A Twisting Electronic Nanoswitch Made of DNA

Single‐stranded DNAs and RNAs that are rich in the nucleobase guanine form four‐stranded G‐quadruplexes, which are held together by hydrogen‐bonded guanine quartets. In aqueous solution, both DNA duplexes and G‐quadruplexes are modest conductors of electrical charge. A tight, topologically constrained DNA construct called twDNA is now reported, in which a core of four guanine‐rich single strands structurally and electronically links together four DNA double helices. The addition and removal of K⁺ or Sr²⁺ cations promote alternative conformers of twDNA, which have strikingly distinct electronic properties. Unlike DNA mechano‐electronic switches that require large conformational changes, twDNA requires only modest twisting/untwisting structural attenuations to achieve electronic switching.     

Tracking the Dynamic Folding and Unfolding of RNA G‐Quadruplexes in Live Cells

Because of the absence of methods for tracking RNA G‐quadruplex dynamics, especially the folding and unfolding of this attractive structure in live cells, understanding of the biological roles of RNA G‐quadruplexes is so far limited. Herein, we report a new red‐emitting fluorescent probe, QUMA‐1 , for the selective, continuous, and real‐time visualization of RNA G‐quadruplexes in live cells. The applications of QUMA‐1 in several previously intractable applications, including live‐cell imaging of the dynamic folding, unfolding, and movement of RNA G‐quadruplexes and the visualization of the unwinding of RNA G‐quadruplexes by RNA helicase have been demonstrated. Notably, our real‐time results revealed the complexity of the dynamics of RNA G‐quadruplexes in live cells. We anticipate that the further application of QUMA‐1 in combination with appropriate biological and imaging methods to explore the dynamics of RNA G‐quadruplexes will uncover more information about the biological roles of RNA G‐quadruplexes.     

Carbohydrate–DNA Interactions at G‐Quadruplexes: Folding and Stability Changes by Attaching Sugars at the 5′‐End

Quadruplex DNA structures are attracting an enormous interest in many areas of chemistry, ranging from chemical biology, supramolecular chemistry to nanoscience. We have prepared carbohydrate–DNA conjugates containing the oligonucleotide sequences of G‐quadruplexes (thrombin binding aptamer (TBA) and human telomere (TEL)), measured their thermal stability and studied their structure in solution by using NMR and molecular dynamics. The solution structure of a fucose–TBA conjugate shows stacking interactions between the carbohydrate and the DNA G‐tetrad in addition to hydrogen bonding and hydrophobic contacts. We have also shown that attaching carbohydrates at the 5′‐end of a quadruplex telomeric sequence can alter its folding topology. These results suggest the possibility of modulating the folding of the G‐quadruplex by linking carbohydrates and have clear implications in molecular recognition and the design of new G‐quadruplex ligands.     

Photocrosslinking of G‐Quadruplex‐Forming Sequences found in Human Promoters

While is it well known that human telomeric DNA sequences can adopt G‐quadruplex structures, some promoters sequences have also been found to form G‐quadruplexes, and over 40% of promoters contain putative G‐quadruplex‐forming sequences. Because UV light has been shown to crosslink human telomeric G‐quadruplexes by cyclobutane pyrimidine dimer (CPD) formation between T's on adjacent loops, UV light might also be able to photocrosslink G‐quadruplexes in promoters. To investigate this possibility, 15 potentially UV‐crosslinkable G‐quadruplex‐forming sequences found in a search of human DNA promoters were UVB irradiated in vitro, and three were confirmed to have formed nonadjacent CPDs by mass spectrometry. In addition to nonadjacent T=T CPDs found in human telomeric DNA, a nonadjacent T=U CPD was discovered that presumably arose from deamination of a nonadjacent T=C CPD. Analysis of the three sequences by circular dichroism, melting temperature analysis and chemical footprinting confirmed the presence of G‐quadruplexes that could explain the formation of the nonadjacent CPDs. The formation of nonadjacent CPDs from the sequences in vitro suggests that they might be useful probes for the presence of non‐B DNA structures, such as G‐quadruplexes, in vivo, and if they were to form in vivo, might also have significant biological consequences.     

Fluorescent Bioprobes: Structural Matching in the Docking Processes of Aggregation‐Induced Emission Fluorogens on DNA Surfaces

Whereas most conventional DNA probes are flat disklike aromatic molecules, we explored the possibility of developing quadruplex sensors with nonplanar conformations, in particular, the propeller‐shaped tetraphenylethene (TPE) salts with aggregation‐induced emission (AIE) characteristics. 1,1,2,2‐Tetrakis[4‐(2‐triethylammonioethoxy)phenyl]ethene tetrabromide (TPE‐ 1 ) was found to show a specific affinity to a particular quadruplex structure formed by a human telomeric DNA strand in the presence of K⁺ ions, as indicated by the enhanced and bathochromically shifted emission of the AIE fluorogen. Steady‐state and time‐resolved spectral analyses revealed that the specific binding stems from a structural matching between the AIE fluorogen and the DNA strand in the folding process. Computational modeling suggests that the AIE molecule docks on the grooves of the quadruplex surface with the aid of electrostatic attraction. The binding preference of TPE‐ 1 enables it to serve as a bioprobe for direct monitoring of cation‐driven conformational transitions between the quadruplexes of various conformations, a job unachievable by the traditional G‐quadruplex biosensors. Methyl thiazolyl tetrazolium (MTT) assays reveal that TPE‐ 1 is cytocompatible, posing no toxicity to living cells.     

DNA Triplexes‐Guided Assembly of G‐Quadruplexes for Constructing Label‐free Fluorescent Logic Gates

Assembly of G‐quadruplexes guided by DNA triplexes in a controlled manner is achieved for the first time. The folding of triplex sequences in acidic conditions brings two separated guanine‐rich sequences together and subsequently a G‐quadruplex structure is formed in the presence of K⁺. Based on this novel platform, label‐free fluorescent logic gates, such as AND, INHIBIT, and NOR, are constructed with ions as input and the fluorescence of a G‐quadruplex‐specific fluorescent probe NMM as output.     

Involvement of Long‐Lived Intermediate States in the Complex Folding Pathway of the Human Telomeric G‐Quadruplex

The energy landscapes of human telomeric G‐quadruplexes are complex, and their folding pathways have remained largely unexplored. By using real‐time NMR spectroscopy, we investigated the K⁺‐induced folding of the human telomeric DNA sequence 5′‐TTGGG(TTAGGG)₃A‐3′. Three long‐lived states were detected during folding: a major conformation (hybrid‐1), a previously structurally uncharacterized minor conformation (hybrid‐2), and a partially unfolded state. The minor hybrid‐2 conformation is formed faster than the more stable hybrid‐1 conformation. Equilibration of the two states is slow and proceeds via a partially unfolded intermediate state, which can be described as an ensemble of hairpin‐like structures.     

Direct and Single‐Molecule Visualization of the Solution‐State Structures of G‐Hairpin and G‐Triplex Intermediates

We present the direct and single‐molecule visualization of the in‐pathway intermediates of the G‐quadruplex folding that have been inaccessible by any experimental method employed to date. Using DNA origami as a novel tool for the structural control and high‐speed atomic force microscopy (HS‐AFM) for direct visualization, we captured images of the unprecedented solution‐state structures of a tetramolecular antiparallel and (3+1)‐type G‐quadruplex intermediates, such as G‐hairpin and G‐triplex, with nanometer precision. No such structural information was reported previously with any direct or indirect technique, solution or solid‐state, single‐molecule or bulk studies, and at any resolution. Based on our results, we proposed a folding mechanism of these G‐quadruplexes.     

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.     

Binding Behaviors for Different Types of DNA G‐Quadruplexes: Enantiomers of [Ru(bpy)2(L)]2+ (L=dppz,dppz‐idzo)

Polymorphic DNA G‐quadruplex recognition has attracted great interest in recent years. The strong binding affinity and potential enantioselectivity of chiral [Ru(bpy)₂(L)]²⁺ (L=dipyrido[3,2‐a:2′,3′‐c]phenazine, dppz‐10,11‐imidazolone; bpy=2,2′‐bipyridine) prompted this investigation as to whether the two enantiomers, Δ and Λ, can show different effects on diverse structures with a range of parallel, antiparallel and mixed parallel/antiparallel G‐quadruplexes. These studies provide a striking example of chiral‐selective recognition of DNA G‐quadruplexes. As for antiparallel (tel‐Na⁺) basket G‐quadruplex, the Λ enantiomers bind stronger than the Δ enantiomers. Moreover, the behavior reported here for both enantiomers stands in sharp contrast to B‐DNA binding. The chiral selectivity toward mixed parallel/antiparallel (tel‐K⁺) G‐quadruplex of both compounds is weak. Different loop arrangements can change chiral complex selectivity for both antiparallel and mixed parallel/antiparallel G‐quadruplex. Whereas both Δ and Λ isomers bind to parallel G‐quadruplexes with comparable affinity, no appreciable stereoselective G‐quadruplex binding of the isomers was observed. In addition, different binding stoichiometries and binding modes for Δ and Λ enantiomers were confirmed. The results presented here indicate that chiral selective G‐quadruplex binding is not only related to G‐quadruplex topology, but also to the sequence and the loop constitution.     

Towards the Development of Structure‐Selective G‐Quadruplex‐Binding Indolo[3,2‐b]quinolines

The interaction of phenyl‐substituted indolo[3,2‐b]quinolines with DNA G‐quadruplexes of different topology were studied by using a combination of spectroscopic and calorimetric methodologies. N5‐Methylated indoloquinoline derivatives (^MePIQ) with an aminoalkyl side chain exhibit high affinities for the parallel‐stranded MYC quadruplex and a (3+1)‐hybrid structure combined with an excellent discrimination against the antiparallel thrombin‐binding aptamer (TBA) and the human telomeric (HT) quadruplexes. Dissociation constants for the binding of the ligand to the MYC quadruplex are in the submicromolar range, being below the corresponding dissociation constants for the antiparallel‐stranded quadruplexes by about one order of magnitude. Competition experiments with double‐helical DNA reveal the impact of indoloquinoline structural features on the selectivity for the parallel quadruplex relative to duplex DNA. Based on a calorimetric analysis binding to MYC is shown to be equally driven by favorable enthalpic and entropic contributions with no significant impact on the type of cation present.     

Targeting Non‐B‐Form DNA in Living Cells

Under certain conditions, repetitive DNA motifs have the potential to adopt non‐B‐form DNA structures, such as hairpins, triplexes, Z‐DNA, quadruplexes, and i‐motifs. Some non‐B‐form DNAs have been proposed to cause mutations and, consequently, participate in several biologically important processes, including regulation, evolution, and human disease. Advancement in the knowledge of specific interactions between molecules and non‐B‐form DNAs at the molecular level in living cells is important for understanding their biological functions. In this review, we describe the latest studies on molecules that target non‐B‐form DNAs in vivo, with a focus on Z‐DNA, G‐quadruplexes, triplexes, i‐motifs, and hairpins.     

Solution NMR Structure of a Ligand/Hybrid‐2‐G‐Quadruplex Complex Reveals Rearrangements that Affect Ligand Binding

Telomeric G‐quadruplexes have recently emerged as drug targets in cancer research. Herein, we present the first NMR structure of a telomeric DNA G‐quadruplex that adopts the biologically relevant hybrid‐2 conformation in a ligand‐bound state. We solved the complex with a metalorganic gold(III) ligand that stabilizes G‐quadruplexes. Analysis of the free and bound structures reveals structural changes in the capping region of the G‐quadruplex. The ligand is sandwiched between one terminal G‐tetrad and a flanking nucleotide. This complex structure involves a major structural rearrangement compared to the free G‐quadruplex structure as observed for other G‐quadruplexes in different conformations, invalidating simple docking approaches to ligand–G‐quadruplex structure determination.     

Reversible pH Switch of Two‐Quartet G‐Quadruplexes Formed by Human Telomere

A four‐repeat human telomere DNA sequence without the 3′‐end guanine, d[TAGGG(TTAGGG)₂TTAGG] (htel1‐ΔG23) has been found to adopt two distinct two G‐quartet antiparallel basket‐type G‐quadruplexes, TD and KDH⁺ in presence of KCl. NMR, CD, and UV spectroscopy have demonstrated that topology of KDH⁺ form is distinctive with unique protonated T18?A20⁺?G5 base triple and other capping structural elements that provide novel insight into structural polymorphism and heterogeneity of G‐quadruplexes in general. Specific stacking interactions amongst two G‐quartets flanking base triples and base pairs in TD and KDH⁺ forms are reflected in 10 K higher thermal stability of KDH⁺. Populations of TD and KDH⁺ forms are controlled by pH. The (de)protonation of A20 is the key for pH driven structural transformation of htel1‐ΔG23. Reversibility offers possibilities for its utilization as a conformational switch within different compartments of living cell enabling specific ligand and protein interactions.     

Trianguleniums as Optical Probes for G‐Quadruplexes: A Photophysical,Electrochemical, and Computational Study

Nucleic acids can adopt non‐duplex topologies, such as G‐quadruplexes in vitro. Yet it has been challenging to establish their existence and function in vivo due to a lack of suitable tools. Recently, we identified the triangulenium compound DAOTA‐M2 as a unique fluorescence probe for such studies. This probe's emission lifetime is highly dependent on the topology of the DNA it interacts with opening up the possibility of carrying out live‐cell imaging studies. Herein, we describe the origin of its fluorescence selectivity for G‐quadruplexes. Cyclic voltammetry predicts that the appended morpholino groups can act as intra‐ molecular photo‐induced electron transfer (PET) quenchers. Photophysical studies show that a delicate balance between this effect and inter‐molecular PET with nucleobases is key to the overall fluorescence enhancement observed upon nucleic acid binding. We utilised computational modelling to demonstrate a conformational dependence of intra‐molecular PET. Finally, we performed orthogonal studies with a triangulenium compound, in which the morpholino groups were removed, and demonstrated that this change inverts triangulenium fluorescence selectivity from G‐quadruplex to duplex DNA, thus highlighting the importance of fine tuning the molecular structure not only for target affinity, but also for fluorescence response.     

An Unprecedented Knot‐like G‐Quadruplex Peripheral Motif

A knot‐like G‐quadruplex peripheral structure is formed by a 7‐nt DNA sequence DL7 (TGTTGGT), in which six out of its seven nucleobases participate in compact base‐pairing interactions. Here, the solution NMR structure of a 24‐nt DNA oligonucleotide containing the DL7 sequence shows the interaction between a two‐layer anti‐parallel G‐quadruplex core and the peripheral knot‐like structure, including the construction of two sharp turns in the DNA backbone. The formation of this novel structural element highlights the intricate properties of single‐stranded DNA folding in presence of G‐quadruplex‐forming motifs. We demonstrated the compatibility of the DL7 knot‐like structure with various G‐quadruplexes, which could have implications in drug design and DNA engineering.     

Pyridyl‐Substituted Corrole Isomers: Synthesis and their Regulation to G‐quadruplex Structures

G‐quadruplex DNA plays an important role in the potential therapeutic target for the design and development of anticancer drugs. As various G‐quadruplex sequences in the promoter regions or telomeres can form different secondary structural modes and display a diversity of biology functions, variant G‐quadruplex interactive agents may be necessary to cure different disease by differentiating variant types of G‐quadruplexes. We synthesize five cationic methylpyridylium corroles and compare the interactions of corroles with different types of G‐quadruplexes such as cmyc, htelo, and bcl2 by using surface plasmon resonance. Because of the importance of human telomere G‐quadruplex DNA, we focus on the biological properties of the interactions between human telomere G‐quadruplex DNA and corrole isomers using CD, T_m, PCR‐stop (PCR= polymerase chain reaction), and polymerase‐stop assay, which demonstrate the excellent ability of the corrole to induce and stabilize the G‐quadruplex. This study provides the first experimental insight into how selectivity might be achieved for different G‐quadruplexes by a single group of methylpyridylium corrole isomers that may be optimized for potential selective cancer therapy.