Apurinic/Apyrimidinic Endodeoxyribonuclease 1 modulates RNA G-quadruplex folding of miR-92b and controls its expression in cancer cells.

In the last decade, several novel functions of the mammalian Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APE1) have been discovered, going far beyond its canonical function as DNA repair enzyme and unveiling its potential roles in cancer development. Indeed, it was shown to be involved in DNA G-quadruplex biology and RNA metabolism, most importantly in the miRNA maturation pathway and the decay of oxidized or abasic miRNAs during oxidative stress conditions. In recent years, several noncanonical pathways of miRNA biogenesis have emerged, with a specific focus on guanosine-rich precursors that can form RNA G-quadruplex (rG4) structures. Here, we show that several miRNA precursors, dysregulated upon APE1 depletion, contain an rG4 motif and that their corresponding target genes are up-regulated after APE1 depletion. We also demonstrate, both by in vitro assays and by using different cancer cell lines, that APE1 can modulate the folding of an rG4 structure contained in pre-miR-92b, with a mechanism strictly dependent on lysine residues present in its N-terminal disordered region. Furthermore, APE1 cellular depletion alters the maturation process of miR-92b, mainly affecting the shuttling between the nucleus and cytosol. Bioinformatic analysis of APE1-regulated rG4-containing miRNAs supports the relevance of our findings in cancer biology. Specifically, these miRNAs exhibit high prognostic significance in lung, cervical, and liver tumors, as suggested by their involvement in several cancer-related pathways.

Structural Perspective into the Interaction of an Oncogenesis-Relevant pre-miRNA G-Quadruplex Ligand Carrier with the Protein Nucleolin.

The structural determinants of the interaction of the G-quadruplex (G4) motif found in precursor miRNA 149 (rG4) with the acridine orange derivative C8 , a G4 ligand stabilizer possessing anticancer activity, and the protein nucleolin (overexpressed in cancer cells) were investigated by Nuclear Magnetic Resonance (NMR) spectroscopy. For the rG4/C8 complex, the results revealed a strong stabilizing interaction between the aromatic core and the iodinated ring of the C8 ligand with the rG4 structure. The NMR study revealed also different interaction patterns between nucleolin and rG4 and nucleolin and rG4/C8 complex. In the absence of the ligand, rG4 establishes interactions with polar residues of the protein while for the rG4/C8 complex, these contacts are mainly established with amino acids that have hydrophobic side chains. However, nucleolin chemical shift perturbation studies in the presence of rG4 or rG4/C8 reveal the same location between domains 1 and 2 of the protein, which suggests that the rG4 and rG4/C8 complex bind in this region. This puzzling structural study opens a new framework to study rG4/ligand/nucleolin complexes that might impact the biogenesis of miRNA 149.

The beginning and the end: flanking nucleotides induce a parallel G-quadruplex topology.

Genomic sequences susceptible to form G-quadruplexes (G4s) are always flanked by other nucleotides, but G4 formation in vitro is generally studied with short synthetic DNA or RNA oligonucleotides, for which bases adjacent to the G4 core are often omitted. Herein, we systematically studied the effects of flanking nucleotides on structural polymorphism of 371 different oligodeoxynucleotides that adopt intramolecular G4 structures. We found out that the addition of nucleotides favors the formation of a parallel fold, defined as the 'flanking effect' in this work. This 'flanking effect' was more pronounced when nucleotides were added at the 5'-end, and depended on loop arrangement. NMR experiments and molecular dynamics simulations revealed that flanking sequences at the 5'-end abolish a strong syn-specific hydrogen bond commonly found in non-parallel conformations, thus favoring a parallel topology. These analyses pave a new way for more accurate prediction of DNA G4 folding in a physiological context.

Structure of two G-quadruplexes in equilibrium in the KRAS promoter.

KRAS is one of the most mutated oncogenes and still considered an undruggable target. An alternative strategy would consist in targeting its gene rather than the protein, specifically the formation of G-quadruplexes (G4) in its promoter. G4 are secondary structures implicated in biological processes, which can be formed among G-rich DNA (or RNA) sequences. Here we have studied the major conformations of the commonly known KRAS 32R, or simply 32R, a 32 residue sequence within the KRAS Nuclease Hypersensitive Element (NHE) region. We have determined the structure of the two major stable conformers that 32R can adopt and which display slow equilibrium (>ms) with each other. By using different biophysical methods, we found that the nucleotides G9, G25, G28 and G32 are particularly implicated in the exchange between these two conformations. We also showed that a triad at the 3' end further stabilizes one of the G4 conformations, while the second conformer remains more flexible and less stable.

The Promoter Region of the Proto-Oncogene MST1R Contains the Main Features of G-Quadruplexes Formation.

MST1R (RON) is a receptor of the MET tyrosine kinase receptor family involved in several cancers such as pancreas, breast, ovary, colon, and stomach. Some studies have shown that overexpression of MST1R increases the migratory and invasive properties of cancer cells. The promoter region of the oncogene MST1R is enriched in guanine residues that can potentially form G-quadruplexes (G4s), as it was observed in other oncogenic promoters such as KRAS and c-MYC. There is abundant literature that links the presence of G4s in promoter regions of oncogenes to diverse gene regulation processes that are not well understood. In this work, we have studied the reverse and forward sequence of MST1R promoter region using the G4Hunter software and performed biophysical studies to characterize the best scored sequences.

Nanoaggregate-forming lipid-conjugated AS1411 aptamer as a promising tumor-targeted delivery system of anticancer agents in vitro.

Nanoparticles offer targeted delivery of drugs with minimal toxicity to surrounding healthy tissue and have great potential in the management of human papillomavirus (HPV)-related diseases. We synthesized lipid-modified AS1411 aptamers capable of forming nanoaggregates in solution containing Mg2+. The nanoaggregates presented suitable properties for pharmaceutical applications such as small size (100 nm), negative charge, and drug release. The nanoaggregates were loaded with acridine orange derivative C8 for its specific delivery into cervical cancer cell lines and HPV-positive tissue biopsies. This improved inhibition of HeLa proliferation and cell uptake without significantly affecting healthy cells. Finally, the nanoaggregates were incorporated in a gel formulation with promising tissue retention properties aiming at developing a local delivery strategy of the nanoaggregates in the female genital tract. Collectively, these findings suggest that the nanoformulation protocol has great potential for the delivery of both anticancer and antiviral agents, becoming a novel modality for cervical cancer management.

Phthalocyanines for G-quadruplex aptamers binding.

The G-quadruplex (G4)-forming sequence within the AS1411 derivatives with alternative nucleobases and backbones can improve the chemical and biological properties of AS1411. Zn(II) phthalocyanine (ZnPc) derivatives have potential as high-affinity G4 ligands because they have similar size and shape to the G-quartets. The interactions of four Zn(II) phthalocyanines with the G4 AS1411 aptamer and its derivatives were determined by biophysical techniques, molecular docking and gel electrophoresis. Cell viability assay was carried out to evaluate the antiproliferative effects of Zn(II) phthalocyanines and complexes. CD experiments showed structural changes after addition of ZnPc 4, consistent with multiple binding modes and conformations shown by NMR and gel electrophoresis. CD melting confirmed that ZnPc 2 and ZnPc 4, both containing eight positive charges, are able to stabilize the AT11 G4 structure (ΔTm > 30 °C and 18.5 °C, respectively). Molecular docking studies of ZnPc 3 and ZnPc 4 suggested a preferential binding to the 3'- and 5'-end, respectively, of the AT11 G4. ZnPc 3 and its AT11 and AT11-L0 complexes revealed pronounced cytotoxic effect against cervical cancer cells and no cytotoxicity to normal human cells. Zn(II) phthalocyanines provide the basis for the development of effective therapeutic agents as G4 ligands.

Design and Structure Determination of a Composite Zinc Finger Containing a Nonpeptide Foldamer Helical Domain.

A number of foldamer backbones have been described as useful mimics of protein secondary structure elements, enabling for example the design of synthetic oligomers with the ability to engage specific protein surfaces. Synthetic folded backbones can also be used to create artificial proteins in which a folded peptide segment (e.g., an α-helix, a loop) is replaced by its unnatural counterpart, with the expectation that the resulting molecule would maintain its ability to fold while manifesting new exploitable features. The similarities in screw sense, pitch, and polarity between peptide α-helices and oligourea 2.5-helices suggest that a tertiary structure could be retained when swapping the two backbones in a protein sequence. In the present work, we move a step toward the creation of such composite proteins by replacing the 10-residue long original α-helical segment in the Cys2His2 zinc finger 3 of transcription factor Egr1 (also known as Zif268) by an oligourea sequence bearing two appropriately spaced imidazole side chains for zinc coordination. We show by spectroscopic techniques and mass spectrometry analysis under native conditions that the ability of the peptide/oligourea hybrid to coordinate the zinc ion is not affected by the foldamer replacement. Moreover, detailed NMR analysis provides evidence that the engineered zinc finger motif adopts a folded structure in which the native ß-sheet arrangement of the peptide region and global arrangement of DNA-binding side chains are preserved. Titration in the presence of the Egr1 target DNA sequence supports binding to GC bases as reported for the wild-type zinc finger.

High-resolution three-dimensional NMR structure of the KRAS proto-oncogene promoter reveals key features of a G-quadruplex involved in transcriptional regulation.

Non-canonical base pairing within guanine-rich DNA and RNA sequences can produce G-quartets, whose stacking leads to the formation of a G-quadruplex (G4). G4s can coexist with canonical duplex DNA in the human genome and have been suggested to suppress gene transcription, and much attention has therefore focused on studying G4s in promotor regions of disease-related genes. For example, the human KRAS proto-oncogene contains a nuclease-hypersensitive element located upstream of the major transcription start site. The KRAS nuclease-hypersensitive element (NHE) region contains a G-rich element (22RT; 5'-AGGGCGGTGTGGGAATAGGGAA-3') and encompasses a Myc-associated zinc finger-binding site that regulates KRAS transcription. The NEH region therefore has been proposed as a target for new drugs that control KRAS transcription, which requires detailed knowledge of the NHE structure. In this study, we report a high-resolution NMR structure of the G-rich element within the KRAS NHE. We found that the G-rich element forms a parallel structure with three G-quartets connected by a four-nucleotide loop and two short one-nucleotide double-chain reversal loops. In addition, a thymine bulge is found between G8 and G9. The loops of different lengths and the presence of a bulge between the G-quartets are structural elements that potentially can be targeted by small chemical ligands that would further stabilize the structure and interfere or block transcriptional regulators such as Myc-associated zinc finger from accessing their binding sites on the KRAS promoter. In conclusion, our work suggests a possible new route for the development of anticancer agents that could suppress KRAS expression.

Phenanthroline polyazamacrocycles as G-quadruplex DNA binders.

Targeting quadruplex DNA structures with small molecules is a promising strategy for anti-cancer drug design. Four phenanthroline polyazamacrocycles were studied for their binding affinity, thermal stabilization, inhibitory effect on the activity of helicase towards human telomeric 22AG and oncogene promoter c-MYC G-quadruplexes (G4s), and their ability to inhibit Taq polymerase-mediated DNA extension. The fluorescence resonance energy transfer (FRET) melting assay indicates that the melting temperature increases (ΔTm values) of c-MYC and 22AG G4s are 17.2 and 20.3 °C, respectively, for the ligand [32]phen2N4 followed by [16]phenN4 (11.3 and 15.0 °C, for c-MYC and 22AG, respectively). Competitive FRET assays show that [32]phen2N4 and [16]phenN4 exhibit G4 selectivity over duplex DNA. Different G4s were compared; no considerable selectivity of the ligands for a specific G4 was found. Circular dichroism (CD) confirms the formation of G4 structures and the melting experiments show that [16]phenN4 and [32]phen2N4 are the most stabilizing ligands with a ΔTm of 19.3 °C and 15.1 °C, respectively, at 5 molar equivalents for the c-MYC G4. The fluorescent intercalator displacement (FID) assay also demonstrates that ligand [32]phen2N4 furnishes very low DC50 values (0.87-1.24 µM), indicating high stabilization of c-MYC and 22AG G4s. These results suggest that the hexyl chain in these compounds plays an important role in regulating the stabilization of these G4s. Binding constants, determined by fluorescence titrations, indicate a moderate ligand-G4 binding with KSV between 105 and 106 M-1 in which [16]phenN4 has a slightly higher apparent binding constant for telomeric 22AG G4 than that for the c-MYC G4. The ligand's ability to inhibit Taq polymerase confirms the biological activity of [16]phenN4 and [32]phen2N4 against the c-MYC G4. In addition, ligands [32]phen2N4 and [16]phenN4 affect the unwinding activity of Pif1 in the presence of DNA systems harboring c-MYC and telomeric G4 motifs.

Unexpected Position-Dependent Effects of Ribose G-Quartets in G-Quadruplexes.

To understand the role of ribose G-quartets and how they affect the properties of G-quadruplex structures, we studied three systems in which one, two, three, or four deoxyribose G-quartets were substituted with ribose G-quartets. These systems were a parallel DNA intramolecular G-quadruplex, d(TTGGGTGGGTTGGGTGGGTT), and two tetramolecular G-quadruplexes, d(TGGGT) and d(TGGGGT). Thermal denaturation experiments revealed that ribose G-quartets have position-dependent and cumulative effects on G-quadruplex stability. An unexpected destabilization was observed when rG quartets were presented at the 5'-end of the G stack. This observation challenges the general belief that RNA residues stabilize G-quadruplexes. Furthermore, in contrast to past proposals, hydration is not the main factor determining the stability of our RNA/DNA chimeric G-quadruplexes. Interestingly, the presence of rG residues in a central G-quartet facilitated the formation of additional tetramolecular G-quadruplex topologies showing positive circular dichroism signals at 295 nm. 2D NMR analysis of the tetramolecular TGgGGT (lowercase letter indicates ribose) indicates that Gs in the 5'-most G-quartet adopt the syn conformation. These analyses highlight several new aspects of the role of ribose G-quartets on G-quadruplex structure and stability, and demonstrate that the positions of ribose residues are critical for tuning G-quadruplex properties.

NMR based model of human telomeric repeat G-quadruplex in complex with 2,4,6-triarylpyridine family ligand.

G-quadruplexes (G4) are one of the several different forms of non-canonical DNA structures that can occur in our genome. Their existence is thought to be implicated in important biological functions such as positive and negative transcription regulation or telomeric extension. The human telomeric sequence G4 formed by repetitive nucleotide sequences (T2AG3) at each chromosome end is an important example of intramolecular G4. Knowing the atomic details for different families of ligands targeting G-quadruplex structures hypothetically found in the telomeric repeat it is an important step for rational drug design. Especially if the aim is to prevent or interfere with telomerase extending the 3' end of telomeres. In this study, we report the structure of the complex formed between the telomeric repeat sequence (d[AG3(T2AG3)3]) intramolecular G-quadruplex and the 2,4,6-Triarylpyridine compound. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

Phenanthroline-bis-oxazole ligands for binding and stabilization of G-quadruplexes.

BACKGROUND: G-quadruplexes (G4) are found at important genome regions such as telomere ends and oncogene promoters. One prominent strategy to explore the therapeutic potential of G4 is stabilized it with specific ligands. METHODS: We report the synthesis of new phenanthroline, phenyl and quinoline acyclic bisoxazole compounds in order to explore and evaluate the targeting to c-myc and human telomeric repeat 22AG G4 using FRET-melting, CD-melting, NMR, fluorescence titrations and FID assays. RESULTS: The design strategy has led to potent compounds (Phen-1 and Phen-2) that discriminate different G4 structures (human telomeric sequences and c-myc promoter) and selectively stabilize G4 over duplex DNA. CD studies show that Phen-2 binds and induces antiparallel topologies in 22AG quadruplex and also binds c-myc promotor, increasing their Tm in about 12°C and 30°C respectively. In contrast, Phen-1 induces parallel topologies in 22AG and c-myc, with a moderate stabilization of 4°C for both sequences. Consistent with a CD melting study, Phen-2 binds strongly (K=106 to 107M-1) to c-myc and 22AG quadruplexes. CONCLUSIONS: Phen-1 and Phen-2 discriminated among various quadruplex topologies and exhibited high selectivity for quadruplexes over duplexes. Phen-2 retains antiparallel topologies for quadruplex 22AG and does not induce conformational changes on the parallel c-myc quadruplex although Phen-1 favors the parallel topology. NMR studies also showed that the Phen-2 binds to the c-myc quadruplex via end stacking. GENERAL SIGNIFICANCE: Overall, the results suggest the importance of Phen-2 as a scaffold for the fine-tuning with substituents in order to enhance binding and stabilization to G4 structures. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

Interaction of a peptide derived from C-terminus of human TRPA1 channel with model membranes mimicking the inner leaflet of the plasma membrane.

The transient receptor potential ankyrin 1 channel (TRPA1) belongs to the TRP cation channel superfamily that responds to a panoply of stimuli such as changes in temperature, calcium levels, reactive oxygen and nitrogen species and lipid mediators among others. The TRP superfamily has been implicated in diverse pathological states including neurodegenerative disorders, kidney diseases, inflammation, pain and cancer. The intracellular C-terminus is an important regulator of TRP channel activity. Studies with this and other TRP superfamily members have shown that the C-terminus association with lipid bilayer alters channel sensitivity and activation, especially interactions occurring through basic residues. Nevertheless, it is not yet clear how this process takes place and which regions in the C-terminus would be responsible for such membrane recognition. With that in mind, herein the first putative membrane interacting region of the C-terminus of human TRPA1, (corresponding to a 29 residue peptide, IAEVQKHASLKRIAMQVELHTSLEKKLPL) named H1 due to its potential helical character was chosen for studies of membrane interaction. The affinity of H1 to lipid membranes, H1 structural changes occurring upon this interaction as well as effects of this interaction in lipid organization and integrity were investigated using a biophysical approach. Lipid models systems composed of zwitterionic and anionic lipids, namely those present in the lipid membrane inner leaflet, where H1 is prone to interact, where used. The study reveals a strong interaction and affinity of H1 as well as peptide structuration especially with membranes containing anionic lipids. Moreover, the interactions and peptide structure adoption are headgroup specific.

Orienting tetramolecular G-quadruplex formation: the quest for the elusive RNA antiparallel quadruplex.

DNA and RNA G-quadruplexes (G4) are unusual nucleic acid structures involved in a number of key biological processes. RNA G-quadruplexes are less studied although recent evidence demonstrates that they are biologically relevant. Compared to DNA quadruplexes, RNA G4 are generally more stable and less polymorphic. Duplexes and quadruplexes may be combined to obtain pure tetrameric species. Here, we investigated whether classical antiparallel duplexes can drive the formation of antiparallel tetramolecular quadruplexes. This concept was first successfully applied to DNA G4. In contrast, RNA G4 were found to be much more unwilling to adopt the forced antiparallel orientation, highlighting that the reason RNA adopts a different structure must not be sought in the loops but in the G-stem structure itself. RNA antiparallel G4 formation is likely to be restricted to a very small set of peculiar sequences, in which other structural features overcome the formidable intrinsic barrier preventing its formation.

Solid-state 2H NMR relaxation illuminates functional dynamics of retinal cofactor in membrane activation of rhodopsin.

Rhodopsin is a canonical member of the family of G protein-coupled receptors, which transmit signals across cellular membranes and are linked to many drug interventions in humans. Here we show that solid-state (2)H NMR relaxation allows investigation of light-induced changes in local ps-ns time scale motions of retinal bound to rhodopsin. Site-specific (2)H labels were introduced into methyl groups of the retinal ligand that are essential to the activation process. We conducted solid-state (2)H NMR relaxation (spin-lattice, T(1Z), and quadrupolar-order, T(1Q)) experiments in the dark, Meta I, and Meta II states of the photoreceptor. Surprisingly, we find the retinylidene methyl groups exhibit site-specific differences in dynamics that change upon light excitation--even more striking, the C9-methyl group is a dynamical hotspot that corresponds to a crucial functional hotspot of rhodopsin. Following 11-cis to trans isomerization, the (2)H NMR data suggest the ß-ionone ring remains in its hydrophobic binding pocket in all three states of the protein. We propose a multiscale activation mechanism with a complex energy landscape, whereby the photonic energy is directed against the E2 loop by the C13-methyl group, and toward helices H3 and H5 by the C5-methyl of the ß-ionone ring. Changes in retinal structure and dynamics initiate activating fluctuations of transmembrane helices H5 and H6 in the Meta I-Meta II equilibrium of rhodopsin. Our proposals challenge the Standard Model whereby a single light-activated receptor conformation yields the visual response--rather an ensemble of substates is present, due to the entropy gain produced by photolysis of the inhibitory retinal lock.

Tuning molecular interactions in lipid-oligonucleotides assemblies via locked nucleic acid (LNA)-based lipids.

Hybrid nucleotide-lipids containing locked nucleic acid (LNA) show enhanced hybridization properties with complementary single strand RNAs compared to DNA lipid analogues. The LNA adenosine lipid features unique binding properties with a high binding affinity for poly-uridine and the entropically driven formation of a stable complex (K(d) ≈ 43 nM). Enhanced hybridization properties of LNA-based lipids should be applicable for the development of oligonucleotide (ON) delivery systems or as small molecule binders to RNA for novel therapeutic strategies.

The interaction of antipsychotic drugs with lipids and subsequent lipid reorganization investigated using biophysical methods.

The interaction of antipsychotic drugs (AP) with lipids and the subsequent lipid reorganization on model membranes was assessed using a combination of several complementary biophysical approaches (calorimetry, plasmon resonance, fluorescence microscopy, X-ray diffraction and molecular modeling). The effect of haloperidol (HAL), risperidone (RIS), and 9-OH-risperidone (9-OH-RIS) was examined on single lipid and mixtures comprising lipids of biological origin. All APs interact with lipids and induced membrane reorganization. APs showed higher affinity for sphingomyelin than for phosphatidylcholine. Cholesterol increased AP affinity for the lipid bilayer and led to the following AP ranking regarding affinity and structural changes: RIS >9-OH-RIS >HAL. Liquid-ordered domain formation and bilayer thickness were differentially altered by AP addition. Docking calculations helped understanding the observed differences between the APs and offer a representation of their conformation in the lipid bilayer. Present results indicate that AP drugs may change membrane compartmentalization which could differentially modulate the signaling cascade of the dopamine D2 receptor for which APs are ligands.

Insights into internal dynamics of 6-phosphogluconolactonase from Trypanosoma brucei studied by nuclear magnetic resonance and molecular dynamics.

Nuclear magnetic resonance is used to investigate the backbone dynamics in 6-phosphogluconolactonase from Trypanosoma brucei (Tb6PGL) with (holo-) and without (apo-) 6-phosphogluconic acid as ligand. Relaxation data were analyzed using the model-free approach and reduced spectral density mapping. Comparison of predictions, based on 77 ns molecular dynamics simulations, with the observed relaxation rates gives insight into dynamical properties of the protein and their alteration on ligand binding. Data indicate dynamics changes in the vicinity of the binding site. More interesting is the presence of perturbations located in remote regions of this well-structured globular protein in which no large-amplitude motions are involved. This suggests that delocalized changes in dynamics that occur upon binding could be a general feature of protein-target interactions.

Nucleolin: a binding partner of G-quadruplex structures.

Nucleolin protein is involved in a plethora of cellular pathways across the nucleolus, nucleus, and cytoplasm. The association of its RNA-binding domain (RBD) and its RGG (arginine-glycine-glycine-rich) domain allows it to interact with G-quadruplex structures in nucleic acids. We highlight evidence that the nucleolin/G-quadruplex partnership is of extensive relevance to neurodegenerative disease, cancer, and viral infections.