Formation of a Ligand‐Assisted Complex of Two RNA Hairpin Loops

The hairpin structure is one of the most common secondary structures in RNA and holds a central position in the stream of RNA folding from a non‐structured RNA to structurally complex and functional ribonucleoproteins. Since the RNA secondary structure is strongly correlated to the function and can be modulated by the binding of small molecules, we have investigated the modulation of RNA folding by a ligand‐assisted formation of loop–loop complexes of two RNA hairpin loops. With a ligand (NCT6), designed based on the ligand binding to the G–G mismatches in double‐stranded DNA, we successfully demonstrated the formation of both inter‐ and intra‐molecular NCT6‐assisted complex of two RNA hairpin loops. NCT6 selectively bound to the two hairpin loops containing (CGG)₃ in the loop region. Native polyacrylamide gel electrophoresis analysis of two doubly‐labeled RNA hairpin loops clearly showed the formation of intermolecular NCT6‐assisted loop–loop complex. Förster resonance energy‐transfer studies of RNA constructs containing two hairpin loops, in which each hairpin was labeled with Alexa488 and Cy3 fluorophores, showed the conformational change of the RNA constructs upon binding of NCT6. These experimental data showed that NCT6 simultaneously bound to two hairpin RNAs at the loop region, and can induce the conformational change of the RNA molecule. These data strongly support that NCT6 functions as molecular glue for two hairpin RNAs.     

Characterization of G‐Quadruplex/Hairpin Transitions of RNAs by 19F NMR Spectroscopy

2′‐O‐[(4‐Trifluoromethyl‐triazol‐1‐yl)methyl] reporter groups have been incorporated into guanosine‐rich RNA models (including a known bistable Qd/Hp RNA and two G‐rich regions of mRNA of human prion protein, PrP) and applied for the ¹⁹F NMR spectroscopic characterization of plausible G‐quadruplex/hairpin (Qd/Hp) transitions in these RNA structures. For the synthesis of the CF₃‐labeled RNAs, phosphoramidite building blocks of 2′‐O‐[(4‐CF₃‐triazol‐1‐yl)methyl] nucleosides (cytidine, adenosine, and guanosine) were prepared and used as an integral part of the standard solid‐phase RNA synthesis. The obtained ¹⁹F NMR spectra supported the usual characterization data (obtained by UV‐ and CD‐melting profiles and by ¹H NMR spectra of the imino regions) and additionally gave more detailed information on the Qd/Hp transitions. The molar fractions of the secondary structural species (Qd, Hp) upon thermal denaturation and under varying ionic conditions could be determined from the intensities and shifts of the ¹⁹F NMR signals. For a well‐behaved Qd/Hp transition, thermodynamic parameters could be extracted.     

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.     

RNA Hairpin Folding in the Crowded Cell

Precise secondary and tertiary structure formation is critically important for the cellular functionality of ribonucleic acids (RNAs). RNA folding studies were mainly conducted in vitro, without the possibility of validating these experiments inside cells. Here, we directly resolve the folding stability of a hairpin‐structured RNA inside live mammalian cells. We find that the stability inside the cell is comparable to that in dilute physiological buffer. On the contrary, the addition of in vitro artificial crowding agents, with the exception of high‐molecular‐weight PEG, leads to a destabilization of the hairpin structure through surface interactions and reduction in water activity. We further show that RNA stability is highly variable within cell populations as well as within subcellular regions of the cytosol and nucleus. We conclude that inside cells the RNA is subject to (localized) stabilizing and destabilizing effects that lead to an on average only marginal modulation compared to diluted buffer.     

Monomer–Dimer Equilibrium for the 5′–5′ Stacking of Propeller‐Type Parallel‐Stranded G‐Quadruplexes: NMR Structural Study

Guanine‐rich sequence motifs, which contain tracts of three consecutive guanines connected by single non‐guanine nucleotides, are abundant in the human genome and can form a robust G‐quadruplex structure with high stability. Herein, by using NMR spectroscopy, we investigate the equilibrium between monomeric and 5′–5′ stacked dimeric propeller‐type G‐quadruplexes that are formed by DNA sequences containing GGGT motifs. We show that the monomer–dimer equilibrium depends on a number of parameters, including the DNA concentration, DNA flanking sequences, the concentration and type of cations, and the temperature. We report on the high‐definition structure of a simple monomeric G‐quadruplex containing three single‐residue loops, which could serve as a reference for propeller‐type G‐quadruplex structures in solution.     

A Quadruplex‐Based,Label‐Free,and Real‐Time Fluorescence Assay for RNase H Activity and Inhibition

We demonstrate a unique quadruplex‐based fluorescence assay for sensitive, facile, real‐time, and label‐free detection of RNase H activity and inhibition by using a G‐quadruplex formation strategy. In our approach, a RNA–DNA substrate was prepared, with the DNA strand designed as a quadruplex‐forming oligomer. Upon cleavage of the RNA strand by RNase H, the released G‐rich DNA strand folds into a quadruplex in the presence of monovalent ions and interacts with a specific G‐quadruplex binder, N‐methyl mesoporphyrin IX (NMM); this gives a dramatic increase in fluorescence and serves as a reporter of the reaction. This novel assay is simple in design, fast in operation, and is more convenient and promising than other methods. It takes less than 30 min to finish and the detection limit is much better or at least comparable to previous reports. No sophisticated experimental techniques or chemical modification for either RNA or DNA are required. The assay can be accomplished by using a common spectrophotometer and obviates possible interference with the kinetic behavior of the catalysts. Our approach offers an ideal system for high‐throughput screening of enzyme inhibitors and demonstrates that the structure of the G‐quadruplex can be used as a functional tool in specific fields in the future.     

Specific Binding of a d‐RNA G‐Quadruplex Structure with an l‐RNA Aptamer

G‐quadruplex (G4) structures are of general importance in chemistry and biology, such as in biosensing, gene regulation, and cancers. Although a large repertoire of G4‐binding tools has been developed, no aptamer has been developed to interact with G4. Moreover, the G4 selectivity of current toolkits is very limited. Herein, we report the first l ‐RNA aptamer that targets a d ‐RNA G‐quadruplex (rG4). Using TERRA rG4 as an example, our results reveal that this l ‐RNA aptamer, Ap3‐7, folds into a unique secondary structure, exhibits high G4 selectivity and effectively interferes with TERRA‐rG4–RHAU53 binding. Our approach and findings open a new door in further developing G4‐specific tools for diverse applications.     

Restricting the conformational heterogeneity of RNA by specific incorporation of 8-bromoguanosine

In an effort to reduce the conformational heterogeneity of RNA, the modified nucleobase 8-bromoguanosine (8BrG) was introduced into oligonucleotides having the hairpin tetraloop motif YNMG (Y = U or C and M = C or A). Purine nucleobases with bromine at position eight are known to preferentially adopt the syn conformation as nucleosides. The hairpin tetraloop motif YNMG was chosen as a model system because it has a syn guanosine at position four of the loop that is essential for thermodynamic stability. Thermodynamic and structural characterization of modified oligonucleotides with the hairpin sequences UUCG, CGCG, and CGAG by UV-melting and NMR spectroscopy revealed that 8BrG substitution has a small effect upon the hairpin conformation, while the duplex conformation is strongly destabilized (DeltaDeltaG degrees 37 approximately +4.7 kcal mol-1), thus inhibiting dimerization. These results support a model in which 8BrG substitution shifts the hairpin-duplex equilibrium constant toward the hairpin conformation by destabilizing the duplex. This methodology should be useful for limiting conformational heterogeneity in large RNAs, with potential applications in structural biology and enzymology.     

A New Tool in Peptide Engineering: A Photoswitchable Stilbene‐type β‐Hairpin Mimetic

Peptide secondary structure mimetics are important tools in medicinal chemistry, as they provide analogues of endogenous peptides with new physicochemical and pharmacological properties. The development, synthesis, photochemical investigation, and conformational analysis of a stilbene‐type β‐hairpin mimetic capable of light‐triggered conformational changes have been achieved. In addition to standard spectroscopic techniques (nuclear Overhauser effects, amide temperature coefficients, circular dichroism spectroscopy), the applicability of self‐diffusion measurements (longitudinal eddy current delay pulsed‐field gradient spin echo (LED‐PGSE) NMR technique) in conformational studies of oligopeptides is demonstrated. The title compound shows photoisomerization of the stilbene chromophore, resulting in a change in solution conformation between an unfolded structure and a folded β‐hairpin.     

Binding of a Telomestatin Derivative Changes the Mechanical Anisotropy of a Human Telomeric G‐Quadruplex

Mechanical anisotropy is an essential property for biomolecules to assume structural and functional roles in mechanobiology. However, there is insufficient information on the mechanical anisotropy of ligand–biomolecule complexes. Herein, we investigated the mechanical property of individual human telomeric G‐quadruplexes bound to telomestatin, using optical tweezers. Stacking of the ligand to the G‐tetrad planes changes the conformation of the G‐quadruplex, which resembles a balloon squeezed in certain directions. Such a squeezed balloon effect strengthens the G‐tetrad planes, but dislocates and weakens the loops in the G‐quadruplex upon ligand binding. These dynamic interactions indicate that the binding between the ligand and G‐quadruplex follows the induced‐fit model. We anticipate that the altered mechanical anisotropy of the ligand–G‐quadruplex complex can add additional level of regulations on the motor enzymes that process DNA or RNA molecules.     

Luminescent Strategies for Label‐Free G‐Quadruplex‐Based Enzyme Activity Sensing

By catalyzing highly specific and tightly controlled chemical reactions, enzymes are essential to maintaining normal cellular physiology. However, aberrant enzymatic activity can be linked to the pathogenesis of various diseases. Therefore, the unusual activity of particular enzymes can represent testable biomarkers for the diagnosis or screening of certain diseases. In recent years, G‐quadruplex‐based platforms have attracted wide attention for the monitoring of enzymatic activities. In this Personal Account, we discuss our group's works on the development of G‐quadruplex‐based sensing system for enzyme activities by using mainly iridium(III) complexes as luminescent label‐free probes. These studies showcase the versatility of the G‐quadruplex for developing assays for a variety of different enzymes.     

An Aggregating Amphiphilic Squaraine: A Light‐up Probe That Discriminates Parallel G‐Quadruplexes

G‐quadruplexes (G4s) are peculiar DNA or RNA tertiary structures that are involved in the regulation of many biological events within mammalian cells, bacteria, and viruses. Although their role as versatile therapeutic targets has been emphasized for 35 years, G4 selectivity over ubiquitous double‐stranded DNA/RNA, as well as G4 differentiation by small molecules, still remains challenging. Here, a new amphiphilic dicyanovinyl‐substituted squaraine, SQgl , is reported to act as an NIR fluorescent light‐up probe discriminating an extensive panel of parallel G4s while it is non‐fluorescent in the aggregated state. The squaraine can form an unconventional sandwich π‐complex binding two quadruplexes, which leads to a strongly fluorescent (Φ _F=0.61) supramolecular architecture. SQgl is highly selective against non‐quadruplex and non‐parallel G4 sequences without altering their topology, as desired for applications in selective in vivo high‐resolution imaging and theranostics.     

Dual Binding of an Antibody and a Small Molecule Increases the Stability of TERRA G‐Quadruplex

In investigating the binding interactions between the human telomeric RNA (TERRA) G‐quadruplex (GQ) and its ligands, it was found that the small molecule carboxypyridostatin (cPDS) and the GQ‐selective antibody BG4 simultaneously bind the TERRA GQ. We previously showed that the overall binding affinity of BG4 for RNA GQs is not significantly affected in the presence of cPDS. However, single‐molecule mechanical unfolding experiments revealed a population (48 %) with substantially increased mechanical and thermodynamic stability. Force‐jump kinetic investigations suggested competitive binding of cPDS and BG4 to the TERRA GQ. Following this, the two bound ligands slowly rearrange, thereby leading to the minor population with increased stability. Given the relevance of G‐quadruplexes in the regulation of biological processes, we anticipate that the unprecedented conformational rearrangement observed in the TERRA‐GQ–ligand complex may inspire new strategies for the selective stabilization of G‐quadruplexes in cells.     

Effects of Six‐Membered Carbohydrate Rings on Structure,Stability, and Kinetics of G‐Quadruplexes

We have evaluated the conformational, thermal, and kinetic properties of d(TGGGGT) analogues with one or five of the ribose nucleotides replaced with the carbohydrate residues hexitol nucleic acid (HNA), cyclohexenyl nucleic acid (CeNA), or altritol nucleic acid (ANA). All of the modified oligonucleotides formed G‐quadruplexes, but substitution with the six‐membered rings resulted in a mixture of G‐quadruplex structures. UV and CD melting analyses showed that the structure formed by d(TGGGGT) modified with HNA was stabilized whereas that modified with CeNA was destabilized, relative to the structure formed by the unmodified oligonucleotide. Substitution at the fourth base of the G‐tract with ANA resulted in a greater stabilization effect than substitution at the first G residue; substitution with five ANA residues resulted in significant stabilization of the G‐quadruplex. A single substitution with CeNA at the first base of the G‐tract or five substitutions with HNA resulted in striking deceleration or acceleration of G‐quadruplex formation, respectively. Our results shed light on the effect of the sugar moiety on the properties of G‐quadruplex structures.     

Template‐Assembled Synthetic G‐Quadruplex (TASQ): A Useful System for Investigating the Interactions of Ligands with Constrained Quadruplex Topologies

A new biomolecular device for investigating the interactions of ligands with constrained DNA quadruplex topologies, using surface plasmon resonance (SPR), is reported. Biomolecular systems containing an intermolecular‐like G‐quadruplex motif 1 (parallel G‐quadruplex conformation), an intramolecular G‐quadruplex 2 , and a duplex DNA 3 have been designed and developed. The method is based on the concept of template‐assembled synthetic G‐quadruplex (TASQ), whereby quadruplex DNA structures are assembled on a template that allows precise control of the parallel G‐quadruplex conformation. Various known G‐quadruplex ligands have been used to investigate the affinities of ligands for intermolecular 1 and intramolecular 2 DNA quadruplexes. As anticipated, ligands displaying a π‐stacking binding mode showed a higher binding affinity for intermolecular‐like G‐quadruplexes 1 , whereas ligands with other binding modes (groove and/or loop binding) showed no significant difference in their binding affinities for the two quadruplexes 1 or 2 . In addition, the present method has also provided information about the selectivity of ligands for G‐quadruplex DNA over the duplex DNA. A numerical parameter, termed the G‐quadruplex binding mode index (G4‐BMI), has been introduced to express the difference in the affinities of ligands for intermolecular G‐quadruplex 1 against intramolecular G‐quadruplex 2 . The G‐quadruplex binding mode index (G4‐BMI) of a ligand is defined as follows: G4‐BMI=K_D^intra/K_D^inter, where K_D^intra is the dissociation constant for intramolecular G‐quadruplex 2 and K_D^inter is the dissociation constant for intermolecular G‐quadruplex 1 . In summary, the present work has demonstrated that the use of parallel‐constrained quadruplex topology provides more precise information about the binding modes of ligands.     

A Multi‐component All‐DNA Biosensing System Controlled by a DNAzyme

We report on a programmable all‐DNA biosensing system that centers on the use of a 4‐way junction (4WJ) to transduce a DNAzyme reaction into an amplified signal output. A target acts as a primary input to activate an RNA‐cleaving DNAzyme, which then cleaves an RNA‐containing DNA substrate that is designed to be a component of a 4WJ. The formation of the 4WJ controls the release of a DNA output that becomes an input to initiate catalytic hairpin assembly (CHA), which produces a second DNA output that controls assembly of a split G‐quadruplex as a fluorescence signal generator. The 4WJ can be configured to produce either a turn‐off or turn‐on switch to control the degree of CHA, allowing target concentration to be determined in a quantitative manner. We demonstrate this approach by creating a sensor for E. coli that could detect as low as 50 E. coli cells mL^?1 within 85 min and offers an amplified bacterial detection method that does not require a protein enzyme.     

Flipping a G‐Tetrad in a Unimolecular Quadruplex Without Affecting Its Global Fold

A unimolecular G‐quadruplex with a hybrid‐type topology and propeller, diagonal, and lateral loops was examined for its ability to undergo structural changes upon specific modifications. Substituting 2′‐deoxy‐2′‐fluoro analogues with a propensity to adopt an anti glycosidic conformation for two or three guanine deoxyribonucleosides in syn positions of the 5′‐terminal G‐tetrad significantly alters the CD spectral signature of the quadruplex. An NMR analysis reveals a polarity switch of the whole tetrad with glycosidic conformational changes detected for all four guanine nucleosides in the modified sequence. As no additional rearrangement of the overall fold occurs, a novel type of G‐quadruplex is formed with guanosines in the four columnar G‐tracts lined up in either an all‐syn or an all‐anti glycosidic conformation.