Here we describe AGCGA-quadruplexes, an unexpected addition to the well-known tetrahelical families, G-quadruplexes and i-motifs, that have been a focus of intense research due to their potential ...biological impact in G- and C-rich DNA regions, respectively. High-resolution structures determined by solution-state nuclear magnetic resonance (NMR) spectroscopy demonstrate that AGCGA-quadruplexes comprise four 5'-AGCGA-3' tracts and are stabilized by G-A and G-C base pairs forming GAGA- and GCGC-quartets, respectively. Residues in the core of the structure are connected with edge-type loops. Sequences of alternating 5'-AGCGA-3' and 5'-GGG-3' repeats could be expected to form G-quadruplexes, but are shown herein to form AGCGA-quadruplexes instead. Unique structural features of AGCGA-quadruplexes together with lower sensitivity to cation and pH variation imply their potential biological relevance in regulatory regions of genes responsible for basic cellular processes that are related to neurological disorders, cancer and abnormalities in bone and cartilage development.
A prolonged expansion of GGGGCC repeat within non-coding region of C9orf72 gene has been identified as the most common cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal ...dementia (FTD), which are devastating neurodegenerative disorders. Formation of unusual secondary structures within expanded GGGGCC repeat, including DNA and RNA G-quadruplexes and R-loops was proposed to drive ALS and FTD pathogenesis. Initial NMR investigation on DNA oligonucleotides with four repeat units as the shortest model with the ability to form an unimolecular G-quadruplex indicated their folding into multiple G-quadruplex structures in the presence of K(+) ions. Single dG to 8Br-dG substitution at position 21 in oligonucleotide d(G4C2)3G4 and careful optimization of folding conditions enabled formation of mostly a single G-quadruplex species, which enabled determination of a high-resolution structure with NMR. G-quadruplex structure adopted by d(G4C2)3GG(Br)GG is composed of four G-quartets, which are connected by three edgewise C-C loops. All four strands adopt antiparallel orientation to one another and have alternating syn-anti progression of glycosidic conformation of guanine residues. One of the cytosines in every loop is stacked upon the G-quartet contributing to a very compact and stable structure.
The potential to affect gene expression via G-quadruplex stabilization has been extended to all domains of life, including viruses. Here, we investigate the polymorphism and structures of ...G-quadruplexes of the human papillomavirus type 52 with UV, CD and NMR spectroscopy and gel electrophoresis. We show that oligonucleotide with five G-tracts folds into several structures and that naturally occurring single nucleotide polymorphisms (SNPs) have profound effects on the structural polymorphism in the context of G-quadruplex forming propensity, conformational heterogeneity and folding stability. With help of SNP analysis, we were able to select one of the predominant forms, formed by G-rich sequence d(G₃TAG₃CAG₄ACACAG₃T). This oligonucleotide termed HPV52
adopts a three G-quartet snap back (3 + 1) type scaffold with four
guanine residues, two edgewise loops spanning the same groove, a no-residue V loop and a propeller type loop. The first guanine residue is incorporated in the central G-quartet and all four-guanine residues from G4 stretch are included in the three quartet G-quadruplex core. Modification studies identified several structural elements that are important for stabilization of the described G-quadruplex fold. Our results expand set of G-rich targets in viral genomes and address the fundamental questions regarding folding of G-rich sequences.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
DNA can form diverse higher-order structures, whose details are greatly dependent on nucleotide sequence. G-rich sequences containing four or more repeats of three guanines are expected to form ...G-quadruplexes. Here we show that DNA sequences with GGGAGCG repeats found in the regulatory region of the PLEKHG3 gene are capable of forming tetrahelical DNA structures that are distinct from G-quadruplexes. The d(GGGAGCGAGGGAGCG) sequence, VK1, forms a dimer. Two VK1 sequences connected by an adenine residue, VK2, fold into a monomer, which shares identical structural characteristics with the VK1 fold. Their four-stranded architectures are stabilized by four G-C, four G-A and six G-G base pairs. No G-quartets or Hoogsteen-type hydrogen-bonded guanine residues are present and the overall topology is conserved in the presence of Li(+), Na(+), K(+) and NH4(+) ions. Unique structural features include two edgewise loops on each side of the structure stabilized by three G-G base pairs in N1-carbonyl symmetric geometry.
By substitution of natural nucleotides by their abasic analogs (i.e., 1',2'-dideoxyribose phosphate residue) at critically chosen positions within 27-bp DNA constructs originating from the first ...intron of
gene, we hindered hybridization within the guanine- and cytosine-rich central region and followed formation of non-canonical structures. The impeded hybridization between the complementary strands leads to time-dependent structural transformations of guanine-rich strand that are herein characterized with the use of solution-state NMR, CD spectroscopy, and native polyacrylamide gel electrophoresis. Moreover, the DNA structural changes involve transformation of intra- into inter-molecular G-quadruplex structures that are thermodynamically favored. Intriguingly, the transition occurs in the presence of complementary cytosine-rich strands highlighting the inability of Watson-Crick base-pairing to preclude the transformation between G-quadruplex structures that occurs via intertwining mechanism and corroborates a role of G-quadruplex structures in DNA recombination processes.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Topologies of G-quadruplexes depend on oligonucleotide sequences and on environmental factors, and the diversity of G-quadruplex topologies complicates investigation of functions of these nucleic ...acid structures. To investigate how metal ions and cosolutes regulate topologies of G-quadruplexes, we stabilized the antiparallel conformation by insertion of 2′-deoxyxanthosine and 8-oxo-2′-deoxyguanosine into selected positions of an oligonucleotide. Thermodynamic analyses of the oligonucleotide revealed that Na+ stabilized the antiparallel G-quadruplex, whereas K+ destabilized this topology. This result suggests that metal ions selectively stabilize G-quadruplex topologies with cavities between G-quartet planes of certain sizes. In the presence of KCl in 20wt% poly(ethylene glycol) with average molecular weight of 200, the antiparallel basket-type G-quadruplex conformation was not stabilized compared with the dilute condition. In the presence of NaCl, the cosolute did stabilize the G-quadruplex with respect to the dilute condition. The presented data show that metal ions and cosolutes regulate topologies of G-quadruplexes through mechanisms that depend on sizes of metal ion cavities and hydration states.
We investigated how metal ions and cosolutes stabilize particular topologies by analyses of oligodeoxynucleotides modified with 2′-deoxyxanthosine and 8-oxo-2′-deoxyguanosine pairs which enable to control G-quadruplex topologies. Thermodynamic analyses show that metal ions and cosolutes regulate G-quadruplex topologies through the sizes of metal ion cavities and hydration states. Display omitted
•Modified bases at designed positions of a G-quadruplex controlled the topology.•Na+ stabilized an antiparallel G-quadruplex, whereas K+ destabilized this topology.•The cosolute did not stabilized the antiparallel G-quadruplex with KCl.•The cosolute did stabilize the G-quadruplexes with NaCl.•How metal ions and cosolutes regulate G-quadruplex topologies was discussed.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
A possible engineering of materials with diverse bio- and nano-applications relies on robust self-assembly of oligonucleotides. Bottom-up approach utilizing guanine-rich DNA oligonucleotides can lead ...to formation of G-wires, nanostructures consisting of continuous stacks of G-quartets. However, G-wire structure and self-assembly process remain poorly understood, although they are crucial for optimizing properties needed for specific applications. Herein, we use nuclear magnetic resonance to get insights at molecular level on how chosen short, guanine-rich oligonucleotides self-assemble into G-wires, whereas complementary methods are used for their characterization. Additionally, unravelling mechanistic details enable us to guide G-wire self-assembly in a controlled manner. MD simulations provide insight why loop residues with considerably different properties, i.e., hydrogen-bond affinity, stacking interactions, electronic effects and hydrophobicity extensively increase or decrease G-wire length. Our results provide fundamental understanding of G-wire self-assembly process useful for future design of nanomaterials with specific properties.
A four‐repeat human telomere DNA sequence without the 3′‐end guanine, dTAGGG(TTAGGG)2TTAGG (htel1‐ΔG23) has been found to adopt two distinct two G‐quartet antiparallel basket‐type G‐quadruplexes, TD ...and KDH+ in presence of KCl. NMR, CD, and UV spectroscopy have demonstrated that topology of KDH+ form is distinctive with unique protonated T18⋅A20+⋅G5 base triple and other capping structural elements that provide novel insight into structural polymorphism and heterogeneity of G‐quadruplexes in general. Specific stacking interactions amongst two G‐quartets flanking base triples and base pairs in TD and KDH+ forms are reflected in 10 K higher thermal stability of KDH+. Populations of TD and KDH+ forms are controlled by pH. The (de)protonation of A20 is the key for pH driven structural transformation of htel1‐ΔG23. Reversibility offers possibilities for its utilization as a conformational switch within different compartments of living cell enabling specific ligand and protein interactions.
Reversible G‐quadruplex pH switch: A human telomere DNA sequence has been found to adopt two distinct two G‐quartet antiparallel basket‐type G‐quadruplexes, a TD and KDH+ form, which can reversibly transform to each other by variation of pH. The protonation and deprotonation of A20 is the key for the transformation. Reversibility offers possibilities for its utilization as conformational switch in a highly polymorphic system.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
The G4C2 hexanucleotide repeat expansion in the c9orf72 gene is a major genetic cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), with the formation ...of G-quadruplexes directly linked to the development of these diseases. Cations play a crucial role in the formation and structure of G-quadruplexes. In this study, we investigated the impact of biologically relevant potassium ions on G-quadruplex structures and utilized 15N-labeled ammonium cations as a substitute for K+ ions to gain further insights into cation binding and exchange dynamics. Through nuclear magnetic resonance spectroscopy and molecular dynamics simulations, we demonstrate that the single d(G4C2) repeat, in the presence of 15NH4+ ions, adopts a tetramolecular G-quadruplex with an all-syn quartet at the 5′-end. The movement of 15NH4+ ions through the central channel of the G-quadruplex, as well as to the bulk solution, is governed by the vacant cation binding site, in addition to the all-syn quartet at the 5′-end. Furthermore, the addition of K+ ions to G-quadruplexes folded in the presence of 15NH4+ ions induces stacking of G-quadruplexes via their 5′-end G-quartets, leading to the formation of stable higher-ordered species.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Abstract
Guanine-rich regions of the human genome can adopt non-canonical secondary structures. Their role in regulating gene expression has turned them into promising targets for therapeutic ...intervention. Ligands based on polyaromatic moieties are especially suitable for targeting G-quadruplexes utilizing their size complementarity to interact with the large exposed surface area of four guanine bases. A predictable way of (de)stabilizing specific G-quadruplex structures through efficient base stacking of polyaromatic functional groups could become a valuable tool in our therapeutic arsenal. We have investigated the effect of pyrene-modified uridine nucleotides incorporated at several positions of the thrombin binding aptamer (TBA) as a model system. Characterization using spectroscopic and biophysical methods provided important insights into modes of interaction between pyrene groups and the G-quadruplex core as well as (de)stabilization by enthalpic and entropic contributions. NMR data demonstrated that incorporation of pyrene group into G-rich oligonucleotide such as TBA may result in significant changes in 3D structure such as formation of novel dimeric topology. Site specific structural changes induced by stacking of the pyrene moiety on nearby nucleobases corelate with distinct thrombin binding affinities and increased resistance against nuclease degradation.
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