Search Results (51)

Tetrahelical DNA structures, such as G-quadruplexes (G4s) or i-motifs (iMs), are adopted by sequences comprising several G/C tracts, exist in equilibria with respective duplexes, and may contribute to genomic instability upon helicase deficiency. To understand genomic rearrangements resulting from the juxtaposition of G/C-rich DNA duplexes, models of possible intermediate structures are needed. In this study, a general strategy for creating and verifying in silico 3D models of tetrahelical DNA was proposed. This strategy was used to investigate contacts of two or more duplexes with n G₃/C₃ tracts (n = 2–6) separated by T/A nucleotides. The revealed viable structures of DNA–DNA contacts include stacks of right-handed and left-handed G-quadruplexes (G4s), Holliday structure-resembling assemblies with the G4 and iM opposite each other on the borders of the central “hole”, etc. Based on molecular dynamic simulations, the most probable variants were determined by estimating the contributions to the free energy. The results may be used to clarify the mechanisms of strand exchange and other rearrangements upon DNA breaks near prolonged G/C-rich sites in living systems. Additionally, they provide a balanced view on the polymorphic versus programmed DNA assemblies in artificial systems. Full article

Cytosine-rich DNA sequences form i-motif structures associated with various cellular functions including gene regulation. DNA sequences containing consecutive C residues are widely deemed essential for i-motif formation; however, some sequences lacking C-tracts have been reported to form minimal i-motif structures. We systematically investigated the variability in the minimal i-motif-forming DNA sequence comprising two TCGTTCCGT sequence units, which forms two C:C⁺ pairs and two G:C:G:T base tetrads. A comprehensive analysis of structural stability by DNA thermal melting temperature measurements revealed that oligonucleotides disrupting the formation of the base tetrad or its stacking interactions with a C:C⁺ pair prevent stable i-motif formation, and modifications to the sequence context and length of the lateral loops are difficult. This study further demonstrated that spermine effectively restores the stability reduction caused by creating a bulge, long loop, or dangling end within the minimal i-motif structure, which is less pronounced in the C-rich i-motif. The results suggest that the formation of minimal i-motifs with various sequences is facilitated in polyamine-rich environments, such as the nucleus of mammalian cells. These findings are valuable for identifying potential i-motif-forming sites lacking C-tracts in genomes and provide insights into the electrostatic interactions between i-motif structures and biological polyamines. Full article

Porphyrins are highly conjugated macrocyclic compounds that possess exceptional photophysical and chemical properties, progressively establishing themselves as versatile tools in the structural investigation of biomolecules. This review explores their role as chiroptical conformational probes, focusing on their interactions with DNA and RNA. The planar electron rich structure of porphyrin macrocycle that promote π–π interactions, their easy functionalization at the meso positions, and their capacity to coordinate metal ions enable their use in probing nucleic acid structures with high sensitivity. Emphasis is placed on their induced circular dichroism (ICD) signals in the Soret region, which provide precise diagnostic insights into binding mechanisms and molecular interactions. The review examines the interactions of porphyrins with various DNA structures, including B-, Z-, and A-DNA, single-stranded DNA, and G-quadruplex DNA, as well as less common structures like I-motif and E-motif DNA. The last part highlights recent advancements in the use of porphyrins to probe RNA structures, emphasizing binding behaviors and chiroptical signals observed with RNA G-quadruplexes, as well as the challenges in interpreting ICD signals with other RNA motifs due to their inherent structural complexity. Full article

G-quadruplexes and i-motifs are four-stranded non-canonical structures of DNA. They exist in the cell, where they are implicated in the conformational regulation of cellular events, such as transcription, translation, DNA replication, telomere homeostasis, and genomic instability. Formation of the G-quadruplex and i-motif conformations in the genome is controlled by their competition with the pre-existing duplex. The fate of that competition depends upon the relative stabilities of the competing conformations, leading ultimately to a distribution of double helical, tetrahelical, and coiled conformations that coexist in dynamic equilibrium with each other. We previously developed a CD spectroscopy-based procedure to characterize the distribution of conformations adopted by equimolar mixtures of complementary G- and C-rich DNA strands from the promoter regions of the c-MYC, VEGF, and Bcl-2 oncogenes. In those bimolecular systems, duplex-to-tetraplex and duplex-to-coil transitions are accompanied by strand separation and an associated entropic cost. This situation is distinct from the pseudo-monomolecular nature of conformational transformations within the genome, where strand separation does not occur. To mimic better the situation in the genome, we here extend our studies to a monomolecular DNA construct—a hairpin—in which complementary G- and C-rich strands featuring sequences from the promoter region of the c-MYC oncogene are linked by a dT₁₁ loop. We used our CD-based procedure to quantify the distribution of conformational states sampled by the hairpin at pH 5.0 and 7.0 as a function of temperature and the concentration of KCl. The data were analyzed according to a thermodynamic model based on equilibria between the different conformational states to evaluate the thermodynamic properties of the duplex-to-coil, G-quadruplex-to-coil, and i-motif-to-coil transitions of the hairpin. The results have implications for the modulation of such transitions as a means of therapeutic intervention. Full article

The retinoblastoma (RB) gene is an important tumor suppressor gene with a higher mutation frequency than other tumor suppressor genes. The mutation or inactivation of RB has been found in various cancers. The discovery of small molecules to promote RB expression is an effective anti-cancer strategy. Special DNA secondary structures with G-quadruplex and i-motif on the RB promoter could act as “molecular switches” for gene transcriptional regulation and are potentially important targets for the development of new anti-cancer drugs. After extensive screening, we found that the bisacridine derivative A06 had selective binding and destabilization for both the G-quadruplex and i-motif on the RB promoter, which significantly up-regulated RB gene transcription and translation, resulting in the inhibition of tumor cell proliferation and metastasis. A06 exhibited potent anti-tumor activity on Hela cells and strongly suppressed tumor growth on the Hela xenograft mice model without significant toxicity. In comparison, A02 exhibited strong binding and destabilization to the RB promoter G-quadruplex only, which showed a much weaker effect than A06 on regulating RB expression and producing anti-tumor activity. As we know, this is the first study for up-regulating a tumor suppressor gene through destabilization of both the G-quadruplex and i-motif on the gene promoter, which provides a new strategy for innovative anti-cancer drug discovery and development. Full article

Acute kidney injury (AKI) is a global public health problem with its pathogenesis not fully understood. Excessive apoptosis of renal tubular epithelial cells is an important feature of AKI patients, and therefore an anti-apoptotic approach could be used in the treatment for AKI. Up-regulation of B-cell lymphoma-2 (BCL-2) gene and protein has been found to be correlated with anti-apoptosis of cells. It has been found that the presence of the C-rich sequence on the upstream region of the BCL-2 gene promoter could form DNA secondary i-motif structure, and its stabilization by small molecules could up-regulate gene transcription and translation. In the present study, we constructed AKI models through folic acid (FA) induction. With these in vitro and in vivo models, we demonstrated that the acridone derivative A22 could up-regulate the expression of BCL-2 by targeting its gene promoter i-motif to reduce renal tubular epithelial cell apoptosis and improve renal function in many ways. A22 could alleviate FA-induced oxidative stress injury, inflammatory response, and endoplasmic reticulum stress in mouse kidneys. Our results provided a potentially new anti-apoptotic approach for the treatment of early stages of AKI. Our employed model focused on its short-term effect on AKI, while its long-term efficacy and safety, particularly regarding the regeneration of renal tubular epithelial cells, require further investigation before clinical application. This study further demonstrated that promoter i-motif could be targeted for up-regulating BCL-2 expression for the treatment of important diseases caused by excessive apoptosis. Full article

This article provides a comprehensive examination of non-canonical DNA structures, particularly focusing on G-quadruplexes (G4s) and i-motifs. G-quadruplexes, four-stranded structures formed by guanine-rich sequences, are stabilized by Hoogsteen hydrogen bonds and monovalent cations like potassium. These structures exhibit diverse topologies and are implicated in critical genomic regions such as telomeres and promoter regions of oncogenes, playing significant roles in gene expression regulation, genome stability, and cellular aging. I-motifs, formed by cytosine-rich sequences under acidic conditions and stabilized by hemiprotonated cytosine–cytosine (C:C+) base pairs, also contribute to gene regulation despite being less prevalent than G4s. This review highlights the factors influencing the stability and dynamics of these structures, including sequence composition, ionic conditions, and environmental pH. Molecular dynamics simulations and high-resolution structural techniques have been pivotal in advancing our understanding of their folding and unfolding mechanisms. Additionally, the article discusses the therapeutic potential of small molecules designed to selectively bind and stabilize G4s and i-motifs, with promising implications for cancer treatment. Furthermore, the structural properties of these DNA forms are explored for applications in nanotechnology and molecular devices. Despite significant progress, challenges remain in observing these structures in vivo and fully elucidating their biological functions. The review underscores the importance of continued research to uncover new insights into the genomic roles of G4s and i-motifs and their potential applications in medicine and technology. This ongoing research promises exciting developments in both basic science and applied fields, emphasizing the relevance and future prospects of these intriguing DNA structures. Full article

DNA requires hydration to maintain its structural integrity. Crystallographic analyses have enabled patterns of water arrangements to be visualized. We survey these water motifs in this review, focusing on left- and right-handed duplex and quadruplex DNAs, together with the i-motif. Common patterns of linear spines of water organization in grooves have been identified and are widely prevalent in right-handed duplexes and quadruplexes. By contrast, a left-handed quadruplex has a distinctive wheel of hydration populating the almost completely circular single groove in this structure. Full article

Due to the advantages of its numerous modification sites, predictable structure, high thermal stability, and excellent biocompatibility, DNA is the ideal choice as a key component of biosensors. DNA biosensors offer significant advantages over existing bioanalytical techniques, addressing limitations in sensitivity, selectivity, and limit of detection. Consequently, they have attracted significant attention from researchers worldwide. Here, we exemplify four foundational categories of functional nucleic acids: aptamers, DNAzymes, i-motifs, and G-quadruplexes, from the perspective of the structure-driven functionality in constructing DNA biosensors. Furthermore, we provide a concise overview of the design and detection mechanisms employed in these DNA biosensors. Noteworthy advantages of DNA as a sensor component, including its programmable structure, reaction predictility, exceptional specificity, excellent sensitivity, and thermal stability, are highlighted. These characteristics contribute to the efficacy and reliability of DNA biosensors. Despite their great potential, challenges remain for the successful application of DNA biosensors, spanning storage and detection conditions, as well as associated costs. To overcome these limitations, we propose potential strategies that can be implemented to solve these issues. By offering these insights, we aim to inspire subsequent researchers in related fields. Full article

Increased expression of the human telomere reverse transcriptase (hTERT) in tumors promotes tumor cell survival and diminishes the survival of patients. Cytosine-to-thymine (C-to-T) transition mutations (C250T or C228T) in the hTERT promoter create binding sites for transcription factors, which enhance transcription. The G-rich strand of the hTERT promoter can form G-quadruplex structures, whereas the C-rich strand can form an i-motif in which multiple cytosine residues are protonated. We considered the possibility that i-motif formation might promote cytosine deamination to uracil and C-to-T mutations. We computationally probed the accessibility of cytosine residues in an i-motif to attack by water. We experimentally examined regions of the C-rich strand to form i-motifs using pH-dependent UV and CD spectra. We then incubated the C-rich strand with and without the G-rich complementary strand DNA under various conditions, followed by deep sequencing. Surprisingly, deamination rates did not vary substantially across the 46 cytosines examined, and the two mutation hotspots were not deamination hotspots. The appearance of mutational hotspots in tumors is more likely the result of the selection of sequences with increased promoter binding affinity and hTERT expression. Full article

I-motifs are non-canonical DNA structures formed by intercalated hemiprotonated (CH·C)${}^{+}$ pairs, i.e., formed by a cytosine (C) and a protonated cytosine (CH${}^{+}$), which are currently drawing great attention due to their biological relevance and promising nanotechnological properties. It is important to characterize the processes occurring in I-motifs following irradiation by UV light because they can lead to harmful consequences for genetic code and because optical spectroscopies are the most-used tools to characterize I-motifs. By using time-dependent DFT calculations, we here provide the first comprehensive picture of the photoactivated behavior of the (CH·C)${}^{+}$ core of I-motifs, from absorption to emission, while also considering the possible photochemical reactions. We reproduce and assign their spectral signatures, i.e., infrared, absorption, fluorescence and circular dichroism spectra, disentangling the underlying chemical–physical effects. We show that the main photophysical paths involve C and CH${}^{+}$ bases on adjacent steps and, using this basis, interpret the available time-resolved spectra. We propose that a photodimerization reaction can occur on an excited state with strong C→CH${}^{+}$ charge transfer character and examine some of the possible photoproducts. Based on the results reported, some future perspectives for the study of I-motifs are discussed. Full article

Four-stranded folded structures, such as G-quadruplexes and i-motifs in the genome, have attracted a growing interest nowadays since they have been discovered in the telomere and in several oncogene promoter regions. Their biological relevance is undeniable since their existence in living cells has been observed. In vivo they take part in the regulation of gene expression, in vitro they are used in the analytical biochemistry. They are attractive and promising targets for cancer therapy. Pressure studies can reveal specific aspects of the molecular processes. Pressure tuning experiments allow the determination of the volumetric parameters of the folded structures and of the folding–unfolding processes. Here, we review the thermodynamic parameters with a special focus on the volumetric ones, which were determined using pressure tuning spectroscopic experiments on the G-quadruplex and i-motif nucleic acid forms. Full article

During the last decade, the evidence for the biological relevance of i-motif DNA (i-DNA) has been accumulated. However, relatively few molecules were reported to interact with i-DNA, and a controversy concerning their binding mode, affinity, and selectivity persists in the literature. In this context, the cholestane derivative IMC-48 has been reported to modulate bcl-2 gene expression by stabilizing an i-motif structure in its promoter. In the present contribution, we report on a novel, more straightforward, synthesis of IMC-48 requiring fewer steps compared to the previous approach. Furthermore, the interaction of IMC-48 with four different i-motif DNA sequences was thoroughly investigated by bio-layer interferometry (BLI) and circular dichroism (CD) spectroscopy. Surprisingly, our results show that IMC-48 is a very weak ligand of i-DNA as no quantifiable interaction or significant stabilization of i-motif structures could be observed, stimulating a quest for an alternative mechanism of its biological activity. Full article

The reliable, readily accessible and label-free measurement of aptamer binding remains a challenge in the field. Recent reports have shown large changes in the intrinsic fluorescence of DNA upon the formation of G-quadruplex and i-motif structures. In this work, we examined whether DNA intrinsic fluorescence can be used for studying aptamer binding. First, DNA hybridization resulted in a drop in the fluorescence, which was observed for A30/T30 and a 24-mer random DNA sequence. Next, a series of DNA aptamers were studied. Cortisol and Hg²⁺ induced fluorescence increases for their respective aptamers. For the cortisol aptamer, the length of the terminal stem needs to be short to produce a fluorescence change. However, caffeine and adenosine failed to produce a fluorescence change, regardless of the stem length. Overall, using the intrinsic fluorescence of DNA may be a reliable and accessible method to study a limited number of aptamers that can produce fluorescence changes. Full article

Tetrads (or quartets) are arrangements of four nucleobases commonly involved in the stability of four-stranded nucleic acids structures. Four-stranded or quadruplex structures have attracted enormous attention in the last few years, being the most extensively studied guanine quadruplex (G-quadruplex). Consequently, the G-tetrad is the most common and well-known tetrad. However, this is not the only possible arrangement of four nucleobases. A number of tetrads formed by the different nucleobases have been observed in experimental structures. In most cases, these tetrads occur in the context of G-quadruplex structures, either inserted between G-quartets, or as capping elements at the sides of the G-quadruplex core. In other cases, however, non-G tetrads are found in more unusual four stranded structures, such as i-motifs, or different types of peculiar fold-back structures. In this report, we review the diversity of these non-canonical tetrads, and the structural context in which they have been found. Full article