Triazoles from N-Alkynylheterocycles and Their Coordination to Iridium

N-alkynylheterocycles (benzimidazole and indazole) are converted to triazoles by click chemistry, and the resulting triazoles react with [IrCl₂Cp*]₂. The benzimidazole-triazole coordinates in a monodentate fashion through the benzimidazole, whereas the indazole-triazole is bidentate through coordination of both heterocycles. Reaction of the benzimidazole-triazole with methyliodide gives a benzimidazolium salt that deprotonates on coordination to afford a rare example of a bidentate NHC–triazole

Highly Efficient Synthesis of DNA-Binding Polyamides Using a Convergent Fragment-Based Approach

Two advances in the synthesis of hairpin pyrrole-imidazole polyamides (PAs) are described. First, the application of a convergent synthetic strategy is shown, involving the Boc-based solid phase synthesis of a <i>C</i>-terminal fragment and the solution phase synthesis of the <i>N</i>-terminal fragment. Second a new hybrid resin is developed that allows for the preparation of hairpin PAs lacking a C-terminal β-alanine tail. Both methods are compatible with a range of coupling reagents and provide a facile, modular route to prepare PA libraries in high yield and crude purity

Sequence-Selective Detection of Double-Stranded DNA Sequences Using Pyrrole–Imidazole Polyamide Microarrays

We describe a microarray format that can detect double-stranded DNA sequences with a high degree of sequence selectivity. Cyclooctyne-derivatized pyrrole–imidazole polyamides were immobilized on azide-modified glass substrates using microcontact printing and a strain-promoted azide–alkyne cycloaddition (SPAAC) reaction. These polyamide-immobilized substrates selectively detected a seven-base-pair binding site incorporated within a double-stranded oligodeoxyribonucleotide sequence even in the presence of an excess of a sequence with a single-base-pair mismatch

Directed Assembly of DNA-Functionalized Gold Nanoparticles Using Pyrrole–Imidazole Polyamides

Traditional methods for the construction of nanoparticle arrays and lattices exploit Watson–Crick base pairing of single-stranded DNA sequences as a proxy for self-assembly. Although this approach has been utilized in a variety of applications in nanoassembly, diagnostics, and biomedicine, the diversity of this recognition lexicon could be considerably increased by developing strategies that recognize the base-pairing landscape of double-stranded DNA (dsDNA) sequences. Herein we describe the first report of programmed gold nanoparticle (GNP) aggregation directed by the recognition of dsDNA sequences using pyrrole–imidazole polyamide–GNP (PA–GNP) conjugates. We demonstrate the reversibility and selectivity of this strategy for forming GNP aggregates in the presence of fully matched dsDNA sequences relative to dsDNA sequences containing one- and two-base-pair mismatches

The mechanisms of a mammalian splicing enhancer.

Exonic splicing enhancer (ESE) sequences are bound by serine & arginine-rich (SR) proteins, which in turn enhance the recruitment of splicing factors. It was inferred from measurements of splicing around twenty years ago that Drosophila doublesex ESEs are bound stably by SR proteins, and that the bound proteins interact directly but with low probability with their targets. However, it has not been possible with conventional methods to demonstrate whether mammalian ESEs behave likewise. Using single molecule multi-colour colocalization methods to study SRSF1-dependent ESEs, we have found that that the proportion of RNA molecules bound by SRSF1 increases with the number of ESE repeats, but only a single molecule of SRSF1 is bound. We conclude that initial interactions between SRSF1 and an ESE are weak and transient, and that these limit the activity of a mammalian ESE. We tested whether the activation step involves the propagation of proteins along the RNA or direct interactions with 3' splice site components by inserting hexaethylene glycol or abasic RNA between the ESE and the target 3' splice site. These insertions did not block activation, and we conclude that the activation step involves direct interactions. These results support a model in which regulatory proteins bind transiently and in dynamic competition, with the result that each ESE in an exon contributes independently to the probability that an activator protein is bound and in close proximity to a splice site

Identification of G-quadruplexes in long functional RNAs using 7-deazaguanine RNA

RNA G-quadruplex (G4) structures are thought to affect biological processes, including translation and pre-mRNA splicing, but it is not possible at present to demonstrate that they form naturally at specific sequences in long functional RNA molecules. We developed a new strategy, footprinting of long 7-deazaguanine-substituted RNAs (FOLDeR), that allows the formation of G4s to be confirmed in long RNAs and under functional conditions

2D-IR Spectroscopy Shows that Optimized DNA Minor Groove Binding of Hoechst33258 Follows an Induced Fit Model

The induced fit binding model describes a conformational change occurring when a small molecule binds to its biomacromolecular target. The result is enhanced noncovalent interactions between the ligand and biomolecule. Induced fit is well-established for small molecule–protein interactions, but its relevance to small molecule–DNA binding is less clear. We investigate the molecular determinants of Hoechst33258 binding to its preferred A-tract sequence relative to a suboptimal alternating A-T sequence. Results from two-dimensional infrared spectroscopy, which is sensitive to H-bonding and molecular structure changes, show that Hoechst33258 binding results in loss of the minor groove spine of hydration in both sequences, but an additional perturbation of the base propeller twists occurs in the A-tract binding region. This induced fit maximizes favorable ligand–DNA enthalpic contributions in the optimal binding case and demonstrates that controlling the molecular details that induce subtle changes in DNA structure may hold the key to designing next-generation DNA-binding molecules

Structural Basis of the Mispairing of an Artificially Expanded Genetic Information System

Relative to naturally occurring Watson-Crick base pairs, the synthetic nucleotide P pairs with Z within DNA duplexes through a unique hydrogen-bond arrangement. The loss of this synthetic genetic information by PCR results in the conversion of P-Z into a G-C base pair. Here, we show structural and spectroscopic evidence that the loss of this synthetic genetic information occurs via G-Z mispairing. Remarkably, the G-Z mispair is both plastic and pH dependent; it forms a double-hydrogen-bonded “slipped” pair at pH 7.8 and a triple-hydrogen-bonded Z-G pair when the pH is above 7.8. This study highlights the need for robust structural and functional methods to elucidate the mechanisms of mutation in the development of next-generation synthetic genetic base pairs

Profiling Sulfur(VI) Fluorides as Reactive Functionalities for Chemical Biology Tools and Expansion of the Ligandable Proteome

Here, we report a comprehensive profiling of sulfur(VI) fluorides (SVI-Fs) as reactive groups for chemical biology applications. SVI-Fs are reactive functionalities that modify lysine, tyrosine, histidine, and serine sidechains. A panel of SVI-Fs were studied with respect to hydrolytic stability and reactivity with nucleophilic amino acid sidechains. The use of SVI-Fs to covalently modify carbonic anhydrase II (CAII) and a range of kinases was then investigated. Finally, the SVI-F panel was used in live cell chemoproteomic workflows, identifying novel protein targets based on the type of SVI-F used. This work highlights how SVI-F reactivity can be used as a tool to expand the liganded proteome

A Targeted Oligonucleotide Enhancer of SMN2 Exon 7 Splicing Forms Competing Quadruplex and Protein Complexes in Functional Conditions

The use of oligonucleotides to activate the splicing of selected exons is limited by a poor understanding of the mechanisms affected. A targeted bifunctional oligonucleotide enhancer of splicing (TOES) anneals to SMN2 exon 7 and carries an exonic splicing enhancer (ESE) sequence. We show that it stimulates splicing specifically of intron 6 in the presence of repressing sequences in intron 7. Complementarity to the 5' end of exon 7 increases U2AF65 binding, but the ESE sequence is required for efficient recruitment of U2 snRNP. The ESE forms at least three coexisting discrete states: a quadruplex, a complex containing only hnRNP F/H, and a complex enriched in the activator SRSF1. Neither hnRNP H nor quadruplex formation contributes to ESE activity. The results suggest that splicing limited by weak signals can be rescued by rapid exchange of TOES oligonucleotides in various complexes and raise the possibility that SR proteins associate transiently with ESEs