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A G‐Quartet Formed in the Absence of a Templating Metal Cation: A New 8‐(N,N‐dimethylaniline)guanosine Derivative
Angewandte Chemie International Edition
Abstract
Contrary to what was assumed previously, self-assembled G-quartets may be obtained even in the absence of a templating alkali metal cation. This observation leads to the suggestion that this structural motif, which has been implicated in a variety of biological functions, might be more widespread than believed. Specifically, it is found that guanosine derivative 1 exists as a tetrameric ensemble both in chlorinated organic solvents and in the solid state (see picture). Ar = p-Me2N-C6H4, Rib = ribose.
... G 10 was shown to selfassemble into a G-quartet even without the assistance of a templating cation. 61 Attachment of sterically bulky groups, a dimethylaniline unit, to the C8 position of the guanine ring gave a conformationally constrained nucleoside that prefers to adopt a syn glycosidic bond conformer in both the solid state and solution. This syn conformation prevents the nucleoside from any hydrogen-bonded ribbon formation and thus favors formation of the macrocyclic G-quartet (Figure 1.12). ...
... 273 Recent studies have described G-quadruplexes made from G derivatives substituted at their N2 or C8 positions. 61,64,[300][301][302][303] In some cases, the N2 or C8 substituents stabilized the G-quadruplex relative to the unmodified derivative. 64,301,302 As outlined in Scheme 4.3, it was reasoned that modification at both N2 and C8 (G 51) would lead to a G-quartet that might then be cross-linked to give a "covalent" macrocycle. ...
... The addition of [2.2.2]cryptand appears to remove the M + . We, 62 and others, 61 have previous reported stable G-quartets without the presence of cations. ...
... 1,2 Self-assemblies of guanosine to form well-defined G-quadruplexes have been extensively studied and applied in functional material development and biomedical research. [3][4][5][6][7][8][9][10][11][12][13][14] One particularly interesting application in G-quadruplexes is the formation of gels for tissue engineering, cell culture, and drug delivery. [15][16][17] The fibrous gel network can be constructed through the dynamic supramolecular self-assembly of guanosine. ...
... None of the self-assembled guanosine complexes we tested matches this spectrum in Figure 1A, implying that the guanosine monomer exists as metal-free guanosine assemblies in non-polar solvents. [3][4][5][6][7][8][9][10] Although the formation of the metal-free H-bond isomers could influence the construction of the desired G 8 -octamers, treating aldG ligand with KPF 6 gave 1 set of signals in the NMR spectra ( 1 H, 31 P, and 19 F). The chemical shift of N1-H at 12.8 ppm suggested the formation of a characteristic H-bond in a G-quartet 49,50 (see Figure S1 for other self-assembling NMR spectra). ...
Gel formation using guanosine self-assembly is an important process in supramolecular chemistry. Here, we report the stepwise construction of circularly polarized luminescent supramolecular organogels from self-assembled guanosine quadruplexes. A lipophilic guanosine derivative (aldG) is designed and synthesized for the formation of a well-defined G8-octamer. The diamine linkers are used to connect G8-octamer units by imine formation to facilitate the construction of the supramolecular gel networks. ¹H NMR experiments show that the pre-assembled aldG8-octamer remains intact and is crucial for transparent and stiff organogel formation. With extended conjugation, the aldG organogels exhibit strong green fluorescence emission and circularly polarized properties without the assistance of any external fluorescent dyes, suggesting an alternative approach to construct molecular probes for biological and material applications.
... The anti-conformation of the aliphatic tail facilitated the growth of a ribbon-like structural arrangement in thec-direction through Hbonding between an amide in the aliphatic tail and guanine (Fig. 2c), resulting in the ribbon-like structure observed in SEM imaging (Fig. 1f). This structural arrangement is consistent with the self-assembly of previously reported guanosine/deoxy-guanosine derivatives in the absence of any templating cation 46,47 . ...
... Due to the presence of the flexible aliphatic tail, the planarity of the overall cyclic arrangement is disturbed to some extent. This non-planar tetrameric arrangement has the look of four-stranded topology obtained by natural/synthetic G-quartets ( Fig. 2d) 47,50,51 . Among the four inter-molecularly hydrogen bonded Fmoc-G-PNA conjugates responsible for tetramer formation, the fluorenylmethyl part of two molecules occupies the space either above or below the tetramer plane, with the hydrophobic tail pointing outwards from the cavity. ...
The variety and complexity of DNA-based structures make them attractive candidates for nanotechnology, yet insufficient stability and mechanical rigidity, compared to polyamide-based molecules, limit their application. Here, we combine the advantages of polyamide materials and the structural patterns inspired by nucleic-acids to generate a mechanically rigid fluorenylmethyloxycarbonyl (Fmoc)-guanine peptide nucleic acid (PNA) conjugate with diverse morphology and photoluminescent properties. The assembly possesses a unique atomic structure, with each guanine head of one molecule hydrogen bonded to the Fmoc carbonyl tail of another molecule, generating a non-planar cyclic quartet arrangement. This structure exhibits an average stiffness of 69.6 ± 6.8 N m⁻¹ and Young’s modulus of 17.8 ± 2.5 GPa, higher than any previously reported nucleic acid derived structure. This data suggests that the unique cation-free “basket” formed by the Fmoc-G-PNA conjugate can serve as an attractive component for the design of new materials based on PNA self-assembly for nanotechnology applications.
... En fonction de la nature des substituants, on peut obtenir des arrangements de type ruban A ou B. L'équipe de J. Sessler montre, en 2000, que des dérivés lipophiles de guanosine contraints en conformation syn peuvent néanmoins former des tétrades en l'absence de cation. 63 L'addition d'un groupement encombrant comme la diméthylaniline en C8 de la guanine permet de bloquer la conformation de la base en syn. L'utilisation de la 2',3',5'-(isobutynoyl)-8-(N,N-diméthylaniline)guanosine permet d'obtenir exclusivement des structures en G-quartet ( Figure 29). ...
... Cette conformation est connue pour empêcher la formation de structure de style ruban de guanines. 63 Deuxièmement le fait d'observer deux corrélations montrent que les bases ne sont pas toutes équivalentes. 74 Ces différentes observations, basées sur l'exploitation de la RMN 1 H des composés 4 et 5, permettent de proposer que les deux composés forment en solution aqueuse une tétrade de guanines. ...
It is known for almost 50 years, that guanosine derivatives can form tetrads under specific conditions. Guanine tetrads, which are formed by the coplanar arrangement of four guanines through the formation of Hoogsteen hydrogen bonds, represent a relevant model for the study of molecular self-assembly processes and non covalent synthesis. In the first part of this manuscript, we will describe how a peptidic scaffold can be used to constrain the formation of a tetrad. The introduction of conformational constraints allows the control of the formation of synthetic guanines tetrads in water in the absence of cations to stabilize the structure. Two architectures have been synthesized and characterized and the results highlight the different phenomena that allow the formation of synthetic tetrads. It is also known that guanine rich nucleic acids sequences can form particular secondary structures, known as G-quadruplexes. These motifs represent relevant target for the development of new anticancer therapies since they are involved in a lot of biological events (telomere maintenance, oncogene activation ...). However the study of the interaction between small organic molecules and such motifs are difficult due to their high polymorphism. We propose in a second chapter the design and the synthesis of G-quadruplexes mimics based on the covalent attachment of short nucleic acids sequences on a peptidic scaffold, which allow the control of the topologies of the so-obtained quadruplexes. The study of the interactions of small organic molecules with these mimics has been performed by SPR studies. This approach appears to be very interesting to understand the interactions that are involved in the recognition of quadruplexes and the selection of new G4 ligands.
... Given that the intracellular space and extracellular spaces in most organisms are dominated by K + and Na + respectively, these two metals typically get the most attention (15). The stabilization of G-quartets by cation binding is understandable, since the G-quartet has four oxygen atoms clustered in its centre, without a bound ion this cyclic arrangement would be electronically unfavorable (16). In spite of this, Sessler et al., have described the formation of a G-quartet without a central cation (16). ...
... The stabilization of G-quartets by cation binding is understandable, since the G-quartet has four oxygen atoms clustered in its centre, without a bound ion this cyclic arrangement would be electronically unfavorable (16). In spite of this, Sessler et al., have described the formation of a G-quartet without a central cation (16). In all other cases to date, cations are required for the stabilization of self-assembled G4 structures. ...
... The bulky pan-shaped bithiophene on the C8 position of guanine, which is composed of -conjugated aromatic rings and long lipophilic chains, induces a strong intra-/interquartet - stacking and lamellar stacking (27). Furthermore, the octyl side chain on the N9 position hinders unwanted hydrogen bonding at the N3 position (28), which could help form the LiGQ instead of ribbon-like polymorphs. ...
Single-ion conductors have garnered attention in energy storage systems as a promising alternative to currently widespread electrolytes that allow migration of cations and anions. However, ion transport phenomena of most single-ion conductors are affected by strong ion (e.g., Li ⁺ )–ion (immobilized anionic domains) interactions and tortuous paths, which pose an obstacle to achieving performance breakthroughs. Here, we present a Li ⁺ -centered G-quadruplex (LiGQ) as a class of single-ion conductor based on directional Li ⁺ slippage at the microscopic level. A guanine derivative with liquid crystalline moieties is self-assembled to form a hexagonal ordered columnar structure in the LiGQ, thereby yielding one-dimensional central channels that provide weak ion-dipole interaction and straightforward ionic pathways. The LiGQ exhibits weak Li ⁺ binding energy and low activation energy for ion conduction, verifying its viability as a new electrolyte design.
... G-quadruplex formation in organic solvents is typically aided by a monovalent cation, 29,30 but assemblies in saltfree environments also exist for template directed assembly 31,32 or aggregation in the solid state. 27,28,33 GTDI self-assembly also occurs in the absence of salt, 14 highlighting the role of pendant TDI moieties in stabilizing such structures. Furthermore, assemblies consisting of two to four G-quartet layers as well as G-quadruplex nanoribbons have also been observed, 34 and in the case of GTDI, the πstacking tendencies of TDI assist in the formation of a large 16 layer assembly (Scheme 1). ...
G-quadruplex assemblies are a promising tool for self-assembling π-stacked chromophore arrays to better understand their photophysics. We have shown that coupling a single guanine moiety to terrylenediimide (TDI) produces a structure (GTDI) that self-assembles in tetrahydrofuran (THF) into a nearly monodisperse guanine-quadruplex structure having 16 π-stacked layers (GTDI4)16. The TDI surfaces were determined to have a high degree of cofacial overlap and underwent quantitative symmetry-breaking charge separation (SB-CS) upon photoexcitation. Here, we more deeply examine the relationship between solvent and aggregate formation and develop insights into structure–function relationships over a variety of solvent polarities and hydrogen-bonding capabilities. At high concentrations, GTDI assembles into guanine-quadruplex structures (GTDI4)16 in THF and toluene, as well as (GTDI4)9 in pyridine and benzonitrile. Transient absorption spectroscopy shows that SB-CS occurs in all solvents, regardless of their static dielectric constants, but the SB-CS yield is determined by structure. Solvent polarity independent SB-CS generation is also observed in GTDI films, where there is a complete absence of solvent.
... Only under acidic conditions, it forms anisotropic acid gels [47]. Secondly, metal cations can bind to the four oxygen atoms of the G-quartet center and are essential for the stabilization of the G-quartet [49,50]. Only certain alkali metal ions can induce self-assembly of 5 -GMP. ...
Guanosine is an important building block for supramolecular gels owing to the unique self-assembly property that results from the unique hydrogen bond acceptors and donor groups. Guanosine-derived supramolecular hydrogels have promise in the fields of drug delivery, targeted release, tissue engineering applications, etc. However, the property of poor longevity and the need for excess cations hinder the widespread applications of guanosine hydrogels. Although guanosine-derived supramolecular hydrogels have been reviewed previously by Dash et al., the structural framework of this review is different, as the modification of guanosine is described at the molecular level. In this review, we summarize the development and lifetime stability improvement of guanosine-based supramolecular hydrogels through optimized structure and elaborate on three aspects: sugar modification, base modification, and binary gels. Additionally, we introduce the concept and recent research progress of self-healing gels, providing inspiration for the development of guanosine-derived supramolecular hydrogels with longer lifespans, unique physicochemical properties, and biological activities.
... While it is commonly accepted that the central cation is to reduce the repulsion between carbonyl oxygen atoms from G-quartets, it is important to point out that it is primarily the repulsions between carbonyl oxygen atoms from adjacent planar G-quartets, not from within the same G-quartet, which requires further stabilization from a cation. The main evidence for this view is the fact that whereas a cation-free or "empty" G-quartet was observed [24], an "empty" G-octamer has never been reported. Now when the helix is made of lock-washer-like G 4 , there is no longer any repulsion between carbonyl oxygen atoms along the helical axis, thus making it unnecessary to have a cation inside the central channel. ...
Early X-ray fiber diffraction studies have established that the spontaneous gel formation of guanosine 5′-monophosphate (5′-GMP) under slightly acidic conditions (e.g., pH 5) results from self-assembly of 5′-GMP into a helical structure in which hydrogen-bonded guanine bases form a continuous helix with 15 nucleotides per 4 turns. For more than five decades, the sense of this helix is believed to be left-handed. Using multinuclear solid-state NMR and IR spectroscopic methods, we have finally determined the long-missing structural details of this helix. First, we found that this 5′-GMP helix is right-handed containing exclusive C3′- endo sugar puckers. Second, we showed that the central channel of this helix is free of Na ⁺ ions, which is in sharp contrast to the helix formed by 5′-GMP at pH 8 where the central channel is filled with Na ⁺ ions.
... The other signal at δ 7.13, the calix [4]arene ArH, shows an NOE to a second ArH (green lines). 283 In that report, Sessler highlighted the critical role of the relative base-to-sugar conformation in Gquartet formation. Compound G 2.65 (Figure 2.36A), due to the bulky N,Ndimethylaniline attached to C8, adopts a syn conformation. ...
Two-dimensional (2D) chirality has been actively studied in view of numerous applications of chiral surfaces such as in chiral resolutions and enantioselective catalysis. Here, we report on the expression and amplification of chirality in hybrid 2D metallosupramolecular networks formed by a nucleobase derivative. Self-assembly of a guanine derivative appended with a pyridyl node was studied at the solution-graphite interface in the presence and absence of coordinating metal ions. In the absence of coordinating metal ions, a monolayer that is representative of a racemic compound was obtained. This system underwent spontaneous resolution upon addition of a coordinating ion and led to the formation of a racemic conglomerate. The spontaneous resolution could also be achieved upon addition of a suitable guest molecule. The mirror symmetry observed in the formation of the metallosupramolecular networks could be broken via the use of an enantiopure solvent, which led to the formation of a globally homochiral surface.
Nucleoside‐lipid conjugates are very useful supramolecular building blocks to construct self‐assembled architectures suited for biomedical and material applications. Such nucleoside derivatives can be further synthetically manipulated to endow additional functionalities that could augment the assembling process and impart interesting properties. Here, we report the design, synthesis and self‐assembling process of multifunctional supramolecular nucleolipid synthons containing an environment‐sensitive fluorescent guanine. The amphiphilic synthons are composed of an 8‐(2‐(benzofuran‐2‐yl)vinyl)‐guanine core and alkyl chains attached to 3′‐O and 5′‐O‐positions of 2′‐deoxyguanosine. The 2‐(benzofuran‐2‐yl)vinyl (BFV) moiety attached at the C8 position of the nucleobase adopted a syn conformation about the glycosidic bond, which facilitated the self‐assembly process through the formation of a G‐tetrad as the basic unit. While 3′,5′‐diacylated BFV‐modified dG analog stabilized the guanosine hydrogel by hampering the crystallization process and imparted fluorescence, BFV‐modified dGs containing longer alkyl chains formed a green fluorescent organogel, which transformed into a yellow fluorescent gel in the presence of a complementary non‐fluorescent cytidine nucleolipid. The ability of the dG analog containing short alkyl chains to modulate the mechanical property of a gel, and interesting fluorescence properties and self‐assembling behavior exhibited by the dG analogs containing long alkyl chains in response to heat and complementary base underscore the potential use of these new supramolecular synthons in material applications.
Extending the structural hierarchy and complexity through small-molecular self-assembly is a powerful way to obtain large discrete, functional molecular architecture. A hydrogen-bonded supramolecular organic framework (HSOF) with nanometer-size pores is constructed in a solid state with simple guanosine-monomer self-assembly. To extend the hierarchy of the G-quartet self-assembly to a higher order than that of the traditional G-quadruplex, H-bond acceptors on the C-8 position of guanosine are introduced to establish inter-quadruplex linkage via H bonding to N(2)-HB from the neighboring G-quartet. After screening different C-8 substitution groups and various synthesis conditions, HSOF-G1a’ is obtained by solvent evaporation under diluted condition. Single-crystal X-ray structure reveals that cubic repeating units formed by G8 are the supermolecule secondary building block (SBU) with large pores (d = 34 Å). To our knowledge, this is the first G-quartet self-assembly with an organized structure beyond cylindrical G-quadruplexes.
The hierarchical self-assembly of various lipophilic guanosines exposing either a phenyl or a ferrocenyl group in the C(8) position was investigated. In a solution, all the derivatives were found to self-assemble primarily into isolated guanine (G)-quartets. In spite of the apparent similar bulkiness of the two substituents, most of the derivatives form disordered structures in the solid state, whereas a specific 8-phenyl derivative self-assembles into an unprecedented, cation-free stacked G-quartet architecture.
G-quadruplex, a unique secondary structure in nucleic acids found throughout human genome, elicited widespread interest in the field of therapeutic research. Being present in key regulatory regions of oncogenes, RNAs and telomere, G-quadruplex structure regulates transcription, translation, splicing etc. Changes in its structure and stability leads to differential expression of oncogenes causing cancer. Thus, targeting G-Quadruplex structures with small molecules/other biologics has shown elevated research interest. Covering previous reports, in this review we try to enlighten the facts on the structural diversity in G-quadruplex ligands aiming to provide newer insights to design first-in-class drugs for the next generation cancer treatment.
Studies on interaction of phosphorus acids with N-heterocycles/nucleobases can unravel supramolecular architectures which can have implications in understanding similar interactions in biological systems. Interaction of tert-butyl phosphonic acid (tBuPO3H2) with nucleobases cytosine (cyt) and adenine (aden) leads to the isolation of hydrogen-bonded supramolecular motifs [(tBuPO3H)(cyt-H)] (1) and [(tBuPO3H)(aden-H)] (2). Solid-state structures of these two adducts established by single-crystal X-ray diffraction studies were found to contain a 1:1 ratio of the phosphorus acid and the nucleobase. Presence of hydrophobic t-butyl groups on phosphorous in compounds 1 and 2 aids the isolation of water-free acid-base complexes. Adducts 1 and 2 exist as hydrogen-bonded ion pair, each possessing eight-membered hydrogen-bonded rings involving nucleobase molecules and PO3H units of the phosphonate anions. A number of N–H···O, C–H···O, and O–H···O hydrogen bonds apart from weak C–H···O interactions are prevalent in the structures of these supramolecular architectures.
Hypothesis: G-quadruplex structure has raised increasing attention in supramolecular chemistry as an effective template for ordered functional materials. Thus, it is of practical significance to advance our understanding regarding G-quadruplex structures. Typically, G-quadruplex structures are formed in the presence of suitable metal ions. New methods to construct such structures need to be explored.
Experiments: The supramolecular assembly between CTAB and a guanosine derivative at different molar ratios was systematically studied, including assembly mechanisms, morphology, and macroscopic properties. Cationic surfactants with different alkyl chains were studied as control experiments.
Findings: A novel strategy to construct G-quadruplex with the promotion of the cationic surfactant CTAB is presented in this work. The structure-property relationships of G-quadruplex gels are characterized by rheology and shrinkage ratio experiments. MacKintosh’s theory was used to rationalize the relationship between gel elasticity and water content. The transition of G-quadruplex structures could be easily enabled by modulating CTAB concentration, which promotes the phase transition from gel/sol biphase to homogeneous sol phase. This work will provide a new viewpoint for the construction and modulation of G-quadruplex structures.
Dynamic and reversible non‐covalent interactions endow synthetic systems and materials with smart adaptive functions that allow them to response to diverse stimuli, interact with external agents, or repair structural defects. Inspired by the outstanding performance and selectivity of DNA in living systems, scientists are increasingly employing Watson−Crick nucleobase pairing to control the structure and properties of self‐assembled materials. Two sets of complementary purine‐pyrimidine pairs (guanine:cytosine and adenine:thymine(uracil)) are available that provide selective and directional H‐bonding interactions, present multiple metal‐coordination sites, and exhibit rich redox chemistry. In this review, we highlight several recent examples that profit from these features and employ nucleobase interactions in functional systems and materials, covering the fields of energy/electron transfer, charge transport, adaptive nanoparticles, porous materials, macromolecule self‐assembly, or polymeric materials with adhesive or self‐healing ability. Inspired by DNA: H‐bonding interactions between complementary nucleobases, namely G : C and A : T(U), as well as their coordination to metals can be employed for the generation of functional systems and materials with potential applications in diverse fields, including photosynthetic models, porous materials, chemical sensing, drug delivery, or self‐healable polymer networks.
In analogy to covalent reactions, the understanding of noncovalent association pathways is fundamental to influence and control any supramolecular process. Following an approach that is reminiscent of covalent methodologies, we study here, for the first time, the mechanism of G‐quadruplex formation in organic solvents. Our results support a reaction pathway in which the cation shifts the equilibrium towards a G‐quartet transient intermediate, which then acts as a template in the formation of the G‐quadruplex product.
G4 on the floor: The unprecedented K⁺ G‐quartet has been detected as a transient intermediate along the course of G‐quadruplex self‐assembly following an approach that is reminiscent of classical covalent mechanistic studies.
Abstract
In analogy to covalent reactions, the understanding of noncovalent association pathways is fundamental to influence and control any supramolecular process. Following an approach that is reminiscent of covalent methodologies, we study here, for the first time, the mechanism of G‐quadruplex formation in organic solvents. Our results support a reaction pathway in which the cation shifts the equilibrium towards a G‐quartet transient intermediate, which then acts as a template in the formation of the G‐quadruplex product.
A reverse‐binding‐selectivity between monovalent and divalent cations was observed for two different self‐assembly G16‐hexadecamer and G8‐octamer systems. The dissociation constant between G4‐quadruplex and monomer was calculated via VT‐¹H NMR experiments. Quantitative energy profiles revealed entropy as the key factor for the weaker binding toward Ba²⁺ compared with K⁺ in the G8‐octamer system despite stronger ion‐dipole interactions. This study is the first direct comparison of the G4‐quartet binding affinity between mono and divalent cations and will benefit future applications of G‐quadruplex‐related research. Further competition experiments between the G8‐octamer and 18‐crown‐6 with K⁺ demonstrated the potential of this G8 system as a new potassium receptor.
Guanine-quadruplex assemblies provide a useful platform for studying the spatial, structural, and photophysical effects of intermolecular interactions. Coupling a single guanine moiety to terrylenediimide (TDI) - a chromophore with a large extended π-surface - produces a structure (GTDI) that assembles in plate-like tetramers, with the potential of undergoing tunable π-stacking. At high concentrations (3 × 10⁻³ M), GTDI self-assembles into a nearly monodisperse guanine-quadruplex structure of 16 layers, with strong π-overlap between TDI moieties, observed by small- and wide-angle X-ray scattering (SAXS/WAXS). Transient absorption spectroscopy reveals that excitation of TDI in the guanine-quadruplex results in symmetry-breaking charge separation to form ion pairs within the structure, owing to the strong π-overlap enforced by the hydrogen-bonding. These assemblies yield important insights into the interplay of noncovalent interactions in the assembly of ordered chromophoric arrays.
The formation of well-defined, discrete self-assembled architectures relies on the interplay between non-covalent interactions and cooperative phenomena. In particular, chelate or intramolecular cooperativity is responsible for the assembly of closed, cyclic structures in competition with open, linear oligomers, and it can be enhanced in several ways to increase the stability of a given cycle size. In this article, we review the work of several researchers in the synthesis of hydrogen-bonded macrocycles from ditopic molecules and analyze the main factors, often interrelated, that influence the equilibrium between ring and chain species. Emphasis will be set on the diverse features that can increase cyclization fidelity, including monomer geometry, template effects, conformational effects, intramolecular interactions and H-bonding pattern.
Supramolecular hydrogels made from guanine derivatives have attracted a great interest for various applications. The gelation of guanine analogues usually involves the formation of guanine/K+ quartets. Functional cargoes such as fluorophores and drugs were usually decorated to the guanine quartet gels via non-covalent π-π interactions or dynamic covalent linkages to render the gel with new functionalities. Here, a class of antiviral guanine quartet hydrogels have been developed via direct gelation of clinically available antiviral drugs such as entecavir, penciclovir, and ganciclovir in the presence of K+ ions. The prepared gels were stable at ambient temperature, non-toxic to normal cell lines, and maintained their inherent antiviral activities against hepatitis B virus and Herpes simplex virus, respectively. This study expands the function of guanine-based supramolecular gels and permits the development of a new class of antiviral gels.
Methanol soluble and stable guanosine octamers were successfully achieved via H-bond self-assembly. Through the structural conformational design, we developed a new class of guanosine derivatives with modification on guanine (8-aryl) and ribose (2’,3’-isopropylidene). This unique design led to the formation of the first discrete G8-octamer with structure characterized by single crystal X-ray diffraction, MS and NMR. The G8-octamer showed unique cation recognition property including the formation of a stable Rb+ templated G-quadruplex. Based on this observation, further modification on the 8-aryl moiety was performed to incorporate cross-layer H-bond and covalent linkage respectively. Similar G-octamers were obtained in both cases with structures confirmed by single crystal X-ray diffraction. Furthermore, the covalently linked G-quadruplex exhibited excellent stability even in MeOH and DMSO, suggesting promising future of this new H-bond self-assembly system in biological and material applications.
We herein report the self-assembly of a lipophilic bromoguanosine derivative (G1) in homogeneous solution, in the solid state, and in planar bilayer membrane. The self-assembly of G1, driven by H-bonding and - stacking interactions can form different nano-structures depending on incubation time. The G1 nanostructure is able to bind a bioactive dye like Rose Bengal. In crystal state, it shows ribbon type H-bonding pattern and exhibits birefringence in polarized light. And further, the self-assembled nanostructure of G1 can form discrete transmembrane ion channels in the biological membrane, enabling transportation of potassium ions.
The synthesis and characterization of a series of 3- or 4-substituted 1,8-naphthalic anhydride compounds, and their organophosphorus analogues, bearing a cyclic diketophosphanyl moiety, are reported. The new anhydrides have been synthesized by Stille coupling with a variety of aryl groups with different donor strengths from the corresponding 3- and 4-bromo substituted anhydrides, respectively. The thienyl- and (dimethylamino)phenyl-substituted anhydrides have moreover been used as precursors for the synthesis of the corresponding diketophosphanyl derivatives by reaction with MesP(SiMe3)2. The photophysical and electrochemical properties of the new compounds have been studied. Density functional theory (DFT) and time-dependent DFT calculations have been carried out in order to rationalize their electronic structures and electronic absorption properties. The low-energy absorption bands exhibit mainly (π → π*) charge-transfer character from the aryl moieties to the anhydride or diketophosphanyl core, respectively. In the thiophene-containing diketophosphanyl compounds the low-energy absorption also shows some electron-transfer features from the phosphorus moiety (MesP) to the core. The charge-transfer nature of the transition has been confirmed by solvatochromic absorption and emission studies.
Derivatives of guanine exhibit diverse supramolecular chemistry, with a variety of distinct hydrogen-bonding motifs reported in the solid state, including ribbons and quartets, which resemble the G-quadruplex found in nucleic acids with sequences rich in guanine. Reflecting this diversity, the solid-state structural properties of 3′,5′-bis-O-decanoyl-2′-deoxyguanosine, reported in this paper, reveal a hydrogen-bonded guanine ribbon motif that has not been observed previously for 2′-deoxyguanosine derivatives. In this case, structure determination was carried out directly from powder XRD data, representing one of the most challenging organic molecular structures (a 90-atom molecule) that has been solved to date by this technique. While specific challenges were encountered in the structure determination process, a successful outcome was achieved by augmenting the powder XRD analysis with information derived from solid-state NMR data and with dispersion-corrected periodic DFT calculations for structure optimization. The synergy of experimental and computational methodologies demonstrated in the present work is likely to be an essential feature of strategies to further expand the application of powder XRD as a technique for structure determination of organic molecular materials of even greater complexity in the future.
Two-dimensional covalent organic frameworks often I € stack into crystalline solids that allow precise spatial positioning of molecular building blocks. Inspired by the hydrogen-bonded G-quadruplexes found frequently in guanine-rich DNA, here we show that this structural motif can be exploited to guide the self-Assembly of naphthalene diimide and perylene diimide electron acceptors end-capped with two guanine electron donors into crystalline G-quadruplex-based organic frameworks, wherein the electron donors and acceptors form ordered, segregated I €-stacked arrays. Time-resolved optical and electron paramagnetic resonance spectroscopies show that photogenerated holes and electrons in the frameworks have long lifetimes and display recombination kinetics typical of dissociated charge carriers. Moreover, the reduced acceptors form polarons in which the electron is shared over several molecules. The G-quadruplex frameworks also demonstrate potential as cathode materials in Li-ion batteries because of the favourable electron-and Li-ion-Transporting capacity provided by the ordered rylene diimide arrays and G-quadruplex structures, respectively. © 2017 Macmillan Publisher Limited part of springer Nature. All rights reserved.
The formation of distinct supramolecular assemblies, including a metastable species, is revealed for a lipophilic guanosine (G) derivative in solution and in the solid state. Structurally different G-quartet based assemblies are formed in chloroform depending on the nature of the cation, anion and salt concentration, as characterized by circular dichroism and time course diffusion-ordered NMR spectroscopy data. Intriguingly, even the presence of potassium ions that stabilize G-quartets in chloroform was insufficient to exclusively retain such assemblies in the solid state, leading to the formation of mixed quartet and ribbon-like assemblies as revealed by fast magic-angle spinning (MAS) NMR spectroscopy. Distinct N-H∙∙∙N and N-H∙∙∙O intermolecular hydrogen bonding interactions drive quartet and ribbon-like self-assembly resulting in markedly different 2D 1H solid-state NMR spectra, thus facilitating a direct identification of mixed assemblies. A dissolution NMR experiment confirmed that the quartet and ribbon interconversion is reversible - further demonstrating the changes that occur in the self-assembly process of a lipophilic nucleoside upon a solid-state to solution-state transition and vice versa. A systematic study for complexation with different cations (K+, Sr2+) and anions (picrate, ethanoate and iodide) emphasises that the existence of a stable solution or solid-state structure may not reflect the stability of the same supramolecular entity in another phase.
Supermacrocycles are circular assemblies held together through noncovalent forces, also known as rosettes. This article discusses the self-assembly of organic rosettes composed of three to six modules. Design features that play a part in the self-assembly of these superstructures and the factors that affect their supramolecular organization are described. In the final section of this article, we discuss the self-assembly of these superstructures into two-dimensional networks on solid substrates.
We are assembling in this article the work of many scientists who have designed and prepared self-assembled systems using Watson–Crick H bonding between the natural purine–pyrimidine nucleobase pairs (G–C and A–T/U). Nucleobase pairing has been exploited in many ways, but the main idea is to profit from the selective and complementary binding between DNA bases to bring together different molecular units for a particular function. In this article, we will cover discrete synthetic systems in solution, soft supramolecular materials, condensed two-dimensional systems on surfaces, and polymeric systems in solution or in bulk phases.
A pyrimido[4,5-d]pyrimidine featuring the hydrogen bond donors and acceptors of both guanine (G) and cytosine (C) in the appropriate geometry undergoes hierarchical self-assembly into nanotubular architectures. Specifically, in solution this heterocycle self-organizes into cyclic hexamers through hydrogen bonding interactions, which then further π–π stack into rosette nanotubes (RNTs). The present work reviews the synthetic strategies used to tune the structure of this class of heterocyclic molecules and ultimately tailor the physical properties of the resulting RNTs for potential applications in nanomedicine, catalysis, and renewable energy.
Supramolecular or molecular gels are attractive for various applications, including diagnostics, tissue scaffolding and targeted drug release. Gelators derived from natural products are of particular interest for biomedical purposes, as they are generally biocompatible and stimuli-responsive. The building blocks of nucleic acids (i.e. nucleobases, nucleosides, and nucleotides) are desirable candidates for supramolecular gelation as they readily engage in reversible, noncovalent interactions. In this review, we describe a number of organo- and hydrogels formed through the assembly of nucleosides, nucleotides, and their derivatives. While natural nucleosides and nucleotides generally require derivatization to induce gelation, guanosine and its corresponding nucleotides are well known gelators. This unique gelating ability is due to propensity of the guanine nucleobase to self-associate into stable higher-order assemblies, such as G-ribbons, G4-quartets, and G-quadruplexes.
Emissive purine analogs can probe DNA and RNA structures by reporting changes in local base-stacking interactions that are characteristic of specific secondary and tertiary structures. Fluorescent nucleobase analogs provide a means for directly probing the unique photophysical and electronic properties of nucleic acid structures. Assays that utilize nucleobase analogs have historically been limited by relatively low sensitivity that results from fluorescence quenching of the probe by neighboring residues. New probes such as 2PyG and StG that remain highly emissive in the context of proper base-stacking interactions have provided new means for differentiating nucleic acid structures according to the differences in energy transfer efficiencies from endogenous residues. Notably, this can be accomplished by introducing a very small modification (e.g., a single styrene or pyridine group) into the oligonucleotide at a strategic location, such as 8 position of guanosine.
Aryl radical species derived from enzymatic transformations of aromatic mutagens preferentially react at the 8-site of the guanine (G) nucleobase to afford carbon-linked 8arylG adducts. The resulting lesions possess altered biophysical and genetic coding properties compared to the precursor G nucleoside in B-form DNA. Unlike other adducts, these lesions also possess useful fluorescent properties, since direct attachment of the 8aryl ring extends the purine π-system to afford G mimics with red-shifted excitation maxima and emission that can be sensitive to the microenvironment of the 8arylG base within nucleic acid structures. In B-form DNA, 8arylG adducts are disruptive to duplex formation because they prefer to adopt the syn-conformation about the bond connecting the nucleobase to the deoxyribose backbone, which perturbs Watson–Crick (WC) H-bonding with the opposing cytosine (C). Thus, in a B-form duplex, the emissive properties of 8arylG adducts can be employed as a tool to provide insight into adduct conformation, which can be related to their biological outcomes. However, since Gs preferentially adopt the syn-conformation in left-handed Z-DNA and antiparallel G-quadruplex (GQ) structures, 8arylG lesions can be inserted into syn-G positions without disrupting H-bonding interactions. In fact, 8arylG lesions can serve as ideal fluorescent probes in an antiparallel GQ because their emission is sensitive to GQ folding. This perspective outlines recent developments in the biological implications of 8arylG formation together with their utility as fluorescent G analogs for use in DNA-based diagnostic systems.
A water-soluble template-assembled synthetic G-quartet (TASQ) based on the use of a macrocyclodecapeptide scaffold was designed to display stable intramolecular folds alone in solution. The preformation of the guanine quartet, demonstrated by NMR and CD investigations, results in enhanced peroxidase-type biocatalytic activities and improved quadruplex-interacting properties. Comparison of its DNAzyme-boosting properties with the ones of previously published TASQ revealed that, nowadays, it is the best DNAzyme-boosting agent.
This communication describes a convenient, direct ring closure of a novel 4-cyano-5-cyanomethyl-1-(β-D-ribofuranosyl)-1,2,3-triazole under basic conditions into 4-amino-6-alkoxy-1-(β-D-ribofuranosyl)-1,2,3-triazolo[4,5- c]pyridine, which can be easily transformed to 8-aza-3-deazaisoguanosine.
An NMR crystallography approach that combines experimental solid-state NMR with calculations, for the full crystal structure and extracted isolated molecules, of NMR chemical shifts using the gauge-including projector-augmented wave (GIPAW) approach probes the role of intermolecular N−H···O, N−H···N, O−H···N, O−H···O interactions in stabilizing distinct ribbon-like supramolecular structures adopted by three different guanosine derivatives.
Cyclization into closed assemblies is the most recurrent approach to realize the noncovalent synthesis of discrete, well-defined nanostructures. This review article particularly focuses on the noncovalent synthesis of monocyclic hydrogen-bonded systems that are self-assembled from a single molecule with two binding-sites. Taking advantage of intramolecular binding events, which are favored with respect to intermolecular binding in solution, can afford quantitative amounts of a given supramolecular species under thermodynamic control. The size of the assembly depends on geometric issues such as the monomer structure and the directionality of the binding interaction, whereas the fidelity achieved relies largely on structural preorganization, low degrees of conformational flexibility, and templating effects. Here, we discuss several examples described in the literature in which cycles of different sizes, from dimers to hexamers, are studied by diverse solution or surface characterization techniques.
The electrochemical genosensors introduced in this chapter are classified into two groups, based on the sensing strategy: genosensors in which direct reduction and oxidation of DNA bases as a recognition event is used; and genosensors in which the sensing strategy is based on complementary base paring between the sensor's nucleic acid sequence and the analyte of interest. The DNA sensing procedures based on direct redox reaction of nucleic bases are fairly sensitive and selective, but their applicability is rather limited. One of the methods is based on direct electroactivity of nucleic bases, which takes into account the limitations of genosensors and improves their application, relies on of the same electrochemical mediators that facilitate electron transfer between the electro-active base and the electrode surface. Today, particularly in medical diagnoses, an ideal biosensor is required not only to be miniaturized and cost effective, but also capable of simultaneous detection of multiple analytes.
At present the origins of the elevated stability of G-quartets remain unclear: when a guanine becomes involved in a quartet, the rearrangement of its electron density is a phenomenon referred to as resonance-assisted hydrogen bonding (RAHB), G-quartet stability is assumed to originate in an electronic redistribution ascribed to interplay between H-bond formation and delocalization of the nucleobase p-electrons (resonance). The external G-quartet– small molecule interaction is a pivotal event in the aim of using quadruplexes as structural switches to control all aforementioned DNA–RNA transactions. The knowledge of the nucleic acid community combined with the techniques of chemistry, molecular biology, surface, and biophysical sciences have unambiguously yielded real biotechnological dividends, as vividly demonstrated by the versatility of the selected examples reported in this chapter. The emerging field of synthetic G-quartets is providing not only preliminary but also invaluable solutions that help address these issues.
We report on the synthesis and self-assembly of three novel lipophilic guanosine derivatives exposing a ferrocene moiety on the C(5’) position of the sugar unit. Their self-association in solution, and at the solid/liquid interface, can be tuned by varying the size and nature of the C(8)-substituent, leading to the generation of either G-ribbons, lamellar G-dimer based arrays or the G4 cation-free architectures.
The intrinsic properties of the alkali metal cationized G-tetrads, M(9eG)4+ (M=Li, Na, K, Rb, Cs) composed of 9-ethylguanine (9eG), were studied by a combination of mass spectrometric techniques. The gas phase structures were probed by infrared multiple photon dissociation (IRMPD) spectroscopy in both the fingerprint region (900 – 1850 cm-1) and the N-H/C-H stretching region (2700 – 3800 cm-1). The gas phase structures were found to be similar for all five complexes and most consistent with the metal-centred G-tetrad structure. Energy-resolved CID was also used to compare the gas phase stabilities of the G-tetrads and showed that Na(9eG)4+ was more stable than Li(9eG)4+ followed by the K+, Rb+, and Cs+ G-tetrads in order. The experimental energy ordering was reproduced by electronic structure calculations of the energies. Furthermore, the computations also showed that the lower stability to loss of 9-ethylguanine for the Li+ complex could be due to a strong destabilization of the neutral G-tetrad due to the persistence toward maximizing the ion-dipole interactions while also maintaining hydrogen bonding interactions.
Although the G-quartet structure has been extensively investigated due to its biological importance, the formation mechanism, in particular, the necessity of metal centers, of an isolated G-quartet on solid surfaces remains ambiguous. Here, by using scanning tunneling microscopy under well-controlled ultra-high-vacuum conditions and density functional theory calculations we have been able to clarify that besides the intraquartet hydrogen bonding a metal center is mandatory for the formation of an isolated G-quartet. Furthermore, by subtly perturbing the local coordination bonding schemes within the formed G-quartet complex via local nanoscale scanning tunneling microscopy manipulations, we succeed in modulating the d orbitals and the accompanying magnetic properties of the metal center. Our results demonstrate the feasibility of forming an isolated G-quartet complex on a solid surface and that the strategy of modulating electronic and magnetic properties of the metal center can be extended to other related systems such as molecular spintronics.
The self-assembly of a lipophilic derivative of (5'S)-5',8-cyclo-2'-deoxyguanosine, a mutagenic product formed by hydroxyl radical attack against DNA, has been investigated. This derivative forms, with high fidelity, a dodecameric complex composed of three stacked G-quartets in the presence of strontium picrate. This is the first example of a fully-anti lipophilic G-quadruplex
The kinetic method has been applied, in its extended form, to the evaluation of the entropy effect in the determination of the proton affinity (PA N) of N-3-para substituted benzoyl 2′-deoxycytidines. The latter, mixed with nucleosides of known PA, afforded the appropriate proton-bound heterodimers when subjected to electrospray ionization. Their collisional induced dissociations were studied in a QqTOF hybrid instrument as a function of both the gas pressure and the collision energy of the dimers entering q. The value of ΔS for each reaction was evaluated and correlated with the effect of the substituent at the para position of the benzoyl group. For each reaction, a Hammett plot of ΔPA N vs. σ values of the substituents on the benzoyl group shows that the carbonyl group participates, in the transition state, to the stabilization/destabilization of the N-3-protonated species. In the cases examined, one can rule out that the formation of intramolecular hydrogen bonds occurs under thermodynamic control. The activation energy for the back reaction can, therefore, be neglected.
Researchers have identified small organic compounds that can accelerate the formation of G-quadruplex structures from single-stranded DNA and the interconversion of one type of G-quadruplex into another. G-quadruplexes are folded conformations of DNA that form from the association of sets of four guanine residues into planar arrays. Several research groups have been studying organic compounds that bind to G-quadruplex DNA in an effort to find bioactive agents such as inhibitors of telomerase—an enzyme with a nucleic acid component that is believed to form G-quadruplexes. Telomerase is expressed primarily in rapidly dividing cells, such as cancer cells, and inhibitors of the enzyme are being sought as potential anticancer agents. Porphyrins, anthraquinones, and other small molecules have in the past been shown to bind to and stabilize already-formed G-quadruplex DNA structures in test-tube experiments (C&EN, Oct. 5, 1998, page 42). But by identifying agents that actually accelerate the assembly and interconversion of G-quadruplexes, scientists ...
The 15-mer oligodeoxynucleotide GGTTGGTGTGGTTGG is a potent inhibitor of thrombin and it forms a stable, highly compact structure in solution. Deletions and substitutions by abasic residues, 2′-deoxyinosine, 7-deaza-2′-deoxyguanosine and 8-methyl-2′-deoxyguanosine show that the structural features of the oligodeoxynucleotide are important for its biological activity.
The nucleoside, 5′-(t-butyl-dimethylsilyl)-2′,3′-O-isopropylidene isoG 1, catalyzes the SN2 reactions of alkali and ammonium iodides with dodecyl mesylate 2 under both liquid-liquid and solid-liquid phase transfer conditions. IsoG 1 self-associates to give a complex that extracts the salts into CDCl3 solution. Sodium iodide, in the presence of isoG 1, reacts faster with 2 than the other iodides under solid-liquid conditions. This reactivity difference is attributed to the open-faced structure of the ionophore-M+ complex under solid-liquid conditions.
The ordered forms of the guanosine 5′-monophosphate dianion in the presence of Na+ as the structure-directing cation (Na+/5′-GMP = 2.0) have been investigated in H2O solution by 1H NMR spectroscopy. The resonances assigned to H-bonded N(1)H (11.1-11.3 ppm) and N(2)H (8.8-10.4 ppm) in the ordered nucleotide have normalized intensities of 0.96 ± 0.12 and 1.1 ± 0.1 protons per ordered 5′-GMP, respectively. This results is compatible with the interbase H-bonding scheme expected for planar tetramer units (I) and supports the proposal that the ordered forms are isomeric octamers formed by stacking of tetramer units. An additional resonance at 7.69 ppm has been assigned on the basis of chemical shift, line width, and spin saturation transfer results to a ribose OH involved in extratetramer H bonding. The normalized intensity of the ribose OH proton (0.33 ± 0.06) is equal within experimental uncertainty to the normalized intensity of an unusually high-field line at 2.2 ppm in the 31P NMR spectrum (0.30 ± 0.06), suggesting that a phosphte oxygen on an adjacent tetramer may be acting as the hydrogen acceptor. Several plausible alternatives for the extratetramer H bond also are discussed. Finally, the merits of the stacked tetramer model are shown to be superior to those of a recently proposed stacked asymmetric dimer model when the two models are compared in light of all the relevant data.
Tetramethylammonium has been found to be a structure-inert cation in the solution ordering of guanosine 5′-monophosphate (5′-GMP) at neutral or slightly basic pH. This finding has made it possible to study quantitatively by H NMR spectroscopy the stoichiometry of nucleotide ordering in the presence of Na+ as a structure-directing counterion. The dependence of the ordered structures on total nucleotide concentration is consistent with the formation of octamers. Independent evidence for octamer formation is provided by the binding of ethidium to the ordered nucleotide. Two octamer-ethidium complexes are observed with octamer to ethidium ratios of 1:1 and 1:2. The dependence of ordered structure formation on Na+ concentration indicates the binding of four Na+ ions per octamer unit. Two types of Na+ binding sites are inferred from mixed Na+-K+ experiments. One binding site is a highly Na+-specific structure-directing site. The second site is less Na+ specific, but it plays an important role in stabilizing the ordered structures. The replacement of Na+ by K+ at the second site dramatically stabilizes the Na+-directed self-structures. A model involving coaxial stacking of planar hydrogen-bonded tetramer units is proposed for the ordered structures. Normal and inverted tetramer stacking arrangements account for the presence of three NMR-observable isomers, provided that twisting about the C4 symmetry axis is rapid. The hole defined by the four carbonyl oxygens of a tetramer unit is believed to be the highly Na+-specific binding site. Chelation of Na+ (or structure-stabilizing K+) by phosphate oxygens on adjacent tetramer plates is proposed as the less specific binding site. Model building studies suggest that interplate hydrogen bonding also may be involved in stabilizing the stacked tetramers.
Molecular mechanical calculations have been carried out on r(CGCGCG)2, r(C-M⁸G-C-m⁸G-C-m⁸G)2, d(CGCGCG)2, and d(C-m⁸G-C-m⁸G-C-m⁸G)2 in A, B, and Z1 forms of polynucleotides. To our knowledge, this is the first atomic level molecular mechanical study of double-stranded RNA in the three polymorphic forms, and detailed structures are presented for the energy-refined models. The calculated energies when corrected for artifacts inherent in a model for RNA and DNA without inclusion of specific hydration are in general agreement with experimental results. Specifically, the B form is more stable than the A form in DNA, the reverse being true in RNA, and the counterion condensation promotes the B to Z transition in DNA and (with more difficulty) an A to Z transition in RNA. Further, 8-methylation of guanine bases potentiates an A to Z transition in RNA and, to a smaller extent, a B to Z transition in DNA. The effect of 8-methylation on promoting the A to Z transition in RNA can be attributed to an unfavorable steric interaction of the 8-methyl group with the backbone in the A structure, reducing favorable base-stacking interactions in this structure.
Experimental molecular weights in the range 200 to 100,000 Daltons have been determined by vapor pressure osmometry using three different solvents. For all values in excess of 10,000, molecular weights were also determined by membrane osmometry. The general agreement found for molecular weights determined under a variety of conditions, leads to the conclusion that values as high as 100,000 Daltons can be determined by at least one type of vapor pressure osmometer when it is calibrated with a material of 200 Daltons. Thermistor self-heating and diffusion in the liquid phase are shown to be unimportant for this particular instrument.
Oligodeoxyribonucleotides containing 7-deaza-2′-deoxy-8-methylguanosine (m8c7Gd; 2b) were prepared. For this purpose, the phosphonate 3a and the phosphoramidite 3b were synthesized and employed in solidphase oligodeoxyribonucleotide synthesis. The structures and the thermodynamic data of duplex formation of oligodeoxyribonucleotides containing 2b were investigated by temperature-dependent CD and UV spectra and compared with those containing 7-deaza-2′-deoxy-7-methylguanosine (m7c7Gd) or 7-deaza-2′-deoxy-guanosine (c7Gd; 2a). In general, compound 2b reduces the duplex stability. In case of the sequence d(m8c7G-C)4(18), the B → Z transition was facilitated by the incorporation of 2b. Moreover, a single 7-deaza-8-methylguanine residue present in an oligodeoxyribonucleotide tract of guanine residues destabilizes the dG quadruplex significantly. This destabilization is more pronounced than in the case of 7-deazaguanine or 7-deaza-7-methyl-guanine.
Guanosine-5′-phosphate crystallizes from neutral solutions of NaCl in bundles of hair-like crystals. Dried bundles of these crystals give a fiber-like X-ray diffraction pattern which indicates that the monomers are arranged in a structure similar to the arrangement of residues in poly(rG). This arrangement consists of monomers grouped into tetramers which then stack upon themselves helically. A second type of diffraction pattern obtained from salt-free samples of guanosine-5′-phosphate is also noted.