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.

Sequence-Selective Minor Groove Recognition of a DNA Duplex Containing Synthetic Genetic Components.

The structural basis of minor groove recognition of a DNA duplex containing synthetic genetic information by hairpin pyrrole-imidazole polyamides is described. Hairpin polyamides induce a higher melting stabilization of a DNA duplex containing the unnatural P·Z base-pair when an imidazole unit is aligned with a P nucleotide. An NMR structural study showed that the incorporation of two isolated P·Z pairs enlarges the minor groove and slightly narrows the major groove at the site of this synthetic genetic information, relative to a DNA duplex consisting entirely of Watson-Crick base-pairs. Pyrrole-imidazole polyamides bind to a P·Z-containing DNA duplex to form a stable complex, effectively mimicking a G·C pair. A structural hallmark of minor groove recognition of a P·Z pair by a polyamide is the reduced level of allosteric distortion induced by binding of a polyamide to a DNA duplex. Understanding the molecular determinants that influence minor groove recognition of DNA containing synthetic genetic components provides the basis to further develop unnatural base-pairs for synthetic biology applications.

Molecular Construction of Sulfonamide Antisense Oligonucleotides.

An efficient and scalable synthesis of new oligonucleotide monomers was developed for replacement of the phosphodiester backbone of RNA by a sulfonamide-containing backbone to enable construction of sulfonamide antisense oligonucleotides (SaASOs). It was shown that by employing these sulfonamide RNA (SaRNA) monomers, it was possible to synthesize oligomers in solution. The properties of a sulfonamide moiety replacement were evaluated by incorporation of a SaRNA-monomer into a DNA strand and performing thermal stability tests of the resulting DNA and RNA-double-strand hybrids. Although sulfonamide modification caused a decrease in melting temperature (Tm) of both hybrids, it was lower for the sulfonamide-containing DNA-RNA hybrid than that for the sulfonamide-containing DNA-DNA hybrid.

Influence of perfluorinated end groups on the SFRD of [Pt(cod)Me(C(n)F(2n+1))] onto porous Al(2)O(3) in CO(2) under reductive conditions.

The optimized synthesis of a range of cyclooctadiene-stabilized Pt complexes that contained different perfluoro-alkane chains, [Pt(cod)Me(Cn F2n+1 )], is presented. These metal-organic compounds were employed in the so-called supercritical fluid reactive deposition (SFRD) in CO2 under reductive conditions to generate metallic nanoparticles on aluminum oxide as a porous support. Thus, Al2 O3 -supported Pt nanoparticles with a narrow particle-size distribution were obtained. At a reduction pressure of 15.5 MPa and a temperature of 353 K, particle diameters of d50 =2.3-2.8 nm were generated. Decreasing the pressure during the reduction reaction led to slightly larger particles whilst decreasing the amount of organometallic precursor in CO2 yielded a decrease in the particle size from x50 =3.2 nm to 2.6 nm and a particle-size distribution of 2.2 nm. Furthermore, substitution of the CH3 end group by the Cn F2n+1 end groups led to a significant drop in Pt loading of about 50 %. Within the series of perfluorinated end groups that were considered, the Pt complex that contained a branched perfluoro-isopropyl group showed the most-interesting results when compared to the control precursor, [Pt(cod)Me2 ] (1).

A flow platform for degradation-free CuAAC bioconjugation.

The Cu-catalyzed azide-alkyne cycloaddition (CuAAC) reaction is a cornerstone method for the ligation of biomolecules. However, undesired Cu-mediated oxidation and Cu-contamination in bioconjugates limits biomedical utility. Here, we report a generic CuAAC flow platform for the rapid, robust, and broad-spectrum formation of discrete triazole bioconjugates. This process leverages an engineering problem to chemical advantage: solvent-mediated Cu pipe erosion generates ppm levels of Cu in situ under laminar flow conditions. This is sufficient to catalyze the CuAAC reaction of small molecule alkynes and azides, fluorophores, marketed drug molecules, peptides, DNA, and therapeutic oligonucleotides. This flow approach, not replicated in batch, operates at ambient temperature and pressure, requires short residence times, avoids oxidation of sensitive functional groups, and produces products with very low ppm Cu contamination.