Evaluation of a new linker system cleaved using samarium(II) iodide. Application in the solid phase synthesis of carbonyl compounds

A new linker design for solid phase synthesis has been developed that is cleaved under mild, neutral conditions using samarium(II) iodide. The feasibility of the linker approach has been illustrated in the solid phase synthesis of ketones and amides using an oxygen linker. Insights into the mechanism of the samarium(II) iodide cleavage reaction are described and the potential of a sequential cleavage carbon-carbon bond forming process is assessed.     

Nanoparticle-DNA conjugates bearing a specific number of short DNA strands by enzymatic manipulation of nanoparticle-bound DNA

Self-assembling of metallic nanoparticles to form well-defined nanostructured structures is a field that has been receiving considerable research interest in recent years. In this field, DNA is a commonly used linker molecule to direct the assembly of the nanoscale building blocks because of its unique recognition capabilities, mechanical rigidity, and physicochemical stability. This study reported our novel approach to generate gold nanoparticle-DNA conjugates bearing specially designed DNA linker molecules that can be used as building blocks to construct nanoassemblies with precisely controlled structure or as nanoprobes for quantitative DNA sequence detection analysis. In our approach, gold nanoparticle-DNA conjugates bearing a specific number of long double-stranded DNA strands were prepared by gel electrophoresis. A restriction endonuclease enzyme was then used to manipulate the length of the nanoparticle-bound DNA. This enzymatic cleavage was confirmed by gel electrophoresis, and digestion efficiency of 90% or more was achieved. With this approach, nanoparticle conjugates bearing a specific number of strands of short DNA with less than 20-base can be achieved.     

Introducing structural flexibility into porphyrin-DNA zipper arrays

A more flexible nucleotide building block for the synthesis of new DNA based porphyrin-zipper arrays is described. Changing the rigid acetylene linker between the porphyrin substituent and the 2'-deoxyuridine to a more flexible propargyl amide containing linkage leads in part to an increased duplex stability. The CD spectra reveal different electronic interactions between the porphyrins depending on the type of linker used. Molecular modelling suggests large variation of the relative orientation of the porphyrins within the major groove of the DNA. The porphyrins can be metallated post-synthetically with different metals as shown with zinc, cobalt and copper. The spectroscopic features do not alter drastically upon metallation apart from the CD spectra, and the stability of the metal complex is highly dependent on the nature of the metal. As shown by CD spectroscopy, the zinc porphyrin is rapidly demetallated at high temperatures. Globular structure determination using SAXS indicates that a molecular assembly comprised of a two to four helical bundle dominates in solution at higher concentrations (≥50 μM) which is not observed by spectroscopy at lower concentrations (≤1 μM).     

Arrested chain growth during magnetic directed particle assembly in yield stress matrix fluids

The process of assembling particles into organized functional structures is influenced by the rheological properties of the matrix fluid in which the assembly takes place. Therefore, tuning these properties represents a viable and as yet unexplored approach for controlling particle assembly. In this Letter, we examine the effect of the matrix fluid yield stress on the directed assembly of polarizable particles into linear chains under a uniform external magnetic field. Using particle-level simulations with a simple yield stress model, we find that chain growth follows the same trajectory as in Newtonian matrix fluids up to a critical time that depends on the balance between the yield stress and the strength of magnetic interactions between particles; subsequently, the system undergoes structural arrest. Appropriate dimensionless groups for characterizing the arresting behavior are determined and relationships between these groups and the resulting structural properties are presented. Since field-induced structures can be indefinitely stabilized by the matrix fluid yield stress and "frozen" into place as desired, this approach may facilitate the assembly of more complex and sophisticated structures.     

Cu2+ -mediated assembly of the minor groove binders on the DNA template with sequence selectivity

The ligands (Bpy-H, 5) have been designed to connect the Hoechst33258 skeleton for DNA binding and 2,2'-bipyridine for Cu(2+) complexation. It has been revealed that the new Hoechst ligand long Bpy-H (5L) having a long linker exhibits Cu(2+)-mediated assembly on the DNA template having two A(3)T(3) sites in a selective manner depending on the length of the linker of the ligand as well as on the distance between the two A(3)T(3) sites of DNA. [reaction: see text]     

"Belt and braces": a peptide-based linker system of de novo design

A new self-assembling peptide-based linker is described. The system comprises three leucine-zipper sequences of de novo design: one peptide, "the belt", templates the co-assembly of the other two-half-sized peptides, "the braces". These basic features were confirmed by circular dichroism spectroscopy and analytical ultracentrifugation: when mixed, the three peptides reversibly formed a predominantly helical and stable 1:1:1 ternary complex. Surface plasmon resonance experiments demonstrated assembly of the complex on gold surfaces, while the ability of the system to bring together peptide-bound cargo was demonstrated using colloidal gold nanoparticles. In the latter experiments, the nanoparticles were derivatized with the brace peptides prior to the addition of the belt. Transmission electron microscopy images of the resulting networks revealed regular approximately 7 nm separations between adjacent particles, consistent with the 42-amino acid helical design of the belt and braces. To our knowledge, belt and braces is a novel concept in leucine-zipper assembly and the first example of employing peptides to guide nanoparticle assembly.     

Folding induced assembly of polypeptide decorated gold nanoparticles

Reversible assembly of gold nanoparticles controlled by the homodimerization and folding of an immobilized de novo designed synthetic polypeptide is described. In solution at neutral pH, the polypeptide folds into a helix-loop-helix four-helix bundle in the presence of zinc ions. When immobilized on gold nanoparticles, the addition of zinc ions induces dimerization and folding between peptide monomers located on separate particles, resulting in rapid particle aggregation. The particles can be completely redispersed by removal of the zinc ions from the peptide upon addition of EDTA. Calcium ions, which do not induce folding in solution, have no effect on the stability of the peptide decorated particles. The contribution from folding on particle assembly was further determined utilizing a reference peptide with the same primary sequence but containing both D and L amino acids. Particles functionalized with the reference peptide do not aggregate, as the peptides are unable to fold. The two peptides, linked to the nanoparticle surface via a cysteine residue located in the loop region, form submonolayers on planar gold with comparable properties regarding surface density, orientation, and ability to interact with zinc ions. These results demonstrate that nanoparticle assembly can be induced, controlled, and to some extent tuned, by exploiting specific molecular interactions involved in polypeptide folding.     

Supramolecular Polyphenol-DNA Microparticles for In Vivo Adjuvant and Antigen Co-Delivery and Immune Stimulation

DNA-based materials have attracted interest due to the tunable structure and encoded biological functionality of nucleic acids. A simple and general approach to synthesize DNA-based materials with fine control over morphology and bioactivity is important to expand their applications. Here, we report the synthesis of DNA-based particles via the supramolecular assembly of tannic acid (TA) and DNA. Uniform particles with different morphologies are obtained using a variety of DNA building blocks. The particles enable the co-delivery of cytosine-guanine adjuvant sequences and the antigen ovalbumin in model cells. Intramuscular injection of the particles in mice induces antigen-specific antibody production and T cell responses with no apparent toxicity. Protein expression in cells is shown using capsules assembled from TA and plasmid DNA. This work highlights the potential of TA as a universal material for directing the supramolecular assembly of DNA into gene and vaccine delivery platforms.     

A Hot‐Segment‐Based Approach for the Design of Cross‐Amyloid Interaction Surface Mimics as Inhibitors of Amyloid Self‐Assembly

The design of inhibitors of protein–protein interactions mediating amyloid self‐assembly is a major challenge mainly due to the dynamic nature of the involved structures and interfaces. Interactions of amyloidogenic polypeptides with other proteins are important modulators of self‐assembly. Here we present a hot‐segment‐linking approach to design a series of mimics of the IAPP cross‐amyloid interaction surface with Aβ (ISMs) as nanomolar inhibitors of amyloidogenesis and cytotoxicity of Aβ, IAPP, or both polypeptides. The nature of the linker determines ISM structure and inhibitory function including both potency and target selectivity. Importantly, ISMs effectively suppress both self‐ and cross‐seeded IAPP self‐assembly. Our results provide a novel class of highly potent peptide leads for targeting protein aggregation in Alzheimer’s disease, type 2 diabetes, or both diseases and a chemical approach to inhibit amyloid self‐assembly and pathogenic interactions of other proteins as well.     

Formation and stability of a Janus-Wedge type of DNA triplex

A new type of DNA targeting with the formation of a Janus-Wedge (J-W) triple helix is described. The "wedge" residue (W) attached to a PNA backbone is designed to insert itself into double-stranded DNA and base pair with both Watson-Crick faces. To study the stability of such an assembly, we have examined the formation of the J-W triplex with dC8 - T8 target sequence. The use of this target sequence permits the study of this new helix form without competing Watson-Crick interactions between the two target residues. Studies indicate that the W strand binds to both target strands, with defined polarity and a stability (-15.2 kcal/mol) that is roughly the sum of the two independent duplex interactions.     

Strong stacking between FH--N hydrogen-bonded foldamers and fullerenes: formation of supramolecular nano networks

The stacking interactions between FH--N hydrogen-bonded foldamers 1-3, bis-foldamer 4, and tris-foldamer 5 and C(60) and C(70) are described. Compound 4 contains two folded units, which are connected by an isophthalamide linker, whereas 5 has a C(3)-symmetrical discotic structure, in which three folded units are connected by a benzene-1,3,5-tricarboxamide unit. UV/Vis, fluorescence, and NMR experiments have revealed that the foldamers or folded units strongly stack with fullerenes in chloroform. The (apparent) association constants of the respective complexes have been determined by a fluorescence titration method. The strong association is tentatively attributed to intermolecular cooperative fluorophenylpi and solvophobic interactions. A similar but weaker interaction has also been observed between an MeOH--N hydrogen-bonded foldamer and fullerenes. AFM studies have revealed that the surfaces of 3 and 4 show fibrous networks, while the surface of 5 shows particles. In sharp contrast, mixtures of 3 and 4 with C(60) have been shown to generate thinner separated fibrils, whereas a mixture of 5 and C(60) produces honeycomb-like nano networks, for which a columnar cooperative stacking pattern is proposed. The results demonstrate the usefulness of FH--N hydrogen-bonded folded structures in the construction of nanoscaled materials.     

Length-dependent DNA separations using multiple end-attached peptide nucleic acid amphiphiles in micellar electrokinetic chromatography

End-labeled free-solution electrophoresis (ELFSE) is an alternative approach to gel-based methods for size-based electrophoretic separation of DNA. In ELFSE, an electrically neutral "drag-tag" is appended to DNA to add significant hydrodynamic drag, thereby breaking its constant charge-to-friction ratio. Current drag-tag architecture relies on covalent attachment of polymers to each DNA molecule. We have recently proposed the use of micellar drag-tags in conjunction with sequence-specific hybridization of peptide nucleic acid amphiphiles (PNAAs). This work investigates the effect of multiple PNAA attachment on DNA resolution using MEKC. Simultaneous PNAA hybridization allows for the separation of long DNA targets, up to 1012 bases, using micellar drag-tags. Each PNAA handle independently interacts with the micellar phase, reducing the overall mobility of this complex relative to individual PNAA binding. The sequence- and size-based dependence of this separation technique is maintained with multiple PNAA binding over a range of DNA sizes. Results are accurately described by ELFSE theory, yielding alpha=54 for single-micelle tagging and alpha=142 for dual-micelle tagging. This method is the first example of a non-covalent drag-tag used to separate DNA of 1000 bases based on both size and sequence.     

Assembly of DNA triangles mediated by perylene bisimide caps

Perylene bisimides (PBI) have been synthetically incorporated as caps onto a Y-shaped DNA triple strand. These PBI caps serve as "sticky" ends in the spontaneous assembly of larger DNA ensembles, linking the triangular DNA through stacking interactions. This, in turn, yields a hypsochromic shift in the absorption and a red shift in the fluorescence as characteristic optical readouts. This assembly occurs spontaneously without any enzymatic ligation process and without the use of overhanging DNA as sticky ends. Instead, dimerizations of the PBI chromophores in the assembly are controlled by the DNA as a structural scaffold. Thereby, the PBI-driven assembly is fully reversible. Due to the fact that PBI dimerization does not occur in the single strand, the aggregates can be destroyed by thermal dehybridization of the DNA scaffold and reassembled by reannealing of the DNA construct. In view of the fact that PBI forms stable radical anions, the presented DNA architectures are not only interesting optical biomaterials, but are also promising materials for molecular electronics with DNA.     

Patterned assembly of colloidal particles by confined dewetting lithography

We report the assembly of colloidal particles into confined arrangements and patterns on various cleaned and chemically modified solid substrates using a method which we term "confined dewetting lithography" or CDL for short. The experimental setup for CDL is a simple deposition cell where an aqueous suspension of colloidal particles (e.g., polystyrene spheres) is placed between a floating deposition template (i.e., metal microgrid) and the solid substrate. The voids of the deposition template serve as an array of micrometer-sized reservoirs where several hydrodynamic processes are confined. These processes include water evaporation, meniscus formation, convective flow, rupturing, dewetting, and capillary-bridge formation. We discuss the optimal conditions where the CDL has a high efficiency to deposit intricate patterns of colloidal particles using polystyrene spheres (PS; 4.5, 2.0, 1.7, 0.11, 0.064 microm diameter) and square and hexagonal deposition templates as model systems. We find that the optimization conditions of the CDL method, when using submicrometer, sulfate-functionalized PS particles, are primarily dependent on minimizing attractive particle-substrate interactions. The CDL methodology described herein presents a relatively simple and rapid method to assemble virtually any geometric pattern, including more complex patterns assembled using PS particles with different diameters, from aqueous suspensions by choosing suitable conditions and materials.     

Direct measurement of interactions between stimulation-responsive drug delivery vehicles and artificial mucin layers by colloid probe atomic force microscopy

A novel thermo- and pH-sensitive nanogel particle, which is a core-shell structured particle with a poly(N-isopropylacrylamide) (p(NIPAAm)) hydrogel core and a poly(ethylene glycol) monomethacrylate grafted poly(methacrylic acid) (p(MMA-g-EG)) shell, is of interest as a vehicle for the controlled release of peptide drugs. The interactions between such nanogel particles and artificial mucin layers during both approach and separation were successfully measured by using colloid probe atomic force microscopy (AFM) under various compression forces, scan velocities, and pH values. While the magnitudes of the compression forces and scan velocities did not affect the interactions during the approach process, the adhesive force during the separation process increased with these parameters. The pH values significantly influenced the interactions between the nanogel particles and a mucin layer. A large steric repulsive force and a long-range adhesive force were measured at neutral pH due to the swollen p(MMA-g-EG) shell. On the other hand, at low pH values, the steric repulsive force disappeared and a short-range adhesive force was detected, which resulted from the collapse of the shell layer. The nanogel particles possessed a pH response that was sufficient to protect the incorporated peptide drug under the harsh acidic conditions in the stomach and to effectively adhere to the mucin layer of the small intestine, where the pH is neutral. The relationships among the nanogel particle-mucin layer interactions, pH conditions, scan velocities, and compression forces were systemically investigated and discussed.     

Binding of hairpin polyamides to DNA studied by fluorescence correlation spectroscopy for DNA nanoarchitectures

We have recently constructed a “DNA strut” consisting of two DNA-binding hairpin polyamides of Dervan-type connected via a long flexible linker and were able to show that this strut can be used to sequence-selectively connect DNA helices. This approach provides a second structural element (besides the Watson–Crick base pairing) for the assembly of higher-order DNA nanoarchitectures from smaller DNA building blocks. Since none of the existing analytical techniques for studying this kind of system were found suitable for detection and quantification of the formation of the resulting complexes, we chose fluorescence correlation spectroscopy (FCS). In the present study we show that FCS allowed us in a versatile and fast way to investigate the binding of Dervan polyamides to DNA. In particular it also shows its power in the quantitative detection of the formation of multimeric complexes and the in investigation of binding under nonphysiological conditions. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Efficient bioactive oligonucleotide-protein conjugation for cell-targeted cancer therapy

Oligonucleotide-protein conjugates have important applications in biomedicine. Simple and efficient methods are described for the preparation of these conjugates. Specifically, we describe a new method in which a bifunctional linker is attached to thiol-oligonucleotide to generate a reactive intermediate that is used to link to the protein. Having similar conjugation efficacy compared with the classical method in which the bifunctional linker is attached first to the protein, this new approach produces significantly more active conjugates with higher batch to batch reproducibility. In a second approach, direct conjugation is proposed using oligonucleotides carrying carboxyl groups. These methodologies have been applied to prepare nanoconjugates of an engineered nanoparticle protein carrying a T22 peptide with affinity for the CXCR4 chemokine receptor and oligomers of the antiproliferative nucleotide 2’-deoxy-5-fluorouridine in a very efficient way. The protocols have potential uses for the functionalization of proteins, amino-containing polymers or amino-lipids in order to produce complex therapeutic nucleic acid delivery systems.     

Nucleosome Assembly Alters the Accessibility of the Antitumor Agent Duocarmycin B2 to Duplex DNA

To evaluate the reactivity of antitumor agents in a nucleosome architecture, we conducted in vitro studies to assess the alkylation level of duocarmycin B₂ on nucleosomes with core and linker DNA using sequencing gel electrophoresis. Our results suggested that the alkylating efficiencies of duocarmycin B₂ were significantly decreased in core DNA and increased at the histone‐free linker DNA sites when compared with naked DNA conditions. Our finding that nucleosome assembly alters the accessibility of duocarmycin B₂ to duplex DNA could advance its design as an antitumor agent.