NMR structure of a G-quadruplex formed by four d(G4C2) repeats: insights into structural polymorphism.

Most frequent genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), is a largely increased number of d(G4C2)n•(G2C4)n repeats located in the non-coding region of C9orf72 gene. Non-canonical structures, including G-quadruplexes, formed within expanded repeats have been proposed to drive repeat expansion and pathogenesis of ALS and FTD. Oligonucleotide d[(G4C2)3G4], which represents the shortest oligonucleotide model of d(G4C2) repeats with the ability to form a unimolecular G-quadruplex, forms two major G-quadruplex structures in addition to several minor species which coexist in solution with K+ ions. Herein, we used solution-state NMR to determine the high-resolution structure of one of the major G-quadruplex species adopted by d[(G4C2)3G4]. Structural characterization of the G-quadruplex named AQU was facilitated by a single substitution of dG with 8Br-dG at position 21 and revealed an antiparallel fold composed of four G-quartets and three lateral C-C loops. The G-quadruplex exhibits high thermal stability and is favored kinetically and under slightly acidic conditions. An unusual structural element distinct from a C-quartet is observed in the structure. Two C•C base pairs are stacked on the nearby G-quartet and are involved in a dynamic equilibrium between symmetric N3-amino and carbonyl-amino geometries and protonated C+•C state.

ALS and FTD linked GGGGCC-repeat containing DNA oligonucleotide folds into two distinct G-quadruplexes.

BACKGROUND: The most common genetic cause of neurological disorders ALS and FTD is a largely increased number of GGGGCC repeats in C9orf72 gene. Non-canonical structures including G-quadruplexes adopted by expanded repeats are hypothesized to be crucial in pathogenesis. Recently, we have shown that structural polymorphism of oligonucleotide d(G4C2)3G4 is reduced by dG to 8Br-dG substitution. High-resolution structure of one of the two major G-quadruplexes adopts antiparallel topology comprising of four G-quartets. Herein, we describe the topology of the second major G-quadruplex structure and influence of folding conditions on relative populations of the two folds. METHODS: Influence of folding conditions was explored by 1H 1D NMR. Determination of topology was achieved by 2D NMR complemented with PAGE and CD. UV melting experiment was used to explore thermal stability of structures. RESULTS: Two structures adopted by oligonucleotide d(G4C2)3GGBrGG denoted AQU and NAN coexist in solution and ratio of their populations is determined by pH and rate of cooling when folding from thermally denatured state in the presence of K+ ions. CONCLUSIONS: AQU is kinetically favored and forms by folding at low pH, while NAN is favored thermodynamically and at neutral pH. AQU and NAN share similar antiparallel topology with four G-quartets and three edgewise loops, however they exhibit distinct structural and dynamic properties. GENERAL SIGNIFICANCE: Novel G-quadruplex topology adds insight into diverse polymorphism of DNA sequences comprising potentially pathological GGGGCC repeat. Relative populations of the two structures and their dependence on folding conditions contribute to understanding of factors that govern G-quadruplex folding. This article is part of a Special Issue entitled "Gquadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

Solution structure of a DNA quadruplex containing ALS and FTD related GGGGCC repeat stabilized by 8-bromodeoxyguanosine substitution.

A prolonged expansion of GGGGCC repeat within non-coding region of C9orf72 gene has been identified as the most common cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), which are devastating neurodegenerative disorders. Formation of unusual secondary structures within expanded GGGGCC repeat, including DNA and RNA G-quadruplexes and R-loops was proposed to drive ALS and FTD pathogenesis. Initial NMR investigation on DNA oligonucleotides with four repeat units as the shortest model with the ability to form an unimolecular G-quadruplex indicated their folding into multiple G-quadruplex structures in the presence of K(+) ions. Single dG to 8Br-dG substitution at position 21 in oligonucleotide d[(G4C2)3G4] and careful optimization of folding conditions enabled formation of mostly a single G-quadruplex species, which enabled determination of a high-resolution structure with NMR. G-quadruplex structure adopted by d[(G4C2)3GG(Br)GG] is composed of four G-quartets, which are connected by three edgewise C-C loops. All four strands adopt antiparallel orientation to one another and have alternating syn-anti progression of glycosidic conformation of guanine residues. One of the cytosines in every loop is stacked upon the G-quartet contributing to a very compact and stable structure.

Vancomycin-Polyguanidino Dendrimer Conjugates Inhibit Growth of Antibiotic-Resistant Gram-Positive and Gram-Negative Bacteria and Eradicate Biofilm-Associated S. aureus.

The global challenge of antibiotic resistance necessitates the introduction of more effective antibiotics. Here we report a potentially general design strategy, exemplified with vancomycin, that improves and expands antibiotic performance. Vancomycin is one of the most important antibiotics in use today for the treatment of Gram-positive infections. However, it fails to eradicate difficult-to-treat biofilm populations. Vancomycin is also ineffective in killing Gram-negative bacteria due to its inability to breach the outer membrane. Inspired by our seminal studies on cell penetrating guanidinium-rich transporters (e.g., octaarginine), we recently introduced vancomycin conjugates that effectively eradicate Gram-positive biofilm bacteria, persister cells and vancomycin-resistant enterococci (with V-r8, vancomycin-octaarginine), and Gram-negative pathogens (with V-R, vancomycin-arginine). Having shown previously that the spatial array (linear versus dendrimeric) of multiple guanidinium groups affects cell permeation, we report here for the first time vancomycin conjugates with dendrimerically displayed guanidinium groups that exhibit superior efficacy and breadth, presenting the best activity of V-r8 and V-R in single broad-spectrum compounds active against ESKAPE pathogens. Mode-of-action studies reveal cell-surface activity and enhanced vancomycin-like killing. The vancomycin-polyguanidino dendrimer conjugates exhibit no acute mammalian cell toxicity or hemolytic activity. Our study introduces a new class of broad-spectrum vancomycin derivatives and a general strategy to improve or expand antibiotic performance through combined mode-of-action and function-oriented design studies.

Conjugation of Vancomycin with a Single Arginine Improves Efficacy against Mycobacteria by More Effective Peptidoglycan Targeting.

Drug resistant bacterial infections have emerged as one of the greatest threats to public health. The discovery and development of new antimicrobials and anti-infective strategies are urgently needed to address this challenge. Vancomycin is one of the most important antibiotics for the treatment of Gram-positive infections. Here, we introduce the vancomycin-arginine conjugate (V-R) as a highly effective antimicrobial against actively growing mycobacteria and difficult-to-treat mycobacterial biofilm populations. Further improvement in efficacy through combination treatment of V-R to inhibit peptidoglycan synthesis and ethambutol to inhibit arabinogalactan synthesis underscores the ability to identify compound synergies to more effectively target the Achilles heel of the cell-wall assembly. Moreover, we introduce mechanistic activity data and a molecular model derived from a d-Ala-d-Ala-bound vancomycin structure that we hypothesize underlies the molecular basis for the antibacterial improvement attributed to the arginine modification that is specific to peptidoglycan chemistry employed by mycobacteria and distinct from Gram-positive pathogens.

CPMAS NMR platform for direct compositional analysis of mycobacterial cell-wall complexes and whole cells.

Tuberculosis and non-tuberculosis mycobacterial infections are rising each year and often result in chronic incurable disease. Important antibiotics target cell-wall biosynthesis, yet some mycobacteria are alarmingly resistant or tolerant to currently available antibiotics. This resistance is often attributed to assumed differences in composition of the complex cell wall of different mycobacterial strains and species. However, due to the highly crosslinked and insoluble nature of mycobacterial cell walls, direct comparative determinations of cell-wall composition pose a challenge to analysis through conventional biochemical analyses. We introduce an approach to directly observe the chemical composition of mycobacterial cell walls using solid-state NMR spectroscopy. 13C CPMAS spectra are provided of individual components (peptidoglycan, arabinogalactan, and mycolic acids) and of in situ cell-wall complexes. We assigned the spectroscopic contributions of each component in the cell-wall spectrum. We uncovered a higher arabinogalactan-to-peptidoglycan ratio in the cell wall of M. abscessus, an organism noted for its antibiotic resistance, relative to M. smegmatis. Furthermore, differentiating influences of different types of cell-wall targeting antibiotics were observed in spectra of antibiotic-treated whole cells. This platform will be of value in evaluating cell-wall composition and antibiotic activity among different mycobacteria and in considering the most effective combination treatment regimens.

Detection of intact vancomycin-arginine as the active antibacterial conjugate in E. coli by whole-cell solid-state NMR.

The introduction of new and improved antibacterial agents based on facile synthetic modifications of existing antibiotics represents a promising strategy to deliver urgently needed antibacterial candidates to treat multi-drug resistant bacterial infections. Using this strategy, vancomycin was transformed into a highly active agent against antibiotic-resistant Gram-negative organisms in vitro and in vivo through the addition of a single arginine to yield vancomycin-arginine (V-R). Here, we report detection of the accumulation of V-R in E. coli by whole-cell solid-state NMR using 15N-labeled V-R. 15N CPMAS NMR revealed that the conjugate remained fully amidated without loss of arginine, demonstrating that intact V-R represents the active antibacterial agent. Furthermore, C{N}REDOR NMR in whole cells with all carbons at natural abundance 13C levels exhibited the sensitivity and selectivity to detect the directly bonded 13C-15N pairs of V-R within E. coli cells. Thus, we also present an effective methodology to directly detect and evaluate active drug agents and their accumulation within bacteria without the need for potentially perturbative cell lysis and analysis protocols.