Use of Polyion Complexation for Polymerization-Induced Self-Assembly in Water under Visible Light Irradiation at 25 °C

Polyion complexation (PIC) as the driving force of polymerization-induced self-assembly (PISA), that is, PIC–PISA, is explored. Reversible addition–fragmentation chain transfer (RAFT) dispersion polymerization of NH₃⁺-monomer 2-aminoethylacrylamide hydrochloride (AEAM) can be achieved in water under visible light irradiation at 25 °C, using nonionic poly2-hydroxypropylmethacrylamide (PHPMA) macromolecular chain transfer agent in the presence of anionic poly­(sodium 2-acrylamido-2-methylpropanesulfonate) (PAMPS) PIC-template. Sphere-to-network transition occurs, owing to the PIC of PAMPS with growing chains upon reaction close to isoelectric point (IEP); thereafter, the increase of electrostatic repulsion promotes the split of networks and the rupture of spheres into fragments. Therefore, the free-flowing solution becomes viscous liquid and free-standing physical gel, and then back into viscous and free-flowing liquid. Such a PIC–PISA is appealing for gene delivery because the size and surface charge are variable on demand and at high solids

Chemical structure of related antibiotics (A) and genetic organization of the muraymycin gene cluster (B).

<p>Red arrows indicate the promoters which are probably the approximate binding sites of Mur34, black arrows mean the specific genes selected for quantitative real-time PCR, and the dotted lines show the gene intergenic regions analyzed by EMSA.</p

Synthesis of One-Component Nanostructured Polyion Complexes via Polymerization-Induced Electrostatic Self-Assembly

Nanostructured polyion complexes (PICs) are expected to serve as novel platforms to stabilize and deliver drugs, proteins, and nucleic acids. Yet, traditional self-assembly suffers from lack of scale-up and reproducibility. Particularly for one-component PICs, only spheres are available to date. Here, we report an efficient and scalable strategy to prepare one-component low-dimensional PICs. It involves visible-light-mediated RAFT iterative polymerization of opposite-charge monomers at 25% w/w solids in water at 25 °C. Sphere-film-vesicle transition and charge-/medium-tunable shape selectivity are reported. One-component PIC nanowire, ultrathin film, vesicle, tube, and surface-charged vesicle are easily prepared, and vesicle-polymerization is fulfilled, using this new strategy. This strategy provides a general platform to prepare one-component low-dimensional PICs with tailorable morphologies and high reproducibility on commercially viable scale under eco-friendly conditions

Analysis of the Mur34 binding site by DNase I footprinting assay.

<p>(A) Analysis of antisense strand γ-³²P labeled DNA (left) and the sense strand γ-³²P labeled DNA (right) upstream of <i>mur33</i>. Lanes G (1), A (2), T (3) and C (4) are sequencing ladder. Samples from lands 5–10 contain the same amount of the binding DNA with an increasing amount (0–3.2 µg µl^-1) of purified His₆Mur34. The complexes from the samples were digested by DNase I (0.004U per10 µl) at 30°C for 1 min. The vertical sequences to the right of each gel picture indicate the DNA regions protected from the cleavage of DNase I. The transcription start point (TSP) was shown for each DNA strand. (B) “G” indicates the TSP. The sequences underlined were the protected regions by His₆Mur34 under DNase I, “CAC” indicates the translation initiation codon (TIC), the bold regions upstream of TSP are -10 “TGATAT” and -35 “GTAAAACAG” regions. The bases in the boxes found are palindromes, and the bold and underlined bases near the TIC are supposed to be the Shine-Dalgarno consensus.</p

Gene expression analysis of the <i>mur</i> genes.

<p>(A) Transcription analysis of intergenic region of the selected <i>mur</i> genes. Top, ethidium bromide-stained agarose gels showing RT-PCR fragments from intergenic regions. <i>mur10</i>←<i>mur11</i> means that the detected region between <i>mur10</i> and <i>mur11</i>, and the arrows showed the possible orientation of transcription. In each gel, the left band was positive control using genomic DNA as template, the middle band showed the PCR sample using cDNA as template, the right band is negative control using template from total RNA sample digested with DNase I. (B) Time course of the transcription difference of <i>mur11</i> and <i>mur27</i> for DM-5 and the wild type strain. (C). The transcription difference of DM-5 and the wild type strain for 96 h incubation was used for the comparative analysis.</p

Analysis of <i>mur33</i> promoter by catechol dioxygenase activity assay.

<p>(A) The enzyme activities for the seed cultures of WT/pJTU5034, WT/pJTU5037 and WT/pJTU5038. (B) The enzyme activities for the seed cultures of DM-5/pJTU5034, DM-5/pJTU5037 and DM-5/pJTU5038. (C) The enzyme activities for the seed cultures of WT/pJTU5034 and DM-5/pJTU5034. (D) The enzyme activities for the fermentation cultures of WT/pJTU5034 and DM-5/pJTU5034. All histograms showed the quantitative catechol dioxygenase activity of <i>Streptomyces</i> sp. NRRL30471 and DM-5 independently containing pJTU5034, pJTU5037, pJTU5038 and pJTU3700. WT/pJTU3700 indicates <i>Streptomyces</i> sp. NRRL 30471 containing pJTU3700 (no <i>mur33</i> promoter) is as the negative control. WT/pJTU5034, indicates <i>Streptomyces</i> sp. NRRL 30471 containing pJTU5034 (natural <i>mur33</i> promoter). WT/pJTU5037 indicates <i>Streptomyces</i> sp. NRRL 30471 containing pJTU5037 (the -10 region mutated on <i>mur33</i> promoter). WT/pJTU5038 indicates <i>Streptomyces</i> sp. NRRL 30471 containing pJTU5038 (the -35 region mutated on <i>mur33</i> promoter). Likewise, DM-5 derived strains were designated.</p

EMSA analysis of His₆Mur34.

<p>(A) SDS-PAGE analysis of His₆Mur34, the theoretical molecular mass of His₆Mur34 is 19.6 kDa. The Mur34 protein was loaded into 12% SDS-PAGE for analysis. (B) EMSA analysis of Mur34 and <i>mur33</i> promoter. For the above figure, 50-fold of poly dI-dC was added to the each reaction system with an increasing amount of Mur34. For the competitive assay (below), lower case of all samples contain 2.6×10⁻⁴ M promoter DNA of <i>mur33</i> (90-bp specific DNA), for samples 2, 3 and 4, extra 9×10⁻⁶ M His₆Mur34 was individually contained. Moreover, 50-fold of unlabelled competitive DNA was added to the reaction system (band 3), and 50-fold of unspecific non-competitive DNA to the system (band 4). Band designations, 1, free DNA; 2-4, protein-DNA complexes.</p

Mutational analysis of <i>mur34</i>.

<p>(A) Schematic representation for the construction of DM-5 mutant, as a 0.23-kb of <i>mur34</i> was replaced by the 1.43-kb <i>neo</i> cassette, the DM-5 mutant gives a 1.74-kb PCR product, while the wild type strain is 0.5-kb. (B) Bioassay and LC-MS analysis of the metabolites. Top, the metabolites produced by wild type strain. Bottom, the metabolites produced by DM-5 mutant. Muraymycin C1 and D1 components were selected for LC-MS comparative analysis.</p

Compartmentalization and Unidirectional Cross-Domain Molecule Shuttling of Organometallic Single-Chain Nanoparticles

Compartmentalization and unidirectional cross-domain molecule shuttling are omnipresent in proteins, and play key roles in molecular recognition, enzymatic reaction, and other living functions. Nanomachinery design emulating these biological functions is being considered as one of the most ambitious and challenging tasks in modern chemistry and nanoscience. Here, we present a biomimetic nanomachinery design using single-chain technology. Stepwise complex of the outer blocks of water-soluble linear ABC triblock terpolymer to copper ions yields dumbbell-shaped single-chain nanoparticle. A novel nanomachine capable of compartmentalization and unidirectional cross-domain molecule shuttling has been achieved upon ascorbic acid reduction, leading to synergistically donating/accepting copper centers between discrete double heads, overall dumbbell-to-tadpole configurational transition, and intake of oxidized ascorbic acid into reconstructed head. Subsequent air oxidation results in the inverse molecule shuttling and configurational transition processes. This is the first demonstration of biomimetic nanomachinery design that is capable of compartmentalization and unidirectional cross-domain molecule shuttling, exemplified simply using a new single-chain technology