G-tetrad Research Articles

NMR based structural studies decipher stacking of the alkaloid coralyne to terminal guanines at two different sites in parallel G-quadruplex DNA, [d(TTGGGGT)]4 and [d(TTAGGGT)

BackgroundTelomere elongation by telomerase gets inhibited by G-quadruplex DNA found in its guanine rich region. Stabilization of G-quadruplex DNA upon ligand binding has evolved as a promising strategy to target cancer cells in which telomerase is over expressed. MethodsInteraction of anti-leukemic alkaloid, coralyne, to tetrameric parallel [d(TTGGGGT)]4 (Ttel7), [d(TTAGGGT)]4 (Htel7) and monomeric anti-parallel [dGGGG(TTGGGG)3] (Ttel22) G-quadruplex DNA has been studied using Circular Dichroism (CD) spectroscopy. Titrations of coralyne with Ttel7 and Htel7 were monitored by 1H and 31P NMR spectroscopy. Solution structure of coralyne-Ttel7 complex was obtained by restrained Molecular Dynamics (rMD) simulations using distance restraints from 2D NOESY spectra. Thermal stabilization of DNA was determined by absorption, CD and 1H NMR. Results and conclusionsBinding of coralyne to Ttel7/Htel7 induces negative CD band at 315/300nm. A significant upfield shift in all GNH, downfield shift in T2/T7 base protons and upfield shift (1.8ppm) in coralyne protons indicates stacking interactions. 31P chemical shifts and NOE contacts of G3, G6, T2, T7 protons with methoxy protons reveal proximity of coralyne to T2pG3 and G6pT7 sites. Solution structure reveals stacking of coralyne at G6pT7 and T2pG3 steps with two methoxy groups of coralyne located in the grooves along with formation of a hydrogen bond. Binding stabilizes Ttel7/Htel7 by ~25–35°C in 2:1 coralyne-Ttel7/Htel7 complex. General significanceThe present study is the first report on solution structure of coralyne-Ttel7 complex showing stacking of coralyne with terminal guanine tetrads leading to significant thermal stabilization, which may be responsible for telomerase inhibition.

Modified Guanines as Constituents of Smart Ligands for Nucleic Acid Quadruplexes.

Repetitive guanine-rich nucleic acid sequences play a crucial role in maintaining genome stability and the cell life cycle and represent potential targets for regulatory drugs. Recently, it has been demonstrated that guanine-based ligands with a porphyrin core can be used as markers of G-quadruplex assemblies in cell tissues. Herein, model systems of guanine-based ligands are explored by DFT methods. The energies of formation of modified guanine tetrads and those of modified tetrads stacked on the top of natural guanine tetrads have been calculated. The interaction energy has been decomposed into contributions from hydrogen bonding, stacking, and ion coordination and a twist-rise potential energy scan has been performed to find the individual local minima. Energy decomposition analysis reveals the impact of various substituents (F, Cl, Br, I, Me, NMe2 ) on individual energy terms. In addition, cooperative reinforcement in forming the modified and stacked tetrads, as well as the frontier orbitals participating in the hydrogen-bonding framework involving the HOMO-LUMO gap between the occupied σHOMO on the proton-accepting C=O and =N- groups and unoccupied σLUMO on the N-H groups, has been studied. The investigated systems are demonstrated to have a potential in ligand development, mainly due to stacking enhancement compared with natural guanine, which is used as a reference.

Molecular dynamics simulations reveal the balance of forces governing the formation of a guanine tetrad-a common structural unit of G-quadruplex DNA.

G-quadruplexes (G4) are nucleic acid conformations of guanine-rich sequences, in which guanines are arranged in the square-planar G-tetrads, stacked on one another. G4 motifs form in vivo and are implicated in regulation of such processes as gene expression and chromosome maintenance. The structure and stability of various G4 topologies were determined experimentally; however, the driving forces for their formation are not fully understood at the molecular level. Here, we used all-atom molecular dynamics to probe the microscopic origin of the G4 motif stability. By computing the free energy profiles governing the dissociation of the 3′-terminal G-tetrad in the telomeric parallel-stranded G4, we examined the thermodynamic and kinetic stability of a single G-tetrad, as a common structural unit of G4 DNA. Our results indicate that the energetics of guanine association alone does not explain the overall stability of the G-tetrad and that interactions involving sugar–phosphate backbone, in particular, the constrained minimization of the phosphate–phosphate repulsion energy, are crucial in providing the observed enthalpic stabilization. This enthalpic gain is largely compensated by the unfavorable entropy change due to guanine association and optimization of the backbone topology.

Another step toward DNA selective targeting: Ni(II) and Cu(II) complexes of a Schiff base ligand able to bind gene promoter G-quadruplexes.

DNA G-rich sequences are able to form four-stranded structures organized in stacked guanine tetrads. These structures, called G-quadruplexes, were found to have an important role in the regulation of oncogenes expression and became, for such a reason, appealing targets for anticancer drugs. Aiming at finding selective G-quadruplex binders, we have designed, synthesized and characterized a new water soluble Salen-like Schiff base ligand and its Ni(II) and Cu(II) metal complexes. UV-Vis, circular dichroism and FRET measurements indicated that the nickel complex can stabilize oncogene promoter G-quadruplexes with high selectivity, presenting no interactions with duplex DNA at all. The same compound exhibited dose-dependent cytotoxic activity in MCF-7 breast cancer cells when combined with lipofectamine as lipophilic carrier.

G-quadruplex DNA structures can interfere with uracil glycosylase activity in vitro.

Genome sequences that contain tandem repeats of guanine can form stable four-stranded structures known as G-quadruplex, or G4 DNA. While the molecular mechanisms are not fully defined, such guanine-rich loci are prone to mutagenesis and recombination. Various repair pathways function to reduce the potential for genome instability by correcting base damage and replication errors; however, it is not yet fully defined how well these processes function at G4 DNA. One frequent form of base damage occurs from cytidine deamination, resulting in deoxyuracil and UG mismatches. In duplex and single-stranded DNA, uracil bases are recognised and excised by uracil glycosylases. Here, we tested the efficiency of uracil glycosylase activity in vitro on uracil bases located directly adjacent to guanine repeats and G4 DNA. We show that uracil excision by bacterial UDG and human hUNG2 is reduced at uracils positioned directly 5' or 3' of a guanine tetrad. Control reactions using oligonucleotides disrupted for G4 formation or reaction conditions that do not favour G4 formation resulted in full uracil excision activity. Based on these in vitro results, we suggest that folding of guanine-rich DNA into G4 DNA results in a DNA conformation that is resistant to uracil glycosylase-initiated repair and this has the potential to increase the risk of instability at guanine repeats in the genome.

Inverting the G‐Tetrad Polarity of a G‐Quadruplex by Using Xanthine and 8‐Oxoguanine

AbstractG‐quadruplexes are four‐stranded nucleic acid structures that are built from consecutively stacked guanine tetrad (G‐tetrad) assemblies. The simultaneous incorporation of two guanine base lesions, xanthine (X) and 8‐oxoguanine (O), within a single G‐tetrad of a G‐quadruplex was recently shown to lead to the formation of a stable G⋅G⋅X⋅O tetrad. Herein, a judicious introduction of X and O into a human telomeric G‐quadruplex‐forming sequence is shown to reverse the hydrogen‐bond polarity of the modified G‐tetrad while preserving the original folding topology. The control exerted over G‐tetrad polarity by joint X⋅O modification will be valuable for the design and programming of G‐quadruplex structures and their properties.

Inverting the G-Tetrad Polarity of a G-Quadruplex by Using Xanthine and 8-Oxoguanine.

G-quadruplexes are four-stranded nucleic acid structures that are built from consecutively stacked guanine tetrad (G-tetrad) assemblies. The simultaneous incorporation of two guanine base lesions, xanthine (X) and 8-oxoguanine (O), within a single G-tetrad of a G-quadruplex was recently shown to lead to the formation of a stable G⋅G⋅X⋅O tetrad. Herein, a judicious introduction of X and O into a human telomeric G-quadruplex-forming sequence is shown to reverse the hydrogen-bond polarity of the modified G-tetrad while preserving the original folding topology. The control exerted over G-tetrad polarity by joint X⋅O modification will be valuable for the design and programming of G-quadruplex structures and their properties.

Aven recognition of RNA G-quadruplexes regulates translation of the mixed lineage leukemia protooncogenes.

G-quadruplexes (G4) are extremely stable secondary structures forming stacks of guanine tetrads. DNA G4 structures have been extensively studied, however, less is known about G4 motifs in mRNAs, especially in their coding sequences. Herein, we show that Aven stimulates the mRNA translation of the mixed lineage leukemia (MLL) proto-oncogene in an arginine methylation-dependent manner. The Aven RGG/RG motif bound G4 structures within the coding regions of the MLL1 and MLL4 mRNAs increasing their polysomal association and translation, resulting in the induction of transcription of leukemic genes. The DHX36 RNA helicase associated with the Aven complex and was required for optimal translation of G4 mRNAs. Depletion of Aven led to a decrease in synthesis of MLL1 and MLL4 proteins resulting in reduced proliferation of leukemic cells. These findings identify an Aven-centered complex that stimulates the translation of G4 harboring mRNAs, thereby promoting survival of leukemic cells.

Influence of base stacking geometry on the nature of excited states in G-quadruplexes: a time-dependent DFT study.

G-quadruplexes are four-stranded structures of nucleic acids that are formed from the association of guanine nucleobases into cyclical arrangements known as tetrads. G-quadruplexes are involved in a host of biological processes and are of interest in nanomaterial applications. However, not much is known about their electronic properties. In this paper, we analyze electronic excited states of G-quadruplexes using a combination of time-dependent DFT calculations and molecular dynamics simulations. We systematically consider experimentally observed arrangements of stacked guanine tetrads. The effects of structural features on exciton delocalization and photoinduced charge separation are explored using a quantitative analysis of the transition electron density. It is shown that collective coherent excitations shared between two guanine nucleobases dominate in the absorption spectrum of stacked G-tetrads. These excitations may also include a significant contribution of charge transfer states. Large variation in exciton localization is also observed between different structures with a general propensity toward localization between two bases. We reveal large differences in how charge separation occurs within different nucleobase arrangements, with some geometries favoring separation within a single tetrad and others favoring separation between tetrads. We also investigate the effects of the coordinating K(+) ion located in the central cavity of G-quadruplexes on the relative excited state properties of such systems. Our results demonstrate how the nature of excited states in G-quadruplexes depends on the nucleobase stacking geometry resulting from the mutual arrangement of guanine tetrads.

Guanine tetrads: an IRMPD spectroscopy, energy resolved SORI-CID, and computational study of M(9-ethylguanine)(4)(+) (M = Li, Na, K, Rb, Cs) in the gas phase.

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.

Group 9 organometallic compounds for therapeutic and bioanalytical applications.

CONSPECTUS: Compared with organic small molecules, metal complexes offer several distinct advantages as therapeutic agents or biomolecular probes. Carbon atoms are typically limited to linear, trigonal planar, or tetrahedral geometries, with a maximum of two enantiomers being formed if four different substituents are attached to a single carbon. In contrast, an octahedral metal center with six different substituents can display up to 30 different stereoisomers. While platinum- and ruthenium-based anticancer agents have attracted significant attention in the realm of inorganic medicinal chemistry over the past few decades, group 9 complexes (i.e., iridium and rhodium) have garnered increased attention in therapeutic and bioanalytical applications due to their adjustable reactivity (from kinetically liable to substitutionally inert), high water solubility, stability to air and moisture, and relative ease of synthesis. In this Account, we describe our efforts in the development of group 9 organometallic compounds of general form [M(C(∧)N)2(N(∧)N)] (where M = Ir, Rh) as therapeutic agents against distinct biomolecular targets and as luminescent probes for the construction of oligonucleotide-based assays for a diverse range of analytes. Earlier studies by researchers had focused on organometallic iridium(III) and rhodium(III) half-sandwich complexes that show promising anticancer activity, although their precise mechanisms of action still remain unknown. More recently, kinetically-inert group 9 complexes have arisen as fascinating alternatives to organic small molecules for the specific targeting of enzyme activity. Research in our laboratory has shown that cyclometalated octahedral rhodium(III) complexes were active against Janus kinase 2 (JAK2) or NEDD8-activating enzyme (NAE) activity, or against NO production leading to antivasculogenic activity in cellulo. At the same time, recent interest in the development of small molecules as modulators of protein-protein interactions has stimulated our research group to investigate whether kinetically-inert metal complexes could also be used to target protein-protein interfaces relevant to the pathogenesis of certain diseases. We have recently discovered that cyclometalated octahedral iridium(III) and rhodium(III) complexes bearing C(∧)N ligands based on 2-phenylpyridine could function as modulators of protein-protein interactions, such as TNF-α, STAT3, and mTOR. One rhodium(III) complex antagonized STAT3 activity in vitro and in vivo and displayed potent antitumor activity in a mouse xenograft model of melanoma. Notably, these studies were among the first to demonstrate the direct inhibition of protein-protein interfaces by kinetically-inert group 9 metal complexes. Additionally, we have discovered that group 9 solvato complexes carrying 2-phenylpyridine coligands could function as inhibitors and probes of β-amyloid fibrillogenesis. Meanwhile, the rich photophysical properties of iridium complexes have made them popular tools for the design of luminescent labels and probes. Luminescent iridium(III) complexes benefit from a high quantum yield, responsive emissive properties, long-lived phosphorescence lifetimes, and large Stokes shift values. Over the past few years, our group has developed a number of kinetically-inert, organometallic iridium(III) complexes bearing various C(∧)N and N(∧)N ligands that are selective for G-quadruplex DNA, which is a DNA secondary structure formed from planar stacks of guanine tetrads stabilized by Hoogsteen hydrogen bonding. These complexes were then employed to develop G-quadruplex-based, label-free luminescence switch-on assays for nucleic acids, enzyme activity, small molecules, and metal ions.

Guanine-centric self-assembly of nucleotides in water: an important consideration in prebiotic chemistry.

Investigations of plausible prebiotic chemistry on early Earth must consider not only chemical reactions to form more complex products such as proto-biopolymers but also reversible, molecular self-assembly that would influence the availability, organization, and sequestration of reactant molecules. The self-assembly of guanosine compounds into higher-order structures and lyotropic liquid crystalline "gel" phases through formation of hydrogen-bonded guanine tetrads (G-tetrads) is one such consideration that is particularly relevant to an RNA-world scenario. G-tetrad-based gelation has been well studied for individual guanosine compounds and was recently observed in mixtures of guanosine with 5'-guanosine monophosphate (GMP) as well. The present work investigates the self-assembly of GMP in the presence of the other RNA nucleotides. Effects of the total concentration and relative proportion of the nucleotides in the mixtures, the form (disodium salt vs. free acid) of the nucleotides, temperature, pH, and salt concentration were determined by visual observations and circular dichroism (CD) spectroscopy. The results show that formation of cholesteric G-tetrad phases is influenced by interactions with other nucleotides, likely through association (e.g., intercalation) of the nucleotides with the G-tetrad structures. These interactions affect the structure and stability of the G-tetrad gel phase, as well as the formation of alternate self-assembled GMP structures such as a continuous, hydrogen-bonded GMP helix or dimers and aggregates of GMP. These interactions and multiple equilibria are influenced by the presence of cations, especially in the presence of K(+). This work could have important implications for the emergence of an RNA or proto-RNA world, which would require mixtures of nucleotides at sufficiently high, local concentrations for abiotic polymerization to occur.

G-quadruplex formation of FXYD1 pre-mRNA indicates the possibility of regulating expression of its protein product

G-quadruplexes are higher-order nucleic acid structures formed of square-planar arrangements of four guanine bases held together by Hoogsteen-type hydrogen bonds. Stacks of guanine tetrads are stabilised by intercalating potassium ions. FXYD1 encodes for phospholemman, a regulatory subunit of the cardiac Na+/K+-ATPase. Computational sequence analysis of FXYD1 pre-mRNA predicted the formation of stable intramolecular G-quadruplexes in human and orthologue sequences. Multiple sequence alignment indicated that G-rich sequences are conserved in evolution suggesting a potential role of G-quadruplexes in FXYD1 gene expression. The existence of a non-functional alternative splicing product indicated that the G-quadruplex formation may control alternative splicing. Quadruplex formation of human and bovine oligonucleotides was confirmed in vitro by native polyacrylamide gel electrophoresis and intrinsic fluorescence emission spectroscopy. Taking together the evolutionary conservation of G-quadruplex forming sequences with the confirmation of G-quadruplex formation in vitro by two FXYD1 homologues the results point to a potential role of these structures in regulating the expression of FXYD1 and thus regulate indirectly the activity of the cardiac Na+/K+-ATPase.

Conformational switch of insulin-binding aptamer into G-quadruplex induced by K+ and Na+: an experimental and theoretical approach

Guanine-rich sequences can form the G-quadruplex structure in the presence of specific metal ions. Here, circular dichroism, UV–vis absorption, fluorescence, and molecular dynamics simulation studies revealed that insulin-binding aptamer (IBA) could form an intramolecular G-quadruplex structure after binding K+. Circular dichroism (CD) spectra demonstrated that IBA could fold into a parallel G-quadruplex with a strong positive peak at 263 nm. Analysis of equilibrium titration data revealed that cation binding was cooperative with the Hill coefficient of 2.01 in K+ and 1.90 in Na+. Thermal denaturation assays indicated that K+-induced G-quadruplex is more stable than Na+-induced structure. Folding of IBA into G-quadruplex leading to the contact quenching occurs as a result of the formation of a nonfluorescent complex between donor and acceptor. Based on fluorescence quenching of IBA folding, a potassium-sensing aptasensor in the range of 0–1.4 mM was proposed. Since the quenching process was predominantly static, the binding constant and the number of binding sites were determined. In this research, based on the experimental data, the initial model of IBA G-quadruplex was constructed by molecular modeling method. The modeling structure of IBA is an intramolecular parallel-strand quadruplex conformation with two guanine tetrads. The extended molecular dynamics simulation for the model indicated that the G-quadruplex maintains its structure very well in aqueous solution in presence of K+ in the central cavity. In contrast, it was demonstrated that the G-quadruplex structure of model in the water collapses in absence of this cation.

Advancement in the Structural Polymorphism of G-Quadruplexes

Genomes contain a large number of putative guanine-rich sequences, specifically on promoter regions, untranslated regions (UTR’s) and telomeres etc. that could form guanine-quadruplexes, and may serve as important structural and regulatory elements. They can also be the source of genomic instability which may lead to cancer, aging and human genetic diseases. Four guanines in the same plane, joined together with Hoogsteen hydrogen bonding, and stacked over one another resulting in guanine tetrads, give rise to an incredible class of G-quadruplexes. An extensive range of G-quadruplex structures is well documented, where they differ in number of strands (uni, bi, or tetramolecular), conformations (parallel, antiparallel or mixed), shapes (chair or basket form), or types of loops (edgewise/ lateral, diagonal, double chain reversal/ propeller, or V-shaped loops) etc. With advancements in the techniques, various new multistranded G-rich DNA structures are enriching the DNA structural databases. The most recent ones are (3+1) G-quadruplex, Tri-G-quadruplex, G-triplex DNA etc. which actually add to the diversity of G-quadruplex structures. Exploring their polymorphism with various biophysical and biochemical techniques has hence become extremely important. This review mainly focuses on the discussion of these unusual and comparatively new polymorphic G-quadruplex DNA structures. The robustness of these unique (3+1) G-quadruplex or G-triplex or tri-G-quadruplex structures actually can be exploited for providing a strong foundation for the designing of structure-specific drugs. Recently, G-quadruplex structures have been quantitatively visualized in human cells by engineering structure-specific antibody. Considering these developments, the mapping of G-quadruplex structures in genome may now be possible, with a goal of controlling the gene functions or other cellular processes, which might be involved in diseases like cancer.

Binding of G-quadruplexes to the N-terminal Recognition Domain of the RNA Helicase Associated with AU-rich Element (RHAU)

Polynucleotides containing consecutive tracts of guanines can adopt an intramolecular G-quadruplex structure where multiple planar tetrads of hydrogen-bound guanines stack on top of each other. Remodeling of G-quadruplexes impacts numerous aspects of nucleotide biology including transcriptional and translational control. RNA helicase associated with AU-rich element (RHAU), a member of the ATP-dependent DEX(H/D) family of RNA helicases, has been established as a major cellular quadruplex resolvase. RHAU contains a core helicase domain responsible for ATP binding/hydrolysis/helicase activity and is flanked on either side by N- and C-terminal extensions. The N-terminal extension is required for quadruplex recognition, and we have previously demonstrated complex formation between this domain and a quadruplex from human telomerase RNA. Here we used an integrated approach that includes small angle x-ray scattering, nuclear magnetic resonance spectroscopy, circular dichroism, and dynamic light scattering methods to demonstrate the recognition of G-quadruplexes by the N-terminal domain of RHAU. Based on our results, we conclude that (i) quadruplex from the human telomerase RNA and its DNA analog both adopt a disc shape in solution, (ii) RHAU53-105 adopts a defined and extended conformation in solution, and (iii) the N-terminal domain mediates an interaction with a guanine tetrad face of quadruplexes. Together, these data form the foundation for understanding the recognition of quadruplexes by the N-terminal domain of RHAU.

Binding to G-quadruplex RNA activates the mitochondrial GTPase NOA1

NOA1 is an evolutionary conserved, nuclear encoded GTPase essential for mitochondrial function and cellular survival. The function of NOA1 for assembly of mitochondrial ribosomes and regulation of OXPHOS activity depends on its GTPase activity, but so far no ligands have been identified that regulate the GTPase activity of NOA1. To identify nucleic acids that bind to the RNA-binding domain of NOA1 we employed SELEX (Systemic Evolution of Ligands by EXponential Enrichment) using recombinant mouse wildtype NOA1 and the GTPase mutant NOA1-K353R. We found that NOA1 binds specifically to oligonucleotides that fold into guanine tetrads (G-quadruplexes). Binding of G-quadruplex oligonucleotides stimulated the GTPase activity of NOA1 suggesting a regulatory link between G-quadruplex containing RNAs, NOA1 function and assembly of mitochondrial ribosomes.

The solvent effect and identification of a weakly emissive state in nonradiative dynamics of guanine nucleosides and nucleotides — a combined femtosecond broadband time-resolved fluorescence and transient absorption study

A combined method of femtosecond broadband time-resolved fluorescence (fs-TRF) and transient absorption (fs-TA) was employed to investigate the excited state dynamics of 2'-deoxyguanosine (dG) and 2'-deoxyguanosine 5'-monophosphate (dGMP). Comparative fs-TRF and fs-TA measurements were conducted on dG and dGMP in neutral water, deuterated water, and methanol with excitation wavelengths of 245, 267 and 285 nm. Very similar results were observed with dG and dGMP. The data provide compelling evidence for the co-existence of two nonradiative pathways. One is the generally recognized Laππ* mediated channel, the other involves an unprecedented weakly emissive state which plays a significant role in the overall deactivation processes. The Laππ* channel features biphasic dynamics with time constants (τ1/τ2) of ~0.2/0.8 ps in water and ~0.25/1.0 ps in methanol. The biphasic decay arises due to a partial transfer with τ1 of the Laππ* population to the newly identified state followed by conversion in τ2 of the remaining Laππ* molecules into the electronic ground state. The channel mediated by the weakly emissive species shows solvent-dependent dynamics with time constants (τ3) of ~2.0 ps in water, ~2.3 ps in deuterated water, and ~4.1 ps in methanol. The species features absorption at UV wavelengths (~300-400 nm) and exhibits deeply red-shifted fluorescence (λmax ~ 520 nm) with polarization direction varied markedly from that of the Laππ* but close to the Lbππ*. This species acts as an effective quenching state to the radiative decay of the brightly emissive Laππ* and Lbππ*. It sets in promptly (<~50 fs) after the photoexcitation and is further populated through nonadiabatic coupling with the Laππ*. The overall involvement of this state is facilitated with excitation at high energy and is favoured in methanol over water. According to the spectral character and the solvent effect in particular the kinetic isotope effect, the species is tentatively associated to the πσ* state with charge transfer (CT) character which is considered to be preferentially stabilized by hydrogen-bonding between the guanine amino and surrounding solvent molecules. The result of this study leads to a dramatically different picture of guanine deactivation. It demonstrates a crucial role of the solvent in shaping the nonradiative dynamics of guanine nucleosides and nucleotides. The data presented are important for understanding the detailed photophysics of not only the monomeric guanine but also DNA assemblies that contain guanine in base pairs or have a guanine tetrad as the structural motif.

The effect of size on the optical properties of guanine nanostructures: a femtosecond to nanosecond study

G-quadruplexes, whose building blocks are guanine tetrads, encounter increasing interest with respect to their potential applications in the field of molecular electronics. Here we study how the size of these nanostructures affects their fluorescence. We compare the properties of thymine capped G-quadruplexes, formed by association of four single DNA strands d(TG3T), d(TG4T) and d(TG5T) and stabilized by K(+) ions. We show that an increase in the number of tetrads induces a narrowing of the fluorescence spectrum, an increase in the fluorescence quantum yield, a lengthening of fluorescence lifetime and a decrease of the anisotropy detected on the femtosecond time-scale. The in-plane depolarization of the fluorescence, occurring in less than 1 ps, is attributed to population of Franck-Condon exciton states and ultrafast intraband scattering, leading to energy transfer. The persistence of excitons with partial J-aggregate character on the picosecond time-scale increases with the G-quadruplex size, which enhances the stiffness of the system.

47 Incorporation of alternative nucleic acid chemistries into G-quadruplex structure

Nucleic acids that form G-quadruplex (G4) structure have found applications in a host of research and technology regimes. Numerous G4 based aptamer drugs have been identified with pharmacological activity against cancer, HIV, prions, and blood coagulation (1). In the field of nanotechnology, G4 based sensors and nano-machines have also received much attention. The ability to synthesize nucleic acid ex-vivo allows for the site-specific incorporation of non-natural chemistries into nucleic acids that can be used to tune their physical and pharmacological properties. We summarize the results of a series of studies investigating the effective incorporation of alternative nucleic acid chemistries into G4 DNA. These modified chemistries include C8-modified guanine bases, as well as 2′-F, 2′-F-ANA, and Locked nucleic acid (LNA) modifications to the ribose sugar. We report primarily on the effect of these modifications on G-quadruplex folding topology, thermal stability, and structure. The substitution of LNA-guanosine into the core guanine tetrads disrupts structure in specific structural environments. On the other hand, 2′-F- and 2′-F-ANA guanosine can generally be incorporated without disrupting the structure when substituted into guanine bases in certain structural conformations. We find that 2′-F-ANA-guanosine and 2′-F-guanosine are powerful tools for controling the conformation of G4 structures (2). Functionalization at the C8 of the guanine base stabilizes in a manner dependent on the glycosidic conformation of the base, with different modification chemistries stabilizing to varying extents (3). The results of these studies provide useful insight on how to effectively incorporate some useful chemical tools from the growing toolbox of modified nucleic acid chemistries into G-quadruplex nucleic acid.