Biophysical characterization of G-quadruplex forming FMR1 mRNA and of its interactions with different fragile X mental retardation protein isoforms

Fragile X syndrome, the most common form of inherited mental impairment in humans, is caused by the absence of the fragile X mental retardation protein (FMRP) due to a CGG trinucleotide repeat expansion in the 5′-untranslated region (UTR) and subsequent translational silencing of the fragile x mental retardation-1 (FMR1) gene. FMRP, which is proposed to be involved in the translational regulation of specific neuronal messenger RNA (mRNA) targets, contains an arginine-glycine-glycine (RGG) box RNA binding domain that has been shown to bind with high affinity to G-quadruplex forming mRNA structures. FMRP undergoes alternative splicing, and the binding of FMRP to a proposed G-quadruplex structure in the coding region of its mRNA (named FBS) has been proposed to affect the mRNA splicing events at exon 15. In this study, we used biophysical methods to directly demonstrate the folding of FMR1 FBS into a secondary structure that contains two specific G-quadruplexes and analyze its interactions with several FMRP isoforms. Our results show that minor splice isoforms, ISO2 and ISO3, created by the usage of the second and third acceptor sites at exon 15, bind with higher affinity to FBS than FMRP ISO1, which is created by the usage of the first acceptor site. FMRP ISO2 and ISO3 cannot undergo phosphorylation, an FMRP post-translational modification shown to modulate the protein translation regulation. Thus, their expression has to be tightly regulated, and this might be accomplished by a feedback mechanism involving the FMRP interactions with the G-quadruplex structures formed within FMR1 mRNA.     

Recognition of different base tetrads by RHAU (DHX36): X-ray crystal structure of the G4 recognition motif bound to the 3′-end tetrad of a DNA G-quadruplex

G-quadruplexes (G4) are secondary structures of nucleic acids that can form in cells and have diverse biological functions. Several biologically important proteins interact with G-quadruplexes, of which RHAU (or DHX36) – a helicase from the DEAH-box superfamily, was shown to bind and unwind G-quadruplexes efficiently. We report a X-ray co-crystal structure at 1.5 Å resolution of an N-terminal fragment of RHAU bound to an exposed tetrad of a parallel-stranded G-quadruplex. The RHAU peptide folds into an L-shaped α-helix, and binds to a G-quadruplex through π-stacking and electrostatic interactions. X-ray crystal structure of our complex identified key amino acid residues important for G-quadruplex-peptide binding interaction at the 3′-end G•G•G•G tetrad. Together with previous solution and crystal structures of RHAU bound to the 5′-end G•G•G•G and G•G•A•T tetrads, our crystal structure highlights the occurrence of a robust G-quadruplex recognition motif within RHAU that can adapt to different accessible tetrads.     

Design,synthesis, and antiprotozoal evaluation of new 2,4-bis[(substituted-aminomethyl)phenyl]quinoline, 1,3-bis[(substituted-aminomethyl)phenyl]isoquinoline and 2,4-bis[(substituted-aminomethyl)phenyl]quinazoline derivatives

Abstract

A series of new 2,4-bis[(substituted-aminomethyl)phenyl]quinoline, 1,3-bis[(substituted-aminomethyl)phenyl]isoquinoline, and 2,4-bis[(substituted-aminomethyl)phenyl]quinazoline derivatives was designed, synthesised, and evaluated in?vitro against three protozoan parasites (Plasmodium falciparum, Leishmania donovani, and Trypanosoma brucei brucei). Biological results showed antiprotozoal activity with IC₅₀ values in the µM range. In addition, the in?vitro cytotoxicity of these original molecules was assessed with human HepG2 cells. The quinoline 1c was identified as the most potent antimalarial candidate with a ratio of cytotoxic to antiparasitic activities of 97 against the P. falciparum CQ-sensitive strain 3D7. The quinazoline 3h was also identified as the most potent trypanosomal candidate with a selectivity index (SI) of 43 on T. brucei brucei strain. Moreover, as the telomeres of the parasites P. falciparum and Trypanosoma are possible targets of this kind of nitrogen heterocyclic compounds, we have also investigated stabilisation of the Plasmodium and Trypanosoma telomeric G-quadruplexes by our best compounds through FRET melting assays.     

Molecular encapsulation of berberine by a modified β-cyclodextrin and binding of host: guest complex to G-quadruplex DNA

Abstract

The capacity to control quadruplex formation, especially in cancer cells, is captivating and entails a reasonable comprehension of the ligand-G-quadruplex binding. Herein, we report an iminopyrenyl-β-cyclodextrin conjugate interacting with duplex and G-quadrulex DNAs. In addition, the host: guest association of the established G-quadruplex binder, berberine, with the β-cyclodextrin derivative is studied employing 2-D ROESY. NMR, UV-visible, and fluorescence spectroscopic techniques are utilized to explore the β-cyclodextrin conjugate's interaction with the quadruplexes. The Binding constants are accounted for the association of the ligands to each of the DNAs viz., calf thymus DNA (duplex), kit22, telo24, and myc22 (quadruplexes). The modulation of the iminopyrenyl-β-cyclodextrin binding to the DNAs are observed when berberine is loaded in the host molecule. A vivid distinction between the interactions of the ligands with duplex and quadruplex structures is inferred. Berberine-loaded iminopyrenyl-β-cyclodextrin shows a higher affinity for binding to kit22.     

The investigation of the G-quadruplex aptamer selectivity to Pb2+ ion: a joint molecular dynamics simulation and density functional theory study

Abstract

The aptamers with the ability to form a G-quadruplex structure can be stable in the presence of some ions. Hence, study of the interactions between such aptamers and ions can be beneficial to determine the highest selective aptamer toward an ion. In this article, molecular dynamics (MD) simulations and quantum mechanics (QM) calculations have been applied to investigate the selectivity of the T30695 aptamer toward Pb²⁺ in comparison with some ions. The Free Energy Landscape (FEL) analysis indicates that Pb²⁺ has remained inside the aptamer during the MD simulation, while the other ions have left it. The Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) binding energies prove that the conformational stability of the aptamer is the highest in the presence of Pb²⁺. According to the compaction parameters, the greatest compressed ion-aptamer complex, and hence, the highest ion-aptamer interaction have been induced in the presence of Pb²⁺. The contact maps clarify the closer contacts between the nucleotides of the aptamer in the presence of Pb²⁺. The density functional theory (DFT) results show that Pb²⁺ forms the most stable complex with the aptamer, which is consistent with the MD results. The QM calculations reveal that the N-H bonds and the O…H distances are the longest and the shortest, respectively, in the presence of Pb²⁺. The obtained results verify that the strongest hydrogen bonds (HBs), and hence, the most compressed aptamer structure are induced by Pb²⁺. Besides, atoms in molecules (AIM) and natural bond orbital (NBO) analyses confirm the results.

Communicated by Ramaswamy H. Sarma     

Polymerase γ efficiently replicates through many natural template barriers but stalls at the HSP1 quadruplex

Faithful replication of the mitochondrial genome is carried out by a set of key nuclear-encoded proteins. DNA polymerase γ is a core component of the mtDNA replisome and the only replicative DNA polymerase localized to mitochondria. The asynchronous mechanism of mtDNA replication predicts that the replication machinery encounters dsDNA and unique physical barriers such as structured genes, G-quadruplexes, and other obstacles. In vitro experiments here provide evidence that the polymerase γ heterotrimer is well-adapted to efficiently synthesize DNA, despite the presence of many naturally occurring roadblocks. However, we identified a specific G-quadruplex–forming sequence at the heavy-strand promoter (HSP1) that has the potential to cause significant stalling of mtDNA replication. Furthermore, this structured region of DNA corresponds to the break site for a large (3,895 bp) deletion observed in mitochondrial disease patients. The presence of this deletion in humans correlates with UV exposure, and we have found that efficiency of polymerase γ DNA synthesis is reduced after this quadruplex is exposed to UV in vitro.     

Clerocidin-mediated DNA footprinting discriminates among different G-quadruplex conformations and detects tetraplex folding in a duplex environment

Background

G-quadruplexes are polymorphic non-canonical nucleic acid conformations involved both in physiological and pathological processes. Given the high degree of folding heterogeneity and comparable conformational stabilities, different G-quadruplex forms can occur simultaneously, hence rendering the use of basic instrumental methods for structure determination, like X-ray diffraction or NMR, hardly useful. Footprinting techniques represent valuable and relatively rapid alternative to characterize DNA folding. The natural diterpenoid clerocidin is an alkylating agent that specifically reacts at single-stranded DNA regions, with different mechanisms depending on the exposed nucleotide.

Methods

Clerocidin was used to footprint G-quadruplex structures formed by telomeric and oncogene promoter sequences (c-myc, bcl-2, c-kit2), and by the thrombin binding aptamer.

Results

The easy modulability of CL reactivity towards DNA bases permitted to discriminate fully and partially protected sites, highlights stretched portions of the G-quadruplex conformation, and discriminate among topologies adopted by one sequence in different environmental conditions. Importantly, CL displayed the unique property to allow detection of G-quadruplex folding within a duplex context.

Conclusions

CL is a finely performing new tool to unveil G-quadruplex arrangements in DNA sequences under genomically relevant conditions.

General significance

Nucleic acid G-quadruplex structures are an emerging research field because of the recent indication of their involvement in a series of key biological functions, in particular in regulation of proliferation-associated gene expression. The use of clerocidin as footprinting agent to identify G-quadruplex structures under genomically relevant conditions may allow detection of new G-quadruplex-based regulatory regions.     

Coexistence of G-quadruplex and duplex domains within the secondary structure of 31-mer DNA thrombin-binding aptamer

A number of thrombin-binding DNA aptamers have been developed during recent years. So far the structure of just a single one, 15-mer thrombin-binding aptamer (15TBA), has been solved as G-quadruplex. Structures of others, showing variable anticoagulation activities, are still not known yet. In this paper, we applied the circular dichroism and UV spectroscopy to characterize the temperature unfolding and conformational features of 31-mer thrombin-binding aptamer (31TBA), whose sequence has a potential to form G-quadruplex and duplex domains. Both structural domains were monitored independently in 31TBA and in several control oligonucleotides unable to form either the duplex region or the G-quadruplex region. The major findings are as follows: (1) both duplex and G-quadruplex domains coexist in intramolecular structure of 31TBA, (2) the formation of duplex domain does not change the fold of G-quadruplex, which is very similar to that of 15TBA, and (3) the whole 31TBA structure disrupts if either of two domains is not formed: the absence of duplex structure in 31TBA abolishes G-quadruplex, and vice versa, the lack of G-quadruplex folding results in disallowing the duplex domain.     

基于核酸适配体的生物分析新方法研究进展

核酸适配体是从随机文库中采用SELEX技术筛选所得的单链短链寡核苷酸片段(通常为15-80个ss DNA或ss RNA)。其能够折叠形成独特稳定的三维结构,通过静电相互作用,氢键,范德华力,碱堆叠或多种作用力组合特异性地与多种靶标结合。适配体因具有构象变化能力而被用作生物分析中的理想识别配体。目前,基于适配体的生物分析新方法得到广泛研究,并用于蛋白多肽类药物分析、疾病标志物诊断、外泌体检测、循环肿瘤细胞检测和病毒检测等方面。本文综述了核酸适配体用于生物分析方法开发的最新进展,比较和讨论不同分析方法,并对基于适配体的生物分析新方法提出了设想和展望,为开发新的生物分析方法和检测技术提供了思路和借鉴。