Chemoselective Modification of Vinyl DNA by Triazolinediones

A new method for the post‐synthetic modification of nucleic acids was developed that involves mixing a phenyl triazolinedione (PTAD) derivative with DNA containing a vinyl nucleobase. The resulting reactions proceeded through step‐wise mechanisms, giving either a formal [4+2] cycloaddition product, or, depending on the context of nucleobase, PTAD addition along with solvent trapping to give a secondary alcohol in water. Catalyst‐free addition between PTAD and the terminal alkene of 5‐vinyl‐2′‐deoxyuridine (VdU) was exceptionally fast, with a second‐order rate constant of 2×10³ m ⁻¹ s⁻¹. PTAD derivatives selectively reacted with VdU‐containing oligonucleotides in a conformation‐selective manner, with higher yields observed for G‐quadruplex versus duplex DNA. These results demonstrate a new strategy for copper‐free bioconjugation of DNA that can potentially be used to probe nucleic acid conformations in cells.     

Subcellular Duplex DNA and G‐Quadruplex Interaction Profiling of a Hexagonal PtII Metallacycle

Metal‐driven self‐assembly afforded a multitude of fascinating supramolecular coordination complexes (SCCs) with applications as catalysts, host–guest, and stimuli‐responsive systems. However, the interest in the biological applications of SCCs is only starting to emerge and thorough characterization of their behavior in biological milieus is still lacking. Herein, we report on the synthesis and detailed in‐cell tracking of a Pt₂L₂ metallacycle. We show that our hexagonal supramolecule accumulates in cancer cell nuclei, exerting a distinctive blue fluorescence staining of chromatin resistant to UV photobleaching selectively in nucleolar G4‐rich regions. SCC co‐localizes with epitopes of the quadruplex‐specific antibody BG4 and replaces other well‐known G4 stabilizers. Moreover, the photophysical changes accompanying the metallacycle binding to G4s in solution (fluorescence quenching, absorption enhancement) also take place intracellularly, allowing its subcellular interaction tracking.     

A Molecular Secret Sharing Scheme

A method for implementing a secret sharing scheme at the molecular level is presented. By creating molecular code generators that are self‐assembled from several molecular components, we established a means for distributing distinct code‐activating elements among several participants. In this way, an authorization code can only be generated when all the participants are present, which ensures that highly secured systems cannot be operated by unauthorized individuals or disloyal users. Additional layers of protection result from the ability to program the security code by replacing one or several molecular components and by subjecting the system to distinct chemical inputs.     

Copper–Alkyne Complexation Responsible for the Nucleolar Localization of Quadruplex Nucleic Acid Drugs Labeled by Click Reactions

G‐Quadruplex(es) (G4) are noncanonical nucleic‐acid structures found in guanine‐rich sequences. They can be targeted with small molecules (G4 ligands) acting as reporters, for tracking both in vitro and in cells. We explored the cellular localization of PhenDC₃, one of the most powerful G4 ligands, by synthesizing two clickable azide and alkyne derivatives (PhenDC₃‐alk, PhenDC₃‐az) and labeling them in situ with the corresponding Cy5 click partners. A careful comparison of the results obtained for the copper‐based CuAAC and copper‐free SPAAC methodologies in fixed cells implicated Cu^I/alkyne intermediates in the nonspecific localization of ligands (and fluorophores) to the nucleoli. By contrast, SPAAC yielded similar nucleoplasmic labeling patterns in fixed and live cells. Our findings demonstrate the need for great care when using CuAAC to localize drugs in cells, and show that SPAAC gives results that are more consistent between fixed and live cells.     

Peroxidyme‐Amplified Radical Chain Reaction (PARCR): Visible Detection of a Catalytic Reporter

Peroxidyme Amplified Radical Chain Reaction (PARCR), a novel enzyme‐free system that achieves exponential amplification of a visible signal, is presented. Typical enzyme‐free amplification systems that produce a visible readout suffer from long reaction times, low sensitivity, and narrow dynamic range. PARCR employs photocatalyzed nonlinear signal generation, enabling unprecedented one‐pot, naked‐eye detection of a catalytic reporter from 1 μm down to 100 pm . In this reaction, hemin‐binding peroxidase‐mimicking DNAzymes (“peroxidymes”) mediate the NADH‐driven oxidation of a colorless, nonfluorescent phenoxazine dye (Amplex Red) to a brightly colored, strongly fluorescent product (resorufin); illumination with green light initiates multiple radical‐forming positive‐feedback loops, rapidly producing visible levels of resorufin. Collectively, these results demonstrate the potential of PARCR as an easy‐to‐use readout for a range of detection schemes, including aptamer labels, hybridization assays, and nucleic acid amplification.     

A Thermophilic Tetramolecular G‐Quadruplex/Hemin DNAzyme

The quadruplex‐based DNAzyme system is one of the most useful artificial enzymes or catalysts; their unique properties make them reliable alternatives to proteins for performing catalytic transformation. The first prototype of a thermally stable DNAzyme system is presented. This thermophilic DNAzyme is capable of oxidizing substrates at high temperatures (up to 95 °C) and long reaction times (up to 18 h at 75 °C). The catalytic activity of the DNAzymes were investigated with the standard peroxidase‐mimicking oxidation of 2,2′‐azino‐bis(3‐ethylbenzothiozoline‐6‐sulfonic acid) (ABTS) by H₂O₂. The step‐by‐step design of this unique heat‐activated G‐quadruplex/hemin catalyst, including the modification of adenines at both ends of G‐tracts, the choice of cation, and its concentration for DNAzyme stabilization, is described. This work investigates thoroughly the molecular basis of these catalytic properties and provides an example of an industrially relevant application.     

Drawing with Iron on a Gel Containing a Supramolecular Siderophore

Guanosine‐5′‐hydroxamic acid ( 3 ) forms hydrogels when mixed with guanosine ( 1 ) and KCl. The 5′‐hydroxamic acid (HA) unit is pH‐responsive and also chelates Fe³⁺. When gels are prepared under basic conditions, the 5′‐HA groups are deprotonated and the anionic hydrogel binds cationic thiazole orange (TO), signaled by enhanced fluorescence. The HA nucleoside 3 , when immobilized in the G‐quartet gel, acts as a supramolecular siderophore to form red complexes with Fe³⁺. We patterned the hydrogel's surface with FeCl₃, by hand and by using a 3D printer. Patterns form instantly, are visible by eye, and can be erased using vitamin C. This hydrogel, combining self‐assembled G‐quartet and siderophore–Fe³⁺ motifs, is strong, can be molded into different shapes, and is stable on the bench or under salt water.     

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.     

A Minimal Sequence for Left‐Handed G‐Quadruplex Formation

Recently, we observed the first example of a left‐handed G‐quadruplex structure formed by natural DNA, named Z‐G4. We analysed the Z‐G4 structure and inspected its primary 28‐nt sequence in order to identify motifs that convey the unique left‐handed twist. Using circular dichroism spectroscopy, NMR spectroscopy, and X‐ray crystallography, we revealed a minimal sequence motif of 12 nt (GTGGTGGTGGTG) for formation of the left‐handed DNA G‐quadruplex, which is found to be highly abundant in the human genome. A systematic analysis of thymine loop mutations revealed a moderate sequence tolerance, which would further broaden the space of sequences prone to left‐handed G‐quadruplex formation.