA employing CD spectroscopy. While LaCl 3 failed to induce B-Z transition in shorter oligonucleotides such as (CG) 3 and (GC) 3 , a smooth B-Z transition was recorded for (CG) 6 , (CG) 12 and (GC) 12 sequences. Interestingly, the phenomenon was reversible (Z-B transition) with addition of EDTA. Particularly, two rounds of cyclic transition (B-Z-B-Z-B) have been noticed in (CG) 6 DNA in presence of LaCl 3 and EDTA which strongly suggest that B-Z transition is reversible in short repeat sequences. Thermal melting and annealing behaviour of B-DNA are reversible while the thermal melting of LaCl 3 -induced Z-DNA is irreversible which suggest a stronger binding of LaCl 3 to the phosphate backbone of Z-DNA. This was further supported by isothermal titration calorimetric study. Molecular dynamics (MD) simulation indicates that the mode of binding of La3+ (of LaCl 3) with d(CG) 8.d(CG) 8 is through the minor groove, wherein, 3 out of 11 La3+ bridge the anionic oxygens of the complementary strands. Such a tight coordination of La3+ with the anionic oxygens at the minor groove surface may be the reason for the experimentally observed irreversibility of LaCl 3 -induced Z-DNA seen in longer DNA fragments. Thus, these results indicate LaCl 3 can easily be adopted as an inducer of left-handed DNA in other short oligonucleotides sequences to facilitate the understanding of the molecular mechanism of B-Z transition. Schematic presentation of LaCl 3 -induced B-Z transition in (CG) 6 DNA and reverse by EDTA. [Display omitted] • Low concentration of LaCl 3 stabilizes left-handed DNA in (CG) n repeats and is entropically and enthalpically favorable • (CG) n DNA sequences are more sensitive to B-Z transition than (GC) n DNA and longer sequences favor the B-Z transition • Two-rounds of cyclic transition of B-Z-B-Z-B in (CG) n DNA suggesting reversibility by LaCl 3 and EDTA • Molecular dynamics simulation study confirms the La3+-induced left-handed DNA (CG) n and (GC) n DNA • Z-DNA specific signature FTIR spectra was identified in (CG) n and (GC) n DNA [ABSTRACT FROM AUTHOR]

African Swine Fever (ASF) is a highly contagious viral haemorrhagic disease of swine, leading to enormous economic losses in the swine industry. However, vaccines and drugs to treat ASF have yet to be developed. African swine fever virus (ASFV) encodes more than 150 proteins, but 50% of them have unknown functions. Here, we present the crystal structure of the ASFV I73R protein at a resolution of 2.0 Å. Similar search tools based solely on amino acid sequence shows that it has no relationships to any proteins of known function. Interestingly, the overall structure of the I73R protein shares a winged helix‐turn‐helix fold, structural similarity with the Z‐DNA binding domain (Zα). In accordance with this result, the I73R is capable of binding to a CpG repeats DNA duplex, which has a high propensity for forming Z‐DNA during the DNA binding assays. In addition, the I73R protein was shown to be expressed at both early and late stages of ASFV post‐infection in PAM cells as an 8.9 kDa protein. Immunofluorescence studies revealed that the I73R protein is expressed in the nucleus at early times post‐infection and gradually translocated from the nucleus to the cytoplasm. Taken together, these data indicate that the I73R could be a member of Zα family that is important in host–pathogen interaction, which paves the way for the design of inhibitors to target this severe pathogen. Further exploring the biological role of I73R during ASFV infection in vitro and in vivo will provide new clues for development of new antiviral strategies. [ABSTRACT FROM AUTHOR]

A complete understanding of DNA double-helical structure discovered by James Watson and Francis Crick in 1953, unveil the importance and significance of DNA. For the last seven decades, this has been a leading light in the course of the development of modern biology and biomedical science. Apart from the predominant B-form, experimental shreds of evidence have revealed the existence of a sequence-dependent structural diversity, unusual non-canonical structures like hairpin, cruciform, Z-DNA, multistranded structures such as DNA triplex, G-quadruplex, i-motif forms, etc. The diversity in the DNA structure depends on various factors such as base sequence, ions, superhelical stress, and ligands. In response to these various factors, the polymorphism of DNA regulates various genes via different processes like replication, transcription, translation, and recombination. However, altered levels of gene expression are associated with many human genetic diseases including neurological disorders and cancer. These non-B-DNA structures are expected to play a key role in determining genetic stability, DNA damage and repair etc. The present review is a modest attempt to summarize the available literature, illustrating the occurrence of non-canonical structures at the molecular level in response to the environment and interaction with ligands and proteins. This would provide an insight to understand the biological functions of these unusual DNA structures and their recognition as potential therapeutic targets for diverse genetic diseases.

The binding of proteins to Z-DNA is hard to analyze, especially for short non-modified DNA, because it is easily transferred to B-DNA. Here, by the hybridization of a larger circular single-stranded DNA (ssDNA) with a smaller one, an LR-chimera (involving a left-handed part and a right-handed one) with an ssDNA loop is produced. The circular ssDNAs are prepared by the hybridization of two ssDNA fragments to form two nicks, followed by nick sealing with T4 DNA ligase. No splint (a scaffold DNA for circularizing ssDNA) is required, and no polymeric byproducts are produced. The ssDNA loop on the LR-chimera can be used to attach it with other molecules by hybridization with another ssDNA. The gel shift binding assay with Z-DNA specific binding antibody (Z22) or Z-DNA binding protein 1 (ZBP1) shows that stable Z-DNA can form under physiological ionic conditions even when the extra ssDNA part is present. Concretely, a 5′-terminal biotin-modified DNA oligonucleotide complementary to the ssDNA loop on the LR-chimera is used to attach it on the surface of a biosensor inlaid with streptavidin molecules, and the binding constant of ZBP1 with Z-DNA is analyzed by BLI (bio-layer interferometry). This approach is convenient for quantitatively analyzing the binding dynamics of Z-DNA with other molecules. [ABSTRACT FROM AUTHOR]

Z-DNA binding protein (ZBP1) very much represents the nuclear option. By initiating inflammatory cell death (ICD), ZBP1 activates host defenses to destroy infectious threats. ZBP1 is also able to induce noninflammatory regulated cell death via apoptosis (RCD). ZBP1 senses the presence of left-handed Z-DNA and Z-RNA (ZNA), including that formed by expression of endogenous retroelements. Viruses such as the Epstein–Barr "kissing virus" inhibit ICD, RCD and other cell death signaling pathways to produce persistent infection. EBV undergoes lytic replication in plasma cells, which maintain detectable levels of basal ZBP1 expression, leading us to suggest a new role for ZBP1 in maintaining EBV latency, one of benefit for both host and virus. We provide an overview of the pathways that are involved in establishing latent infection, including those regulated by MYC and NF-κB. We describe and provide a synthesis of the evidence supporting a role for ZNA in these pathways, highlighting the positive and negative selection of ZNA forming sequences in the EBV genome that underscores the coadaptation of host and virus. Instead of a fight to the death, a state of détente now exists where persistent infection by the virus is tolerated by the host, while disease outcomes such as death, autoimmunity and cancer are minimized. Based on these new insights, we propose actionable therapeutic approaches to unhost EBV. [ABSTRACT FROM AUTHOR]

Summary: Alternative DNA conformations, termed non-B DNA structures, can affect transcription, but the underlying mechanisms and their functional impact have not been systematically characterized. Here, we used computational genomic analyses coupled with massively parallel reporter assays (MPRAs) to show that certain non-B DNA structures have a substantial effect on gene expression. Genomic analyses found that non-B DNA structures at promoters harbor an excess of germline variants. Analysis of multiple MPRAs, including a promoter library specifically designed to perturb non-B DNA structures, functionally validated that Z-DNA can significantly affect promoter activity. We also observed that biophysical properties of non-B DNA motifs, such as the length of Z-DNA motifs and the orientation of G-quadruplex structures relative to transcriptional direction, have a significant effect on promoter activity. Combined, their higher mutation rate and functional effect on transcription implicate a subset of non-B DNA motifs as major drivers of human gene-expression-associated phenotypes.

Z-DNA has attracted interest due to its distinctive left-handed helical structure. This non-canonical DNA structure is able to form transiently and plays an important role in cellular processes such as transcriptional regulation and DNA recombination. Alternating purine–pyrimidine sequences are well known to form Z-DNA under high-salt conditions, but the detailed mechanism of B-to-Z transition of DNA containing BZ junctions under these conditions is not well understood. Here, using single-molecule FRET and circular dichroism experiments, we studied the effect of BZ junctions on Z-DNA formation under high-salt conditions. Further thermodynamic analysis revealed that a discrepancy of different DNA substrates in the presence and absence of BZ junctions in Z-DNA formation can be attributed mainly to the competition between enthalpy and entropy. Salt-induced B-to-Z transition is entropically favored in the presence of BZ junctions and is enthalpically favored in their absence. This thermodynamic information provides a deeper understanding of Z-DNA formation of DNA containing BZ junctions. [Display omitted] • Energetics of Z-DNA formation of DNA with or without BZ junction is revealed. • In the presence of BZ junction, Z-DNA formation induced by high salt is entropically favored. • Z-DNA formation is enthalpically favored in the absence of BZ junction. [ABSTRACT FROM AUTHOR]

Scheme 1. PrCl 3 mediated B-Z transition in (CG) n DNA and reverse back by EDTA. [Display omitted] • Praseodymium shows sequence specific B-Z transition in pyrimidine-purine repeats. • PrCl 3 stabilizes left-handed DNA in (CG)n repeats but condensation in (GC)n DNA. • EDTA-based chelation of Pr results in Z-B reverse transition in CG-repeat. • Pr-induced Z-DNA in (CG)n exhibits close to neutral zeta potential and lower Tm. • Molecular dynamics simulation verifies the ability of Pr3+ to produce left-handed DNA. The present study investigates the impact of praseodymium chloride on the conformational transition between the B-form and Z-form of DNA. In the current communication, we employ a combination of biophysical techniques, including circular dichroism, isothermal titration calorimetry, fourier transform infrared, zeta potential and molecular dynamics simulations to elucidate the mechanism of B-Z transition. Our results reveal a praseodymium-dependent modulation of the DNA helical structure, leading to the conversion from the canonical B-DNA to the left-handed Z-DNA in (CG) n repeat and condensation in (GC) n repeat. Altered helical parameters, base pair stacking and overall DNA geometry upon praseodymium binding has been characterized using spectroscopic analysis. The decrease in Gibbs free energy during ITC indicates a spontaneous reaction and provides the specific binding sites and coordination geometry of praseodymium ions within the DNA double helix. Molecular dynamics simulation further complements our experimental findings, offering a dynamic perspective on the temporal evolution of the B-Z transition process. We observe that praseodymium plays a crucial role in stabilizing the Z-DNA conformation in (CG) n repeat, providing a foundation for understanding the thermodynamics and kinetics of this transition. [ABSTRACT FROM AUTHOR]

Summary: Loss of function of adenosine deaminase acting on double-stranded RNA (dsRNA)-1 (ADAR1) causes the severe autoinflammatory disease Aicardi-Goutières syndrome (AGS). ADAR1 converts adenosines into inosines within dsRNA. This process called A-to-I editing masks self-dsRNA from detection by the antiviral dsRNA sensor MDA5. ADAR1 binds to dsRNA in both the canonical A-form and the poorly defined Z conformation (Z-RNA). Mutations in the Z-RNA-binding Zα domain of ADAR1 are common in patients with AGS. How loss of ADAR1/Z-RNA interaction contributes to disease development is unknown. We demonstrate that abrogated binding of ADAR1 to Z-RNA leads to reduced A-to-I editing of dsRNA structures formed by base pairing of inversely oriented short interspersed nuclear elements. Preventing ADAR1 binding to Z-RNA triggers an MDA5/MAVS-mediated type I interferon response and leads to the development of lethal autoinflammation in mice. This shows that the interaction between ADAR1 and Z-RNA restricts sensing of self-dsRNA and prevents AGS development.

The X‐ray crystal structure of the d(CGCGCG)2/putrescine(2+)/K+ complex has been determined at 0.60 Å resolution. Stereochemical restraints were used only for the putrescinium dication, and 23 bonds and 18 angles of the Z‐DNA nucleotides with dual conformation. The N atoms of the putrescine(2+) dication form three direct hydrogen bonds with the N7_G atoms of three different Z‐DNA molecules, plus three water‐mediated hydrogen bonds with cytosine, guanine and phosphate acceptors. A unique potassium cation was also unambiguously identified in the structure, albeit at a ∼0.5 occupation site shared with a water molecule, providing the first example of such a complex with Z‐DNA. The K+ cation has coordination number of eight and an irregular coordination sphere, formed by four water molecules and four O atoms from two phosphate groups of the Z‐DNA, including ligands present at fractional occupancy. The structural disorder of the Z‐DNA duplex is manifested by the presence of alternate conformations along the DNA backbone. Comparison of the position and interactions of putrescine(2+) in the present structure with other ultra‐high‐resolution structures of Z‐DNA in complexes with Mn2+ and Zn2+ ions shows that the dicationic putrescinium moiety can effectively substitute these metal ions for stabilization of Z‐type DNA duplexes. Furthermore, this comparison also suggests that the spermine(4+) tetracation has a higher affinity for Z‐DNA than K+. [ABSTRACT FROM AUTHOR]

ALUs contribute to genetic diversity by altering DNA's linear sequence through retrotransposition, recombination and repair. ALUs also have the potential to form alternative non-B-DNA conformations such as Z-DNA, triplexes and quadruplexes that alter the read-out of information from the genome. I suggest here these structures enable the rapid reprogramming of cellular pathways to offset DNA damage and regulate inflammation. The experimental data supporting this form of genetic encoding is presented. ALU sequence motifs that form non-B-DNA conformations under physiological conditions are called flipons. Flipons are binary switches. They are dissipative structures that trade energy for information. By efficiently targeting cellular machines to active genes, flipons expand the repertoire of RNAs compiled from a gene. Their action greatly increases the informational capacity of linearly encoded genomes. Flipons are programmable by epigenetic modification, synchronizing cellular events by altering both chromatin state and nucleosome phasing. Different classes of flipon exist. Z-flipons are based on Z-DNA and modify the transcripts compiled from a gene. T-flipons are based on triplexes and localize non-coding RNAs that direct the assembly of cellular machines. G-flipons are based on G-quadruplexes and sense DNA damage, then trigger the appropriate protective responses. Flipon conformation is dynamic, changing with context. When frozen in one state, flipons often cause disease. The propagation of flipons throughout the genome by ALU elements represents a novel evolutionary innovation that allows for rapid change. Each ALU insertion creates variability by extracting a different set of information from the neighbourhood in which it lands. By elaborating on already successful adaptations, the newly compiled transcripts work with the old to enhance survival. Systems that optimize flipon settings through learning can adapt faster than with other forms of evolution. They avoid the risk of relying on random and irreversible codon rewrites.

PKZ is a novel and unique eIF2α protein kinase identified in fish. Although PKZ is most homologous to PKR, particularly in the C-terminal catalytic domain, it contains two N-terminal Z-DNA-binding domains (Zα1 and Zα2) instead of the dsRNA binding domains (dsRBDs) in PKR. As a novel member of eIF2α kinase family, the available data suggest that PKZ has some distinct mechanisms for recognition, binding, and B-Z DNA transition. Functionally, PKZ seems to be activated by the binding of Zα to Z-DNA and participates in innate immune responses. In this review, we summarize the recent progress on fish PKZ.

Structural transformation of the canonical right-handed helix, B-DNA, to the non-canonical left-handed helix, Z-DNA, can be induced by the Zα domain of the human RNA editing enzyme ADAR1 (hZα ADAR1). To characterize the site-specific preferences of binding and structural changes in DNA containing the 2′-O-methyl guanosine derivative (m G), titration of the imino proton spectra and chemical shift perturbations were performed on hZα ADAR1 upon binding to Z-DNA. The structural transition between B–Z conformation as the changing ratio between DNA and protein showed a binding affinity of the modified DNA onto the Z-DNA binding protein similar to wild-type DNA or RNA. The chemical shift perturbation results showed that the overall structure and environment of the modified DNA revealed DNA-like properties rather than RNA-like characteristics. Moreover, we found evidence for two distinct regimes, "Z-DNA Sensing" and "Modification Sensing", based on the site-specific chemical shift perturbation between the DNA (or RNA) binding complex and the modified DNA–hZα ADAR1 complex. Thus, we propose that modification of the sugar backbone of DNA with 2′-O-methyl guanosine promotes the changes in the surrounding α3 helical structural segment as well as the non-perturbed feature of the β-hairpin region. Image 1 • Zα ADAR1 prefers to bind to DNA duplex containing a 2′-OMe-guanosine. • Zα ADAR1 exhibits Z-DNA-like binding mode to 2′-OMe-G-containing DNA. • Zα ADAR1 senses 2′-OMe guanosine in DNA duplex through α3 helix. [ABSTRACT FROM AUTHOR]

In this work, we have characterized the interactions of monospermine porphyrin derivative with calf thymus DNA (ct‐DNA) and poly (dG‐dC)2 in both B and Z conformation. By several spectroscopic techniques (UV–vis, electronic circular dichroism and resonance light scattering), the binding modes of monospermine porphyrin derivative with different DNA sequences have been elucidated. In the presence of ct‐DNA, the porphyrin binds along the external double helix as well as in the presence of B conformation of poly (dG‐dC)2. Whilst when the Z form of the poly (dG‐dC)2 is induced, a slight intercalation of the porphyrin between the basis has been detected. [ABSTRACT FROM AUTHOR]

Current interest in lone‐pair⋅⋅⋅π (lp⋅⋅⋅π) interactions is gaining momentum in biochemistry and (supramolecular) chemistry. However, the physicochemical origin of the exceptionally short (ca. 2.8 Å) oxygen‐to‐nucleobase plane distances observed in prototypical Z‐DNA CpG steps remains unclear. High‐level quantum mechanical calculations, including SAPT2+3 interaction energy decompositions, demonstrate that lp⋅⋅⋅π contacts do not result from n→π* orbital overlaps but from weak dispersion and electrostatic interactions combined with stereochemical effects imposed by the locally strained structural context. They also suggest that the carbon van der Waals (vdW) radii, originally derived for sp3 carbons, should not be used for smaller sp2 carbons attached to electron‐withdrawing groups. Using a more adapted carbon vdW radius results in these lp⋅⋅⋅π contacts being no longer of the sub‐vdW type. These findings challenge the whole lp⋅⋅⋅π concept that refers to elusive orbital interactions that fail to explain short interatomic contact distances. [ABSTRACT FROM AUTHOR]

Keywords: Z-DNA; B-Z transition; Z ; Overhang; Loop; Activation energy EN Z-DNA B-Z transition Z Overhang Loop Activation energy 480 483 4 04/20/20 20200401 NES 200401 The presence of single-stranded regions leads to faster B-Z transition possibly by lowering the activation energy. Although both 5'- and 3'-dT overhang ZFDs showed similar kinetic properties, the B-Z transition rate of 3'-dT-overhang ZFDs was little slower than that of 5'-dT-overhang ZFDs. The dA-overhangs at the 3'-ends of ZFDs (ZFD-As) showed a somewhat different outcome on the length-dependency of overhangs in DNA, although all tested 3'-dA-overhang ZFDs have enhanced B-Z transition rates than that of the blunt-ended ZFD. The I k i SB obs sb values of ZFD-hTs become larger as the length of the internal loops increases: ZFD-hT6 > ZFD-hT4 > ZFD-hT2 > ds ZFD-hT1 > ZFD. [Extracted from the article]

PKZ is a novel and unique eIF2α protein kinase identified in fish. Although PKZ is most homologous to PKR, particularly in the C-terminal catalytic domain, it contains two N-terminal Z-DNA-binding domains (Zα1 and Zα2) instead of the dsRNA binding domains (dsRBDs) in PKR. As a novel member of eIF2α kinase family, the available data suggest that PKZ has some distinct mechanisms for recognition, binding, and B-Z DNA transition. Functionally, PKZ seems to be activated by the binding of Zα to Z-DNA and participates in innate immune responses. In this review, we summarize the recent progress on fish PKZ. [ABSTRACT FROM AUTHOR]

Zeta potential is commonly referred as surface charge density and is a key factor in modulating the structural and functional properties of nucleic acids. Although the negative charge density of B-DNA is well understood, there is no prior description of the zeta potential measurement of Z-DNA. In this study, for the first time we discover the zeta potential difference between B-DNA and lanthanum chloride-induced Z-DNA. A series of linear repeat i.e. (CG) n and (GC) n DNA as well as branched DNA (bDNA) structures was used for the B-to-Z DNA transition. Herein, the positive zeta potential of Z-DNA has been demonstrated as a powerful tool to discriminate between B-form and Z-form of DNA. The generality of the approach has been validated both in linear and bDNA nanostructures. Thus, we suggest zeta potential can be used as an ideal signature for the left-handed Z-DNA. • Lanthanum-mediated B-to-Z DNA transition is associated with change in zeta potential. • Zeta potential of B-DNA is highly negative while zeta potential is positive for Z-DNA. • Zeta potential measurement is new signature for differentiation of B- and Z-form DNA. • EDTA-based chelation of La results in Z-B transition and reverse zeta potential. • Lower melting temperature of Z-DNA in comparison to B-DNA and condensed DNA [ABSTRACT FROM AUTHOR]

Bacterial biofilms contribute significantly to the antibiotic resistance, pathogenesis, chronicity and recurrence of bacterial infections. Critical to the stability and survival of extant biofilms is the extracellular DNA (eDNA)-dependent matrix which shields the resident bacteria from hostile environments, allows a sessile metabolic state, but also encourages productive interactions with biofilm-inclusive bacteria. Given the importance of the eDNA, approaches to this area of research have been to target not just the eDNA, but also the additional constituent structural components which appear to be widespread. Chief among these is a ubiquitous two-member family of bacterial nucleoid associated proteins (the DNABII proteins) responsible for providing structural integrity to the eDNA and thereby the biofilm. Moreover, this resultant novel eDNA-rich secondary structure can also be targeted for disruption. Here, we provide an overview of both what is known about the eDNA-dependent matrix, as well as the resultant means that have resulted in biofilm resolution. Results obtained to date have been highly supportive of continued development of DNABII-targeted approaches, which is encouraging given the great global need for improved methods to medically manage, or ideally prevent biofilm-dependent infections, which remains a highly prevalent burden worldwide.

Förster resonance energy transfer (FRET) between a donor nucleobase analogue and an acceptor nucleobase analogue, base–base FRET, works as a spectroscopic ruler and protractor. With their firm stacking and ability to replace the natural nucleic acid bases inside the base-stack, base analogue donor and acceptor molecules complement external fluorophores like the Cy-, Alexa- and ATTO-dyes and enable detailed investigations of structure and dynamics of nucleic acid containing systems. The first base–base FRET pair, tCO–tCnitro, has recently been complemented with among others the adenine analogue FRET pair, qAN1–qAnitro, increasing the flexibility of the methodology. Here we present the design, synthesis, photophysical characterization and use of such base analogues. They enable a higher control of the FRET orientation factor, κ2, have a different distance window of opportunity than external fluorophores, and, thus, have the potential to facilitate better structure resolution. Netropsin DNA binding and the B-to-Z-DNA transition are examples of structure investigations that recently have been performed using base–base FRET and that are described here. Base–base FRET has been around for less than a decade, only in 2017 expanded beyond one FRET pair, and represents a highly promising structure and dynamics methodology for the field of nucleic acids. Here we bring up its advantages as well as disadvantages and touch upon potential future applications.

The classical genetic code maps nucleotide triplets to amino acids. The associated sequence composition is complex, representing many elaborations during evolution of form and function. Other genomic elements code for the expression and processing of RNA transcripts. However, over 50% of the human genome consists of widely dispersed repetitive sequences. Among these are simple sequence repeats (SSRs), representing a class of flipons, that under physiological conditions, form alternative nucleic acid conformations such as Z-DNA, G4 quartets, I-motifs, and triplexes. Proteins that bind in a structure-specific manner enable the seeding of condensates with the potential to regulate a wide range of biological processes. SSRs also encode the low complexity peptide repeats to patch condensates together, increasing the number of combinations possible. In situations where SSRs are transcribed, SSR-specific, single-stranded binding proteins may further impact condensate formation. Jointly, flipons and patches speed evolution by enhancing the functionality of condensates. Here, the focus is on the selection of SSR flipons and peptide patches that solve for survival under a wide range of environmental contexts, generating complexity with simple parts.