Recent advances in DNA-based medicine (gene therapy, genetic vaccination) have intensified the necessity for pharmaceutical-grade plasmid DNA purification at comparatively large scales. In this contribution triple-helix affinity precipitation is introduced for this purpose. A short, single-stranded oligonucleotide sequence (namely (CTT)(7)), which is capable of recognizing a complementary sequence in the double-stranded target (plasmid) DNA, is linked to a thermoresponsive N-isopropylacrylamide oligomer to form a so-called affinity macroligand (AML). At 4 degrees C, i.e., below its critical solution temperature, the AML binds specifically to the target molecule in solution; by raising the temperature to 40 degrees C, i.e., beyond the critical solution temperature of the AML, the complex can be precipitated quantitatively. After redissolution of the complex at lower temperature, the target DNA can be released by a pH shift to slightly alkaline conditions (pH 9.0). Yields of highly pure (plasmid) DNA were routinely between 70% and 90%. Non-specific co- precipitation of either the target molecule by the non-activated AML precursor or of contaminants by the AML were below 7% and presumably due to physical entrapment of these molecules in the wet precipitate. Ligand efficiencies were at least 1 order of magnitude higher than in triple-helix affinity chromatography.

The question of how ligand binding to certain receptor proteins eventually gates ion channels is of central importance in cellular signaling but still unresolved at a molecular level. In order to enhance our understanding of the molecular mechanisms, we used a combined biomolecular and biophysical approach to study a serotonin-gated ion channel.The 5-hydroxytryptamine (5-HT 1 ; serotonin) type 3 receptor (5-HT 3 receptor (5-HT 3 R)) is the only ligand-gated ion channel found among the serotonin receptors. Its gene structure (1) and amino acid sequence are similar to those of other members of the Cys loop receptor family including the nicotinic acetylcholine (nAChR), ionotropic ␥-aminobutyric acid, and glycine receptors. They are composed of five homologous or identical subunits, each comprising four predicted transmembrane regions and a large extracellular N-terminal domain containing the ligand-binding site. Among them, the nAChR is most closely related to the 5-HT 3 receptor. Both receptors form ion channels that are permeable to cations and share about 20 -30% amino acid sequence identity.So far, three different 5-HT 3 receptor subunits, A, B, and C, have been cloned as well as a short splicing variant of the A subunit. Expression of only A subunits results in functional ion channels of similar properties as for 5-HT 3 receptors in native tissues, suggesting that this receptor is active as a homopentamer (2). Expression of solely the B (3) or C (4) subunits did not result in functional receptors; however, their coexpression with the A subunit yields in functional receptor proteins with slightly different channel properties.Site-directed mutagenesis and biochemical studies, combined with amino acid sequence alignments, have identified amino acid residues and sequence regions (the so-called loops A-F; see Fig. 1) in the N-terminal extracellular domain implicated in the ligand-binding site of the nicotinic acetylcholine and 5-HT 3 receptors (for reviews, see Refs. 5-7 and references therein).For the nAChR, these experiments together with the recently resolved three-dimensional structure of the acetylcholine-binding protein (AChBP) (8) indicate that the binding site is located at the interface between two adjacent subunits and that the binding loops A-F are forming the binding pocket. According to this model, loops A-C would form the binding site on one subunit, whereas loops D-F of the adjacent subunit would contribute to a lesser extent to ligand binding. In the case of the 5-HT 3 receptor, only few details are known about the ligand-binding site.

The telomerization (chain transfer polymerization) kinetics of N-isopropylacrylamide were investigated in various (hydro)organic solvents using 3-mercaptopropionic hydrazide as chain transfer agent (telogen). Except for the dioxane/water system telogen consumption rates were similar for all cases, while solvent effects could be observed for the monomer consumption rates. Chain transfer constants, as defined by the ratio of the rate constants for chain transfer and chain propagation (C T = k tr,/k p), were highest in DMF (10.3), a solvent unable to form hydrogen bonds or dipole−dipole interactions with the monomer, while a more promising value of C T = 1.7 was found for the 6:4 methanol/water mixture. The highest monomer consumption rates were observed for the 1:1 dioxane/water mixture. However, in this particular case the telogen consumption was also found to increase, as we propose due to a “hydrophobic effect” whereby polymer microaggregates serve to locally increase the concentration of telogen and/or monomer once a certain water concentration has been passed. As a result a comparatively high C T of 3.2 characterizes the dioxane/water system.

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Affinity precipitation uses reversibly water-soluble affinity macroligands, i.e. stimulus-responsive polymers bearing one or several affinity ligands, to first capture and then co-precipitate a target molecule. Conventional, polymeric AML tend to suffer from a pronounced heterogeneity in both the structure of the polymer backbone and the affinity constants. In this paper a novel type of homogeneous, oligomeric AML is proposed, which carries one affinity ligand per AML in terminal position. The homogeneity of the AML translates into a very uniform precipitation behavior. Oligomeric AML-precursors prepared by chain or group transfer polymerization show a solubility, which is very similar to that reported previously for polymeric molecules of the same chemistry. Oligomers prepared by anionic polymerization are predominantly isotactic and show some deviations from this behavior. The affinity ligand is coupled to the oligomers via a reactive end group (e.g. an amino or carboxylic acid group) created during oligomer synthesis. An iminobiotin activated AML-precursor is used to recover avidin from a cell culture supernatant containing 5% FCS. Over 90% of the avidin are recovered in nearly pure form (residual protein contamination below the detection limit). This is one of the first purifications of a protein other than an enzyme by affinity precipitation with high yields. In a second example, the AML-precursor is activated by a single stranded DNA oligomer tag ((CTT)7) and used to purify double stranded DNA molecules by triple helix affinity precipitation.

To enhance the performance of organic devices, doping and graded mixed‐layer structures, formed by co‐evaporation methods, have been extensively adopted in the formation of organic thin films. Among the criteria for selecting materials systems, much attention has been paid to the materials' energy‐band structure and carrier‐transport behavior. As a result, some other important characteristics may have been overlooked, such as material compatibility or solubility. In this paper, we propose a new doping method utilizing fused organic solid solutions (FOSSs) which are prepared via high‐pressure and high‐temperature processing. By preparing fused solid solutions of organic compounds, the stable materials systems can be selected for device fabrication. Furthermore, by using these FOSSs, doping concentration and uniformity can be precisely controlled using only one thermal source. As an example of application in organic thin films, high‐performance organic light‐emitting diodes with both single‐color and white‐light emission have been prepared using this new method. Compared to the traditional co‐evaporation method, a FOSS provides us with a more convenient way to optimize the doping system and fabricate relatively complicated organic devices.

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