Christopher Jacques Lech

Guanine base stacking in G-quadruplex nucleic acids

G-quadruplexes constitute a class of nucleic acid structures defined by stacked guanine tetrads (or G-tetrads) with guanine bases from neighboring tetrads stacking with one another within the G-tetrad core. Individual G-quadruplexes can also stack with one another at their G-tetrad interface leading to higher-order structures as observed in telomeric repeat-containing DNA and RNA. In this study, we investigate how guanine base stacking influences the stability of G-quadruplexes and their stacked higher-order structures. A structural survey of the Protein Data Bank is conducted to characterize experimentally observed guanine base stacking geometries within the core of G-quadruplexes and at the interface between stacked G-quadruplex structures. We couple this survey with a systematic computational examination of stacked G-tetrad energy landscapes using quantum mechanical computations. Energy calculations of stacked G-tetrads reveal large energy differences of up to 12 kcal/mol between experimentally observed geometries at the interface of stacked G-quadruplexes. Energy landscapes are also computed using an AMBER molecular mechanics description of stacking energy and are shown to agree quite well with quantum mechanical calculated landscapes. Molecular dynamics simulations provide a structural explanation for the experimentally observed preference of parallel G-quadruplexes to stack in a 5′–5′ manner based on different accessible tetrad stacking modes at the stacking interfaces of 5′–5′ and 3′–3′ stacked G-quadruplexes.

Effects of site-specific guanine C8-modifications on an intramolecular DNA G-quadruplex.

Understanding the fundamentals of G-quadruplex formation is important both for targeting G-quadruplexes formed by natural sequences and for engineering new G-quadruplexes with desired properties. Using a combination of experimental and computational techniques, we have investigated the effects of site-specific substitution of a guanine with C8-modified guanine derivatives, including 8-bromo-guanine, 8-O-methyl-guanine, 8-amino-guanine, and 8-oxo-guanine, within a well-defined (3 + 1) human telomeric G-quadruplex platform. The effects of substitutions on the stability of the G-quadruplex were found to depend on the type and position of the modification among different guanines in the structure. An interesting modification-dependent NMR chemical-shift effect was observed across basepairing within a guanine tetrad. This effect was reproduced by ab initio quantum mechanical computations, which showed that the observed variation in imino proton chemical shift is largely influenced by changes in hydrogen-bond geometry within the guanine tetrad.

Electron-hole transfer in G-quadruplexes with different tetrad stacking geometries: a combined QM and MD study.

G-quadruplex nucleic acids represent a unique avenue for the building of electrically conductive wires. These four-stranded structures are formed through the stacking of multiple planar guanine assemblies termed G-tetrads. The diverse folding patterns of G-quadruplexes allow for several geometries to be adopted by stacked guanine bases within the core and at the dimeric interface of these structures. It is currently not clear how different G-tetrad stacking arrangements affect electron hole mobility through a G-quadruplex wire. Using a combined quantum mechanics and molecular dynamics approach, we demonstrate that the electron-hole transfer rates within the G-tetrad stacks vary greatly for different stacking geometries. We identify a distinguished structure that allows for strong electronic coupling and thus enhanced molecular electric conductance. We also demonstrate the importance of sampling a large number of geometries when considering the bulk properties of such systems. Hole hopping within single G-tetrads is slower by at least two orders of magnitude than between stacked guanines; therefore, hole jumping within individual tetrads should not affect the hole mobility in G-quadruplexes. The results of this study suggest engineering G-tetrads with continuous 5/6-ring stacking from an assembly of single guanosine analogs or through modification of the backbone in G-rich DNA sequences.

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.

Sugar-modified G-quadruplexes: effects of LNA-, 2′F-RNA– and 2′F-ANA-guanosine chemistries on G-quadruplex structure and stability

G-quadruplex-forming oligonucleotides containing modified nucleotide chemistries have demonstrated promising pharmaceutical potential. In this work, we systematically investigate the effects of sugar-modified guanosines on the structure and stability of a (4+0) parallel and a (3+1) hybrid G-quadruplex using over 60 modified sequences containing a single-position substitution of 2′-O-4′-C-methylene-guanosine (LNAG), 2′-deoxy-2′-fluoro-riboguanosine (FG) or 2′-deoxy-2′-fluoro-arabinoguanosine (FANAG). Our results are summarized in two parts: (I) Generally, LNAG substitutions into ‘anti’ position guanines within a guanine-tetrad lead to a more stable G-quadruplex, while substitutions into ‘syn’ positions disrupt the native G-quadruplex conformation. However, some interesting exceptions to this trend are observed. We discover that a LNAG modification upstream of a short propeller loop hinders G-quadruplex formation. (II) A single substitution of either FG or FANAG into a ‘syn’ position is powerful enough to perturb the (3+1) G-quadruplex. Substitution of either FG or FANAG into any ‘anti’ position is well tolerated in the two G-quadruplex scaffolds. FANAG substitutions to ‘anti’ positions are better tolerated than their FG counterparts. In both scaffolds, FANAG substitutions to the central tetrad layer are observed to be the most stabilizing. The observations reported herein on the effects of LNAG, FG and FANAG modifications on G-quadruplex structure and stability will enable the future design of pharmaceutically relevant oligonucleotides.

Xanthine and 8-oxoguanine in G-quadruplexes: formation of a G·G·X·O tetrad

G-quadruplexes are four-stranded structures built from stacked G-tetrads (G·G·G·G), which are planar cyclical assemblies of four guanine bases interacting through Hoogsteen hydrogen bonds. A G-quadruplex containing a single guanine analog substitution, such as 8-oxoguanine (O) or xanthine (X), would suffer from a loss of a Hoogsteen hydrogen bond within a G-tetrad and/or potential steric hindrance. We show that a proper arrangement of O and X bases can reestablish the hydrogen-bond pattern within a G·G·X·O tetrad. Rational incorporation of G·G·X·O tetrads in a (3+1) G-quadruplex demonstrated a similar folding topology and thermal stability to that of the unmodified G-quadruplex. pH titration conducted on X·O-modified G-quadruplexes indicated a protonation-deprotonation equilibrium of X with a pKa ∼6.7. The solution structure of a G-quadruplex containing a G·G·X·O tetrad was determined, displaying the same folding topology in both the protonated and deprotonated states. A G-quadruplex containing a deprotonated X·O pair was shown to exhibit a more electronegative groove compared to that of the unmodified one. These differences are likely to manifest in the electronic properties of G-quadruplexes and may have important implications for drug targeting and DNA-protein interactions.

Structure and possible function of a G-quadruplex in the long terminal repeat of the proviral HIV-1 genome

The long terminal repeat (LTR) of the proviral human immunodeficiency virus (HIV)-1 genome is integral to virus transcription and host cell infection. The guanine-rich U3 region within the LTR promoter, previously shown to form G-quadruplex structures, represents an attractive target to inhibit HIV transcription and replication. In this work, we report the structure of a biologically relevant G-quadruplex within the LTR promoter region of HIV-1. The guanine-rich sequence designated LTR-IV forms a well-defined structure in physiological cationic solution. The nuclear magnetic resonance (NMR) structure of this sequence reveals a parallel-stranded G-quadruplex containing a single-nucleotide thymine bulge, which participates in a conserved stacking interaction with a neighboring single-nucleotide adenine loop. Transcription analysis in a HIV-1 replication competent cell indicates that the LTR-IV region may act as a modulator of G-quadruplex formation in the LTR promoter. Consequently, the LTR-IV G-quadruplex structure presented within this work could represent a valuable target for the design of HIV therapeutics.

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.

Ball with hair: modular functionalization of highly stable G-quadruplex DNA nano-scaffolds through N2-guanine modification.

Abstract Functionalized nanoparticles have seen valuable applications, particularly in the delivery of therapeutic and diagnostic agents in biological systems. However, the manufacturing of such nano-scale systems with the consistency required for biological application can be challenging, as variation in size and shape have large influences in nanoparticle behavior in vivo. We report on the development of a versatile nano-scaffold based on the modular functionalization of a DNA G-quadruplex. DNA sequences are functionalized in a modular fashion using well-established phosphoramidite chemical synthesis with nucleotides containing modification of the amino (N2) position of the guanine base. In physiological conditions, these sequences fold into well-defined G-quadruplex structures. The resulting DNA nano-scaffolds are thermally stable, consistent in size, and functionalized in a manner that allows for control over the density and relative orientation of functional chemistries on the nano-scaffold surface. Various chemistries including small modifications (N2-methyl-guanine), bulky aromatic modifications (N2-benzyl-guanine), and long chain-like modifications (N2-6-amino-hexyl-guanine) are tested and are found to be generally compatible with G-quadruplex formation. Furthermore, these modifications stabilize the G-quadruplex scaffold by 2.0–13.3 °C per modification in the melting temperature, with concurrent modifications producing extremely stable nano-scaffolds. We demonstrate the potential of this approach by functionalizing nano-scaffolds for use within the biotin–avidin conjugation approach.

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.