Stability Of Quadruplexes Research Articles

Defined folding pattern of poly(rG) supports inherent ability to encode biological information.

The RNA World hypothesis posits that RNA can represent a primitive life form by reproducing itself and demonstrating catalytic activity. However, this hypothesis is incapable of addressing several major origin-of-life (OoL) questions. A recently described paradox-free alternative OoL hypothesis, the Quadruplex (G4) World, is based on the ability of poly(dG) to fold into a stable architecture with an unambiguous folding pattern using G-tetrads as building elements. Because of the folding pattern of three G-tetrads and single-G loops, dG15 is programmable and has the capability to encode biological information. Here, we address two open questions of the G4 World hypothesis: (1) Does RNA follow the same folding pattern as DNA? (2) How do stable quadruplexes evolve into the present-day system of information transfer, which is based on Watson-Crick base pair complementarity? To address these questions, we systematically studied the thermodynamic and optical properties of both DNA and RNA G15- and G3T (GGGTGGGTGGGTGGG)-derived sequences. Our study revealed that similar to DNA sequences, RNAs adopt quadruplexes with only three G-tetrads. Thus, both poly(dG) and poly(rG) possess inherent ability to fold into 3D quadruplex architecture with strictly defined folding pattern. The study also revealed that despite high stability of both DNA and RNA quadruplexes, they are vulnerable to single-nucleotide substitutions, which drop the thermal stability by ~40°C and can facilitate introduction of the complementarity principle into the G4 World.

Two rhenium compounds with benzimidazole ligands: synthesis and DNA interactions.

Two rhenium compounds: cis-tetrachlorotetrabenzimidazoldirhenium(iii) chloride - I and tetrabenzimidazoldioxorhenium(v) - II have been synthesized and characterized. X-ray data are presented for the new complex II. I and II show strong emission that has been used to investigate their interaction with several non-canonical DNA structures. Both compounds have a quenching effect on the fluorescence intensity upon addition of the investigated oligonucleotides; I was more selective for binding G4-than II. Association constant values obtained for I and II with G-quadruplexes reached 106 M-1, which suggests a strong interaction between both complexes and these sequences. FRET-melting assays show that I and II have a rather high level of stabilization of ckit1 and ckit2 quadruplexes. I is toxic against macrophages RAW267.7 only in high concentrations, while complex II shows no toxicity against these cells. I and II accumulate inside cells in different degrees. Molecular dynamic simulation studies have provided insights into the binding modes of II with ckit1 and ckit2 G-quadruplexes. The results obtained show the DNA binding activity of the rhenium complexes and their ability to be players in the anti-cancer fight since they can bind to non-canonical DNA forms in oncogene promoters, accumulate in some cancer cells, and influence the cancer cells microenvironment.

Long non-coding intergenic RNA, LINC00273 induces cancer metastasis and stemness via miRNA sponging in triple negative breast cancer

LncRNAs and miRNAs, being the master regulators of gene expression, are crucial functional mediators in cancer. Our study unveils the critical regulatory role of the metastatic long non-coding RNA LINC00273 as the master regulator of oncogenes involved in cancer metastasis, stemness, and chemoresistance via its miRNA sponging mechanism. M2 (a salt of bis-Schiff base) mediated G quadruplex (G4) stabilization at the LINC00273 gene promoter remarkably inhibits LINC00273 transcription. Therefore, low-level LINC00273 transcripts are unable to efficiently sponge the miRNAs, which subsequently become available to bind and downregulate their target oncogenes. We have observed significantly different global transcriptomic scenarios in LINC00273 upregulated and downregulated circumstances in MDA-MB-231 triple-negative breast cancer model. Additionally, we have found the G4 sequence in the LINC00273 RNA to play a critical role in miRNA sequestration. miRNAs (miR-6789-5p, miR200b, miR-125b-5p, miR-4268, miR3978) have base pairing complementarity within the G4 region of LINC00273 RNA and the 3′-UTR (untranslated region) of MAPK12, TGF-β1, and SIX-1 transcripts. We have reported TGF-β1, SIX-1, and MAPK12 to be the direct downstream targets of LINC00273. The correlation between abnormal expression of lncRNA LINC00273 and TNBC aggressiveness strongly evidenced in our study shall accelerate the development of lncRNA-based anti-metastatic therapeutics.

Stability of Human Telomeric G-Quadruplexes Complexed with Photosensitive Ligands and Irradiated with Visible Light.

Guanine-rich DNA sequences can fold into non-canonical nucleic acid structures called G-quadruplexes (G4s). These nanostructures have strong implications in many fields, from medical science to bottom-up nanotechnologies. As a result, ligands interacting with G4s have attracted great attention as candidates in medical therapies, molecular probe applications, and biosensing. In recent years, the use of G4-ligand complexes as photopharmacological targets has shown significant promise for developing novel therapeutic strategies and nanodevices. Here, we studied the possibility of manipulating the secondary structure of a human telomeric G4 sequence through the interaction with two photosensitive ligands, DTE and TMPyP4, whose response to visible light is different. The effect of these two ligands on G4 thermal unfolding was also considered, revealing the occurrence of peculiar multi-step melting pathways and the different attitudes of the two molecules on the quadruplex stabilization.

Biophysical Characterization of Nucleolin Domains Crucial for Interaction with Telomeric and TERRA G-Quadruplexes.

Nucleolin (NCL) is a well-characterized nucleic acid-binding protein; it binds to various canonical and noncanonical structures including single- and double-stranded DNA and RNA, hairpin, loops, and G-quadruplex structures. G-quadruplex structures are majorly formed in promoter, telomeric, and untranslated regions of the genome and affect the process of replication, transcription, and translation. One of the widely studied G-quadruplex-forming regions are telomeres, as these are sites for the recruitment for various proteins providing stability or having an effect on the telomerase activity. NCL is known to bind to both single- and double-stranded telomeric regions and its transcribed telomeric RNA (TERRA). In our study, we show that the 21nt G-quadruplex-forming region of telomeric DNA and TERRA RNA binds to NCL and the domains RRM1234 destabilize the telomeric G-quadruplex structure. We also show the preferential binding of the RNA G-quadruplex over the DNA G-quadruplex by two NCL domains, RRM3 and RRM4. Our findings provide insights into the binding preferences of RRM domains toward G-quadruplex structures and their subsequent effect on the quadruplex stability.

G4Boost: a machine learning-based tool for quadruplex identification and stability prediction

BackgroundG-quadruplexes (G4s), formed within guanine-rich nucleic acids, are secondary structures involved in important biological processes. Although every G4 motif has the potential to form a stable G4 structure, not every G4 motif would, and accurate energy-based methods are needed to assess their structural stability. Here, we present a decision tree-based prediction tool, G4Boost, to identify G4 motifs and predict their secondary structure folding probability and thermodynamic stability based on their sequences, nucleotide compositions, and estimated structural topologies.ResultsG4Boost predicted the quadruplex folding state with an accuracy greater then 93% and an F1-score of 0.96, and the folding energy with an RMSE of 4.28 and R2 of 0.95 only by the means of sequence intrinsic feature. G4Boost was successfully applied and validated to predict the stability of experimentally-determined G4 structures, including for plants and humans.ConclusionG4Boost outperformed the three machine-learning based prediction tools, DeepG4, Quadron, and G4RNA Screener, in terms of both accuracy and F1-score, and can be highly useful for G4 prediction to understand gene regulation across species including plants and humans.

Cluster dirhenium(III) cis-dicarboxylates with α-amino acids ligands as mighty selective G4s binders

The synthesis of four dirhenium(III) cis-dicarboxylates with the α-amino acids residues Asp (I), Glu (II), Phe (III) and Tyr (IV) is presented. The G–quadruplex stabilization potential was evaluated by fluorescence resonance energy transfer – melting analysis. All derivatives show specific binding to c-kit1 quadruplex, while II and IV have also strong stabilization activity to HTelo21 quadruplex. At the same time, the compounds do not show any stabilization activity for ds26 DNA, which suggests unique mechanisms of molecular DNA recognition for these complexes.

HIV-1 genomic RNA U3 region forms a stable quadruplex-hairpin structure

The U3 promoter region of the HIV-1 long terminal repeat (LTR) has previously been shown to fold into a series of dynamic G-quadruplex structures. Among the G-quadruplexes identified in the LTR sequence, LTR-III was shown to be the most stable in vitro. NMR studies of this 28-nucleotide (nt) DNA revealed a unique quadruplex−hairpin structure. Whether the hairpin forms in RNA element is unknown and the role of the hairpin in the structure and stability of quadruplexes has not been characterized. Here, we used optical and thermodynamic studies to address these questions. The wild-type LTR-III RNA formed a monomolecular quadruplex with a parallel topology using only propeller loops, including the hairpin loop element. By comparison to the WT and variant RNAs, LTR-III DNA structures were more heterogeneous and less stable. Increased stability of the RNA suggests that the RNA quadruplex-hairpin structure may be a more attractive therapeutic target than the analogous DNA element.

Assessing G4-Binding Ligands In Vitro and in Cellulo Using Dimeric Carbocyanine Dye Displacement Assay.

G-quadruplexes (G4) are the most actively studied non-canonical secondary structures formed by contiguous repeats of guanines in DNA or RNA strands. Small molecule mediated targeting of G-quadruplexes has emerged as an attractive tool for visualization and stabilization of these structures inside the cell. Limited number of DNA and RNA G4-selective assays have been reported for primary ligand screening. A combination of fluorescence spectroscopy, AFM, CD, PAGE, and confocal microscopy have been used to assess a dimeric carbocyanine dye B6,5 for screening G4-binding ligands in vitro and in cellulo. The dye B6,5 interacts with physiologically relevant DNA and RNA G4 structures, resulting in fluorescence enhancement of the molecule as an in vitro readout for G4 selectivity. Interaction of the dye with G4 is accompanied by quadruplex stabilization that extends its use in primary screening of G4 specific ligands. The molecule is cell permeable and enables visualization of quadruplex dominated cellular regions of nucleoli using confocal microscopy. The dye is displaced by quarfloxin in live cells. The dye B6,5 shows remarkable duplex to quadruplex selectivity in vitro along with ligand-like stabilization of DNA G4 structures. Cell permeability and response to RNA G4 structures project the dye with interesting theranostic potential. Our results validate that B6,5 can serve the dual purpose of visualization of DNA and RNA G4 structures and screening of G4 specific ligands, and adds to the limited number of probes with such potential.

Ligands of G-quadruplex nucleic acids

The review summarizes advances in the development of low-molecular-weight ligands for G-quadruplex (G4) nucleic acid structures published in the past five years. Main chemical classes of these ligands (polyaromatic compounds, macrocyclic systems, metal complexes and heterocyclic derivatives) are considered. The chemical structures of the ligands and their influence on the stabilization of quadruplexes are analyzed. Future prospects of design of G4 ligands and their applications are discussed.The bibliography includes 188 references.

Human DNA Telomeres in Presence of Oxidative Lesions: The Crucial Role of Electrostatic Interactions on the Stability of Guanine Quadruplexes.

By using all atom molecular dynamics simulations, we studied the behavior of human DNA telomere sequences in guanine quadruplex (G4) conformation and in the presence of oxidative lesions, namely abasic sites. In particular, we evidenced that while removing one guanine base induces a significant alteration and destabilization of the involved leaflet, human telomere oligomers tend, in most cases, to maintain at least a partial quadruplex structure, eventually by replacing the empty site with undamaged guanines of different leaflets. This study shows that (i) the disruption of the quadruplex leaflets induces the release of at least one of the potassium cations embedded in the quadruplex channel and that (ii) the electrostatic interactions of the DNA sequence with the aforementioned cations are fundamental to the maintenance of the global quadruplex structure.

Stability Factors of the Parallel Quadruplexes: DNA Versus RNA.

One of the most stable quadruplexes is formed by the G3T sequence (GGGTGGGTGGGTGGG) that folds into a parallel quadruplex with three G-tetrads and chain-reversal T-loops. For example, in 1 mM K+, it unfolds at 75 °C and at physiological conditions, it unfolds above 100 °C. The RNA analogue, ggguggguggguggg (g3u), which employs exactly same folding topology, demonstrates even higher thermal stability. Here, we performed melting experiments of G3T, g3u, and more than 30 chimeric constructs (G3T with RNA nucleotides at certain positions). Although the g3u quadruplex is 13 °C more stable than G3T, majority of G → g (DNA-for-RNA) substitutions destabilize G3T. Only three G → g and loop T → u substitutions stabilize the structure. However, stabilization effects of these six substitutions overcome destabilization of other nine G → g, resulting in higher stability of all-RNA g3u. The present work clearly indicates that the stacking interactions are more favorable in parallel DNA quadruplexes, whereas the chain-reversal loops play an important role in higher stability of RNA quadruplexes. In addition, we have shown that the 5'-end of RNA quadruplexes represents a more favorable target for stacking interactions than the 3'-end. Based on the current study, rational design of the quadruplexes for particular biotechnological applications and drugs, targeting the quadruplexes, may be envisaged.

Formation of G-quadruplex structure in supercoiled DNA under molecularly crowded conditions.

G-quadruplex is a secondary structure of nucleic acids that plays crucial roles in many significant biological processes. Potential G-quadruplex-forming sequences exist widely in various regions of the genome such as telomeres and gene promoters. In spite of the fact that G-quadruplex can be readily assembled from a single-stranded segment of DNA, its formation from duplex DNA is very difficult under physiological conditions because Watson–Crick interactions in guanine rich segments need to be weakened first. It is demonstrated in our studies that intrastrand G-quadruplex generated from a perfectly matched guanine-rich duplex in a circular DNA as a result of significant quadruplex stabilization and duplex destabilization created by the combined actions of negative DNA supercoiling and molecular crowding conditions.

Nonconventional C-H···F Hydrogen Bonds Support a Tetrad Flip in Modified G-Quadruplexes.

A G-quadruplex adopting a (3 + 1)-hybrid structure was substituted at its 5'-tetrad by 2'-deoxy-2'-fluoro-arabinoguanosine (FaraG) analogs. Incorporation of anti-favoring FaraG at syn-positions of the 5'-outer tetrad induced a reversal of the tetrad polarity without noticeably compromising the quadruplex stability. This conformational change is shown to be promoted by nonconventional C-H···F hydrogen bonds acting within the anti-FaraG residues.

DNA-Damaging Anticancer Drugs - A Perspective for DNA Repair- Oriented Therapy.

DNA-damaging drugs in cancer present two main problems: therapeutic resistance and side effects and both can associate with DNA repair, which can be targeted in cancer therapy. Bleomycin (BLM) induces complex DNA damages, including strand breaks, base loss and 3'-phosphoglycolate (3'PG) residues repaired by several pathways, but 3'PGs must be processed to the 3'-OH ends, usually by tyrosyl-DNA phosphodiesterase 1 (Tdp1). Therefore, targeting Tdp1 can improve anticancer therapy with BLM. Mitomycin C (MMC) produces a variety of adducts with DNA, including inter-strand cross-links (ICLs) and Xeroderma pigmentosum (XP) proteins, including XPG, XPE and XPF can be crucial for the initial stage of ICL repair, so they can be targeted by inhibitors to increase toxicity of MMC in cancer cells. Although these proteins are essential for nucleotide excision repair (NER), their decreased activity may not be fatal in normal cells as almost all NER substrates can be repaired by other pathways. Four-stranded DNA, resulted mainly from guanine quadruplexes (G-4s), are highly overexpressed at the end of telomeres, where they can inhibit telomerase, hence stabilization G-4s at the telomeres ends can hamper proliferation of cancer cells. Quadruplexes are also found in the promoters of genes important for cancer and are resolved by DNA helicases, which can be targeted in cancer along with stabilization of quadruplexes. As cancer cells often have defects in DNA repair pathway(s), they can be subjected by synthetic lethality, with the most promising results with poly(ADP-ribose) polymerase 1 (PARP-1) and DNA-dependent protein kinase, catalytic subunit (DNA-PKCS).

Clustered abasic lesions profoundly change the structure and stability of human telomeric G-quadruplexes.

Ionizing radiation produces clustered damage to DNA which is difficult to repair and thus more harmful than single lesions. Clustered lesions have only been investigated in dsDNA models. Introducing the term ‘clustered damage to G-quadruplexes’ we report here on the structural effects of multiple tetrahydrofuranyl abasic sites replacing loop adenines (A/AP) and tetrad guanines (G/AP) in quadruplexes formed by the human telomere d[AG3(TTAG3)3] (htel-22) and d[TAG3(TTAG3)3TT] (htel-25) in K+ solutions. Single to triple A/APs increased the population of parallel strands in their structures by stabilizing propeller type loops, shifting the antiparallel htel-22 into hybrid or parallel quadruplexes. In htel-25, the G/APs inhibited the formation of parallel strands and these adopted antiparallel topologies. Clustered G/AP and A/APs reduced the thermal stability of the wild-type htel-25. Depending on position, A/APs diminished or intensified the damaging effect of the G/APs. Taken together, clustered lesions can disrupt the topology and stability of the htel quadruplexes and restrict their conformational space. These in vitro results suggest that formation of clustered lesions in the chromosome capping structure can result in the unfolding of existing G-quadruplexes which can lead to telomere shortening.

The effect of l-thymidine, acyclic thymine and 8-bromoguanine on the stability of model G-quadruplex structures

BackgroundGuanine-rich oligonucleotides are capable of forming tetrahelical structures known as G-quadruplexes with interesting biological properties. We have investigated the effects of site-specific substitution in the loops and in the tetrads model G-quadruplexes using thymine glycol nucleic acid (GNA) units, l-thymidine and 8-Br-2′-deoxyguanosine. MethodsModified oligonucleotides were chemically synthesized and spectroscopic techniques were used to determine the relative stability of the modified G-quadruplex. The double 8-BrdG-modified quadruplexes were further characterized by Nuclear Magnetic Resonance. Binding to thrombin of selected quadruplex was analyzed by gel electrophoresis retention assay. ResultsThe most interesting results were found with a 8-bromoG substitution that had the larger stabilization of the quadruplex. NMR studies indicate a tight relationship between the loops and the tetrads to accommodate 8-bromoG modifications within the TBA. ConclusionsThe substitutions of loop positions with GNA T affect the TBA stability except for single modification in T7 position. Single l-thymidine substitutions produced destabilization of TBA. Larger changes on quadruplex stability are observed with the use of 8-bromoG finding a single substitution with the highest thermal stabilization found in thrombin binding aptamers modified at the guanine residues and having good affinity for thrombin. Double 8-BrdG modification in anti positions of different tetrads produce a conformational flip from syn to anti conformation of 8-Br-dG to favor loop-tetrad interaction and preserve the overall TBA stability. General significanceModified guanine-rich oligonucleotides are valuable tools for the search for G-quadruplex structures with higher thermal stability and may provide compounds with interesting protein-nucleic acid binding properties. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

Sr(2+) induces unusually stable d(GGGTGGGTGGGTGGG) quadruplex dimers.

Guanine-rich sequences are able to form quadruplexes consisting of G-quartet structural units. Quadruplexes play an important role in the regulation of gene expression and have therapeutic and biotechnological potential. The HIV-1 integrase inhibitor, (GGGT)4 , and its variants demonstrate unusually high thermal stability. This property has been exploited in the use of quadruplex formation to drive various endergonic reactions of nucleic acids such as isothermal DNA amplification. Quadruplex stability is mainly determined by cations, which specifically bind into the inner core of the structure. In the present work, we report a systematic study of a variant of the HIV-1 integrase inhibitor, GGGTGGGTGGGTGGG (G3T), in the presence of alkali and alkaline-earth cations. We show that Sr(2+) -G3T is characterized by the highest thermal stability and that quadruplex formation requires only one Sr(2+) ion that binds with low micromolar affinity. These concentrations are sufficient to drive robust isothermal quadruplex priming DNA amplification reaction. The Sr(2+) -quadruplexes are also able to form unusually stable dimers through end-to-end stacking. The multimerization can be induced by a combination of quadruplex forming cations (i.e., K(+) or Sr(2+) ) and non-specific Mg(2+) .

The role of alkali metal cations in the stabilization of guanine quadruplexes: why K(+) is the best.

The alkali metal ion affinity of guanine quadruplexes has been studied using dispersion-corrected density functional theory (DFT-D). We have done computational investigations in aqueous solution that mimics artificial supramolecular conditions where guanine bases assemble into stacked quartets as well as biological environments in which telomeric quadruplexes are formed. In both cases, an alkali metal cation is needed to assist self-assembly. Our quantum chemical computations on these supramolecular systems are able to reproduce the experimental order of affinity of the guanine quadruplexes for the cations Li(+), Na(+), K(+), Rb(+), and Cs(+). The strongest binding is computed between the potassium cation and the quadruplex as it occurs in nature. The desolvation and the size of alkali metal cations are thought to be responsible for the order of affinity. Until now, the relative importance of these two factors has remained unclear and debated. By assessing the quantum chemical 'size' of the cation, determining the amount of deformation of the quadruplex needed to accommodate the cation and through the energy decomposition analysis (EDA) of the interaction energy between the cation and the guanines, we reveal that the desolvation and size of the alkali metal cation are both almost equally responsible for the order of affinity.

Extended molecular dynamics of a c-kit promoter quadruplex.

The 22-mer c-kit promoter sequence folds into a parallel-stranded quadruplex with a unique structure, which has been elucidated by crystallographic and NMR methods and shows a high degree of structural conservation. We have carried out a series of extended (up to 10 μs long, ∼50 μs in total) molecular dynamics simulations to explore conformational stability and loop dynamics of this quadruplex. Unfolding no-salt simulations are consistent with a multi-pathway model of quadruplex folding and identify the single-nucleotide propeller loops as the most fragile part of the quadruplex. Thus, formation of propeller loops represents a peculiar atomistic aspect of quadruplex folding. Unbiased simulations reveal μs-scale transitions in the loops, which emphasizes the need for extended simulations in studies of quadruplex loops. We identify ion binding in the loops which may contribute to quadruplex stability. The long lateral-propeller loop is internally very stable but extensively fluctuates as a rigid entity. It creates a size-adaptable cleft between the loop and the stem, which can facilitate ligand binding. The stability gain by forming the internal network of GA base pairs and stacks of this loop may be dictating which of the many possible quadruplex topologies is observed in the ground state by this promoter quadruplex.