A novel chair-type G-quadruplex formed by a Bombyx mori telomeric sequence

Recently, the human telomeric d[TAGGG(TTAGGG)₃] sequence has been shown to form in K⁺ solution an intramolecular (3+1) G-quadruplex structure, whose G-tetrad core contains three strands oriented in one direction and the fourth in the opposite direction. Here we present a study on the structure of the Bombyx mori telomeric d[TAGG(TTAGG)₃] sequence, which differs from the human counterpart only by one G deletion in each repeat. We found that this sequence adopted multiple G-quadruplex structures in K⁺ solution. We have favored a major G-quadruplex form by a judicious U-for-T substitution in the sequence and determined the folding topology of this form. We showed by NMR that this was a new chair-type intramolecular G-quadruplex which involved a two-layer antiparallel G-tetrad core and three edgewise loops. Our result highlights the effect of G-tract length on the folding topology of G-quadruplexes, but also poses the question of whether a similar chair-type G-quadruplex fold exists in the human telomeric sequences.     

Crystal structure of the potassium form of an Oxytricha nova G-quadruplex

The crystal structures of the potassium-containing quadruplex formed from the Oxytricha nova sequence d(GGGGTTTTGGGG) are reported, in two space groups, the orthorhombic P2(1)2(1)2(1) and the trigonal P3(2)21, which diffract to 2.0 A and 1.49 A, respectively. The orthorhombic form contains two independent quadruplexes in the asymmetric unit, and the trigonal form contains one. All three of these quadruplexes adopt an identical fold, with two strands forming an antiparallel diagonal arrangement. This is identical with that observed previously in NMR studies of the native sodium and potassium forms, and a crystallographic analysis of it complexed with an O. nova protein. The present analysis demonstrates that the native structure is the same in solution and in the crystalline state and, moreover, that the nature of the counter-ion does not affect the overall fold of this quadruplex. The analysis corrects an earlier crystallographic study of this quadruplex. The conformation of the tetra-thymine loop is described in detail, which involves the third thymine base folding back to interact with the first thymine base. The water networks in the grooves and loops are described and, in particular, the ability of water molecules to form a continuous spine of hydration in the narrow groove is detailed. Each quadruplex has five potassium ions organised in a linear channel, with square antiprismatic coordination to each ion from oxygen atoms.     

Human telomeric sequence forms a hybrid-type intramolecular G-quadruplex structure with mixed parallel/antiparallel strands in potassium solution

Human telomeric DNA consists of tandem repeats of the sequence d(TTAGGG). The formation and stabilization of DNA G-quadruplexes in the human telomeric sequence have been shown to inhibit the activity of telomerase, thus the telomeric DNA G-quadruplex has been considered as an attractive target for cancer therapeutic intervention. However, knowledge of the intact human telomeric G-quadruplex structure(s) formed under physiological conditions is a prerequisite for structure-based rational drug design. Here we report the folding structure of the human telomeric sequence in K⁺ solution determined by NMR. Our results demonstrate a novel, unprecedented intramolecular G-quadruplex folding topology with hybrid-type mixed parallel/antiparallel G-strands. This telomeric G-quadruplex structure contains three G-tetrads with mixed G-arrangements, which are connected consecutively with a double-chain-reversal side loop and two lateral loops, each consisting of three nucleotides TTA. This intramolecular hybrid-type telomeric G-quadruplex structure formed in K⁺ solution is distinct from those reported on the 22 nt Tel22 in Na⁺ solution and in crystalline state in the presence of K⁺, and appears to be the predominant conformation for the extended 26 nt telomeric sequence Tel26 in the presence of K⁺, regardless of the presence or absence of Na⁺. Furthermore, the addition of K⁺ readily converts the Na⁺-form conformation to the K⁺-form hybrid-type G-quadruplex. Our results explain all the reported experimental data on the human telomeric G-quadruplexes formed in the presence of K⁺, and provide important insights for understanding the polymorphism and interconversion of various G-quadruplex structures formed within the human telomeric sequence, as well as the effects of sequence and cations. This hybrid-type G-quadruplex topology suggests a straightforward pathway for the secondary structure formation with effective packing within the extended human telomeric DNA. The hybrid-type telomeric G-quadruplex is most likely to be of pharmacological relevance, and the distinct folding topology of this G-quadruplex suggests that it can be specifically targeted by G-quadruplex interactive small molecule drugs.     

Solution structure of the major G-quadruplex formed in the human VEGF promoter in K+: insights into loop interactions of the parallel G-quadruplexes

Vascular endothelial growth factor (VEGF) proximal promoter region contains a poly G/C-rich element that is essential for basal and inducible VEGF expression. The guanine-rich strand on this tract has been shown to form the DNA G-quadruplex structure, whose stabilization by small molecules can suppress VEGF expression. We report here the nuclear magnetic resonance structure of the major intramolecular G-quadruplex formed in this region in K⁺ solution using the 22mer VEGF promoter sequence with G-to-T mutations of two loop residues. Our results have unambiguously demonstrated that the major G-quadruplex formed in the VEGF promoter in K⁺ solution is a parallel-stranded structure with a 1:4:1 loop-size arrangement. A unique capping structure was shown to form in this 1:4:1 G-quadruplex. Parallel-stranded G-quadruplexes are commonly found in the human promoter sequences. The nuclear magnetic resonance structure of the major VEGF G-quadruplex shows that the 4-nt middle loop plays a central role for the specific capping structures and in stabilizing the most favored folding pattern. It is thus suggested that each parallel G-quadruplex likely adopts unique capping and loop structures by the specific middle loops and flanking segments, which together determine the overall structure and specific recognition sites of small molecules or proteins.LAY SUMMARY: The human VEGF is a key regulator of angiogenesis and plays an important role in tumor survival, growth and metastasis. VEGF overexpression is frequently found in a wide range of human tumors; the VEGF pathway has become an attractive target for cancer therapeutics. DNA G-quadruplexes have been shown to form in the proximal promoter region of VEGF and are amenable to small molecule drug targeting for VEGF suppression. The detailed molecular structure of the major VEGF promoter G-quadruplex reported here will provide an important basis for structure-based rational development of small molecule drugs targeting the VEGF G-quadruplex for gene suppression.     

Intragenic G-quadruplex structure formed in the human CD133 and its biological and translational relevance

Cancer stem cells (CSCs) have been identified in several solid malignancies and are now emerging as a plausible target for drug discovery. Beside the questionable existence of CSCs specific markers, the expression of CD133 was reported to be responsible for conferring CSC aggressiveness. Here, we identified two G-rich sequences localized within the introns 3 and 7 of the CD133 gene able to form G-quadruplex (G4) structures, bound and stabilized by small molecules. We further showed that treatment of patient-derived colon CSCs with G4-interacting agents triggers alternative splicing that dramatically impairs the expression of CD133. Interestingly, this is strongly associated with a loss of CSC properties, including self-renewing, motility, tumor initiation and metastases dissemination. Notably, the effects of G4 stabilization on some of these CSC properties are uncoupled from DNA damage response and are fully recapitulated by the selective interference of the CD133 expression.In conclusion, we provided the first proof of the existence of G4 structures within the CD133 gene that can be pharmacologically targeted to impair CSC aggressiveness. This discloses a class of potential antitumoral agents capable of targeting the CSC subpopulation within the tumoral bulk.     

Structure of a two-G-tetrad intramolecular G-quadruplex formed by a variant human telomeric sequence in K+ solution: insights into the interconversion of human telomeric G-quadruplex structures

Human telomeric DNA G-quadruplex has been considered as an attractive target for cancer therapeutic intervention. The telomeric sequence shows intrinsic structure polymorphism. Here we report a novel intramolecular G-quadruplex structure formed by a variant human telomeric sequence in K⁺ solution. This sequence forms a basket-type intramolecular G-quadruplex with only two G-tetrads but multiple-layer capping structures formed by loop residues. While it is shown that this structure can only be detected in the specifically truncated telomeric sequences without any 5′-flanking residues, our results suggest that this two-G-tetrad conformation is likely to be an intermediate form of the interconversion of different telomeric G-quadruplex conformations.     

Switching G-quadruplex to parallel duplex by molecular rotor clustering

Switching of G-quadruplex (G4) structures between variant types of folding has been proved to be a versatile tool for regulation of genomic expression and development of nucleic acid-based constructs. Various specific ligands have been developed to target G4s in K⁺ solution with therapeutic prospects. Although G4 structures have been reported to be converted by sequence modification or a unimolecular ligand binding event in K⁺-deficient conditions, switching G4s towards non-G4 folding continues to be a great challenge due to the stability of G4 in physiological K⁺ conditions. Herein, we first observed the G4 switching towards parallel-stranded duplex (psDNA) by multimolecular ligand binding (namely ligand clustering) to overcome the switching barrier in K⁺. Purine-rich sequences (e.g. those from the KRAS promoter region) can be converted from G4 structures to dimeric psDNAs using molecular rotors (e.g. thioflavin T and thiazole orange) as initiators. The formed psDNAs provided multiple binding sites for molecular rotor clustering to favor subsequent structures with stability higher than the corresponding G4 folding. Our finding provides a clue to designing ligands with the competency of molecular rotor clustering to implement an efficient G4 switching.     

Solution structure of a two-repeat fragment of major vault protein

Major vault protein (MVP) is the main constituent of vaults, large ribonucleoprotein particles implicated in resistance to cancer therapy and correlated with poor survival prognosis. Here, we report the structure of the main repeat element in human MVP. The approximately 55 amino acid residue MVP domain has a unique, novel fold that consists of a three-stranded antiparallel beta-sheet. The solution NMR structure of a two-domain fragment reveals the interdomain contacts and relative orientations of the two MVP domains. We use these results to model the assembly of 672 MVP domains from 96 MVP molecules into the ribs of the 13MDa vault structure. The unique features include a thin, skin-like structure with polar residues on both the cytoplasmic and internal surface, and a pole-to-pole arrangement of MVP molecules. These studies provide a starting point for understanding the self-assembly of MVP into vaults and their interactions with other proteins. Chemical shift perturbation studies identified the binding site of vault poly(ADP-ribose) polymerase, another component of vault particles, indicating that MVP domains form a new class of interaction-mediating modules.     

Crystal structure of the complementary quadruplex formed by d(GCATGCT) at atomic resolution

Here we report the crystal structure of the DNA heptanucleotide sequence d(GCATGCT) determined to a resolution of 1.1 Å. The sequence folds into a complementary loop structure generating several unusual base pairings and is stabilised through cobalt hexammine and highly defined water sites. The single stranded loop is bound together through the G(N2)–C(O2) intra-strand H-bonds for the available G/C residues, which form further Watson–Crick pairings to a complementary sequence, through 2-fold symmetry, generating a pair of non-planar quadruplexes at the heart of the structure. Further, four adenine residues stack in pairs at one end, H-bonding through their N7–N6 positions, and are additionally stabilised through two highly conserved water positions at the structural terminus. This conformation is achieved through the rotation of the central thymine base at the pinnacle of the loop structure, where it stacks with an adjacent thymine residue within the lattice. The crystal packing yields two halved biological units, each related across a 2-fold symmetry axis spanning a cobalt hexammine residue between them, which stabilises the quadruplex structure through H-bonds to the phosphate oxygens and localised hydration.     

G-quadruplex conformation and dynamics are determined by loop length and sequence

The quadruplex forming G-rich sequences are unevenly distributed throughout the human genome. Their enrichment in oncogenic promoters and telomeres has generated interest in targeting G-quadruplex (GQ) for an anticancer therapy. Here, we present a quantitative analysis on the conformations and dynamics of GQ forming sequences measured by single molecule fluorescence. Additionally, we relate these properties to GQ targeting ligands and G4 resolvase 1 (G4R1) protein binding. Our result shows that both the loop (non-G components) length and sequence contribute to the conformation of the GQ. Real time single molecule traces reveal that the folding dynamics also depend on the loop composition. We demonstrate that GQ-stabilizing small molecules, N-methyl mesoporphyrin IX (NMM), its analog, NMP and the G4R1 protein bind selectively to the parallel GQ conformation. Our findings point to the complexity of GQ folding governed by the loop length and sequence and how the GQ conformation determines the small molecule and protein binding propensity.     

Massively parallel interrogation of aptamer sequence, structure and function

Background

Optimization of high affinity reagents is a significant bottleneck in medicine and the life sciences. The ability to synthetically create thousands of permutations of a lead high-affinity reagent and survey the properties of individual permutations in parallel could potentially relieve this bottleneck. Aptamers are single stranded oligonucleotides affinity reagents isolated by in vitro selection processes and as a class have been shown to bind a wide variety of target molecules.

Methodology/Principal Findings

High density DNA microarray technology was used to synthesize, in situ, arrays of approximately 3,900 aptamer sequence permutations in triplicate. These sequences were interrogated on-chip for their ability to bind the fluorescently-labeled cognate target, immunoglobulin E, resulting in the parallel execution of thousands of experiments. Fluorescence intensity at each array feature was well resolved and shown to be a function of the sequence present. The data demonstrated high intra- and inter-chip correlation between the same features as well as among the sequence triplicates within a single array. Consistent with aptamer mediated IgE binding, fluorescence intensity correlated strongly with specific aptamer sequences and the concentration of IgE applied to the array.

Conclusion and Significance

The massively parallel sequence-function analyses provided by this approach confirmed the importance of a consensus sequence found in all 21 of the original IgE aptamer sequences and support a common stem:loop structure as being the secondary structure underlying IgE binding. The microarray application, data and results presented illustrate an efficient, high information content approach to optimizing aptamer function. It also provides a foundation from which to better understand and manipulate this important class of high affinity biomolecules.     

Crystal structure of a DNA/Ba2+ G-quadruplex containing a water-mediated C-tetrad

We have determined the 1.50 Å crystal structure of the DNA decamer, d(CCA^CNVKGCGTGG) (^CNVK, 3-cyanovinylcarbazole), which forms a G-quadruplex structure in the presence of Ba²⁺. The structure contains several unique features including a bulged nucleotide and the first crystal structure observation of a C-tetrad. The structure reveals that water molecules mediate contacts between the divalent cations and the C-tetrad, allowing Ba²⁺ ions to occupy adjacent steps in the central ion channel. One ordered Mg²⁺ facilitates 3′-3′ stacking of two quadruplexes in the asymmetric unit, while the bulged nucleotide mediates crystal contacts. Despite the high diffraction limit, the first four nucleotides including the ^CNVK nucleoside are disordered though they are still involved in crystal packing. This work suggests that the bulky hydrophobic groups may locally influence the formation of non-Watson–Crick structures from otherwise complementary sequences. These observations lead to the intriguing possibility that certain types of DNA damage may act as modulators of G-quadruplex formation.     

Crystal structure of an 82-nucleotide RNA-DNA complex formed by the 10-23 DNA enzyme

The structure of a large nucleic acid complex formed by the 10-23 DNA enzyme bound to an RNA substrate was determined by X-ray diffraction at 3.0 A resolution. The 82-nucleotide complex contains two strands of DNA and two strands of RNA that form five double-helical domains. The spatial arrangement of these helices is maintained by two four-way junctions that exhibit extensive base-stacking interactions and sharp turns of the phosphodiester backbone stabilized by metal ions coordinated to nucleotides at these junctions. Although it is unlikely that the structure corresponds to the catalytically active conformation of the enzyme, it represents a novel nucleic acid fold with implications for the Holliday junction structure.     

Induction of parallel human telomeric G-quadruplex structures by Sr(2+)

Human telomeric DNA forms G-quadruplex secondary structures, which can inhibit telomerase activity and are targets for anti-cancer drugs. Here we show that Sr(2+) can induce human telomeric DNA to form both inter- and intramolecular structures having characteristics consistent with G-quadruplexes. Unlike Na(+) or K(+), Sr(2+) facilitated intermolecular structure formation for oligonucleotides with 2 to 5 5'-d(TTAGGG)-3' repeats. Longer 5'-d(TTAGGG)-3' oligonucleotides formed exclusively intramolecular structures. Altering the 5'-d(TTAGGG)-3' to 5'-d(TTAGAG)-3' in the 1st, 3rd, or 4th repeats of 5'-d(TTAGGG)(4)-3' stabilized the formation of intermolecular structures. However, a more compact, intramolecular structure was still observed when the 2nd repeat was altered. Circular dichroism spectroscopy results suggest that the structures were parallel-stranded, distinguishing them from similar DNA sequences in Na(+) and K(+). This study shows that Sr(2+), promotes parallel-stranded, inter- and intramolecular G-quadruplexes that can serve as models to study DNA substrate recognition by telomerase.     

The parallel G-quadruplex structure of vertebrate telomeric repeat sequences is not the preferred folding topology under physiological conditions

G-quadruplex topologies of telomeric repeat sequences from vertebrates were investigated in the presence of molecular crowding (MC) mimetics, namely polyethylene glycol 200 (PEG), Ficoll 70 as well as Xenopus laevis egg extract by CD and NMR spectroscopy and native PAGE. Here, we show that the conformational behavior of the telomeric repeats in X. laevis egg extract or in Ficoll is notably different from that observed in the presence of PEG. While the behavior of the telomeric repeat in X. laevis egg extract or in Ficoll resembles results obtained under dilute conditions, PEG promotes the formation of high-order parallel topologies. Our data suggest that PEG should not be used as a MC mimetic.     

G-quadruplex structure and stability illuminated by 2-aminopurine phasor plots

The use of time-resolved fluorescence measurements in studies of telomeric G-quadruplex folding and stability has been hampered by the complexity of fluorescence lifetime distributions in solution. The application of phasor diagrams to the analysis of time-resolved fluorescence measurements, collected from either frequency-domain or time-domain instrumentation, allows for rapid characterization of complex lifetime distributions. Phasor diagrams are model-free graphical representations of transformed time-resolved fluorescence results. Simplification of complex fluorescent decays by phasor diagrams is demonstrated here using a 2-aminopurine substituted telomeric G-quadruplex sequence. The application of phasor diagrams to complex systems is discussed with comparisons to traditional non-linear regression model fitting. Phasor diagrams allow for the folding and stability of the telomeric G-quadruplex to be monitored in the presence of either sodium or potassium. Fluorescence lifetime measurements revealed multiple transitions upon folding of the telomeric G-quadruplex through the addition of potassium. Enzymatic digestion of the telomeric G-quadruplex structure, fluorescence quenching and Förster resonance energy transfer were also monitored through phasor diagrams. This work demonstrates the sensitivity of time-resolved methods for monitoring changes to the telomeric G-quadruplex and outlines the phasor diagram approach for analysis of complex time-resolved results that can be extended to other G-quadruplex and nucleic acid systems.