| POLYMERS Vol.63 No.10 |
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COVER STORY
Re-Programming of Macromolecules |
| COVER STORY: Highlight Reviews |
| Ribosomal Synthesis of Functional Macrocyclic Polypeptides and RaPID Discovery of Bioactive Polypeptides | Hiroaki SUGA, Toru YOSHITOMI, Masanobu NAGANO |
| <Abstract> We have developed an in vitro selection system that enables us to rapidly discover potent macrocyclic polypeptide ligands or inhibitors against various protein targets. The system is referred to as RaPID (Random nonstandard Peptide Integrated Discovery) system, where a macrocyclic polypeptide library comprising more than 1012 different species is translated and displayed on the respective mRNA sequences. Iterative affinity selection of ligands is conducted against a protein target immobilized on magnetic beads to yield desired potent molecules. The identified macrocyclic polypeptides are chemically synthesized and studied for their biological activities, showing their extraoridinary potency and specificity. This review describes the concept of this platform technology and examples of functional macrocyclic polypeptides. Keywords: Genetic Code Reprogramming / Flexizyme / Aminoacylation Ribozymes / Rebosomal Translation / Macrocyclic Polypeptide / FIT System / Nonnatural Amino Acids / RaPID System |
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| Precision Control of Monomer Sequences in Vinyl Copolymers | Kotaro SATOH, Masami KAMIGAITO |
| <Abstract> This short review describes several recent approaches to precision control of monomer sequences in vinyl copolymers mainly using radical polymerizations. They include radical chain copolymerization of a specific pair of monomers for generating 1 : 2 or ABB-monomer sequences in addition to conventional 1 : 1 alternating ones, sequential addition of vinyl monomers into the living radical polymers or oligomers, and metathesis polymerization for constructing equivalent structures of vinyl copolymers. Such precision control will contribute to construction of a novel polymer that may work as not only a bulk material but also a functional single macromolecule. Keywords: Monomer Sequnece / Living Polymerization / Radical Polymerization / Vinyl Polymer / Copolymerization / Atom Transfer Radical Addition / Alternating Copolymerization / Metathesis Polymerization |
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| Reprogramming of Nucleic Acid Ligands: “Aptamers” | Kazunori IKEBUKURO, Nasa SAVORY, Koichi ABE |
| <Abstract> Aptamers are promising ligands for molecular recognition in therapeutic and diagnostic applications. Since aptamers fold into unique structures and display specific binding to targets, optimization of their oligonucleotide sequences can potentially improve and reprogram aptamer functions. Aptamers are usually selected from a combinatorial library through iterative in vitro selection. Re-selection from a library of partially-randomized aptamer sequences or a doped library can be perfomed for further screening. Fusing one or more known aptamers in tandem is also a powerful approach to improve aptamer function based on avidity. Because a complex relationship exists between aptamer sequence and function, sequences associated with a desired function cannot be readily identified by rational approaches alone. To solve such problems, genetic algorithm-based approaches that combine in silico sequence recombination with in vitro functional screening have been developed. These methodologies permit progressive exploration of the sequence space and evolution of aptamers. We review recent trends in aptamer improvement and provide a perspective on future aptamer development. Keywords: Aptamers / Genetic Algorithms / In Silico Maturation / Sequence Optimization |
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| COVER STORY: Topics and Products |
| Expansion of Genetic Code and Protein Function | Takahiro HOHSAKA |
| <Abstract> We have developed a method for site-specific incorporation of non-natural amino acids into proteins through expansion of the genetic code. To encode non-natural amino acids, we have used a four-base codon CGGG which can be successfully decoded into non-natural amino acids by using chemically aminoacylated transfer RNA in a cell-free translation system. This method has been utilized to introduce various non-natural amino acids carrying photo-responsive, fluorescent, and polymer molecules. We have achieved the detection of ligand- or antigen-binding as fluorescence or FRET changes by introducing fluorescent non-natural amino acids into ligand-binding proteins and single-chain antibodies in a site-specific manner. The non-natural amino acid technique will be useful not only for artificial protein design but for polymer reprogramming. Keywords: Protein / Genetic Code / Non-natural amino acid / Cell-free Translation / Fluorescence / FRET / Antibody |
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| Bipedal Walking Template toward Sequence-Controlled Vinyl Polymers | Makoto OUCHI |
| <Abstract> Toward syntheses of sequence-controlled vinyl polymers, a set of orthogonal dynamic covalent bonds, i.e., N-hydroxysuccinimide (NHS)-ester and 2-pyridil disulfide (SS-pyridine), were introduced as linkers between an initiator and a vinyl monomer for living radical polymerization. The sepcial linkers allowed iterative cycles consisting of single monomer addition, cleavage and functionalization, and reinstallation of vinyl groups under a bipedal walking template mechanism. The cycle was successfully repeated twice to demonstrate the adequacy of this protocol toward sequence regulation. Keywords: Sequence / Vinyl Polymer / Living Radical Polymerization / Template / Orthogonal / Cyclization / Cleavage / Dynamic Covalent Bond |
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| Design of Functional Peptides Using Informational Analysis | Hiroyuki HONDA |
| <Abstract> Peptides have attracted attention for their variety of biological functions. Computationally-assisted peptide screening and design have become invaluable tools in overcoming the inefficiency of peptide screening because there are too huge variants of peptides. Correlation between amino acid sequence and peptide function should be revealed by the computational approach. In this column, screening and design of both bile acid binding peptide and cell adhesion peptide will be introduced. The sequence rule was successfully acquired by computational analysis. Peptides designed according to the rule were found to express an active function. Keywords: Peptide / Informatics / Amino Acid Sequence / Biological Function |
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| Preparation of Polymer Aptamer from a Copolymer Library | Yu HOSHINO |
| <Abstract> Synthetic polymer aptamers are expected as robust and inexpensive substitute for biomacromolecular ligands. However, in contrast to biomacromolecular ligands, synthetic polymers, which are prepared through one-pot chain reaction, result in heterogeneous sequences and structures with a distribution of recognition sites. To obtain polymer aptamers with strong and specific binding affinity to target molecules, strategies to design and produce polymer aptamers with defined structure have to be developed. This article provides a brief review about recent efforts to develop the polymer aptamers consisting of combination of functional acrylamides. Keywords: Aptamer / Acrylamide / Molecular Recognition / Multifunctional Polymer |
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| Creation of Nucleic Acid Aptamers That Contain Unnatural Nucleotides | Masayasu KUWAHARA |
| <Abstract> Chemically modified nucleic acid aptamers have recently attracted attention due to the potential expandability of functions as therapeutic drugs, diagnostic agents, and biosensors. The author is engaged in enzymatic syntheses of modified nucleic acids and their applications to SELEX (Systematic Evolution of Ligands by EXponential enrichment) methods to create modified nucleic acid aptamers. To date, the author and coworkers have successfully obtained modified DNA aptamers specific for R-thalidomide derivative, l-glutamic acid, and a camptothecin derivative from base-modified DNA libraries. Furthermore, 2’,4’-BNA/LNA aptamers have first been acquired from DNA-based 2’,4’-BNA/LNA library using a capillary electrophoresis (CE)-SELEX method. Recent studies on nucleic acid aptamer development indicate positive effects on the introduction of functionalities in binding affinity and specificity. Further improvements of polymerase variants and selection methodologies will provide various modified nucleic acid aptamers for practical use. Keywords: Aptamer / Polymerase Variant / Nucleoside Triphosphate / Capillary Electrophoresis / SELEX |
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| Polymer Science and I: A Personal Account |
| Still Growing Up? | Hiromu KASHIDA |
| <Abstract> Nucleic acids are often recognized as homogeneous polymers and the creation of functional base-pairs is one of my passions. This personal account describes my reseach life on nucleic acids that is guided by warm-hearted supervisors and students. |
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| Front-Line Polymer Science |
| Past, Present and Future of Single Polymer Chain Mechanics | Ken NAKAJIMA, Xiaobin LIANG |
| <Abstract> Past, present and future of single polymer chain mechanics based on atomic force microscopy were reviewed, which is generally called as single molecule force spectroscopy (SMFS). Static SMFS can provide information such as persistent length and contour length appearing in a worm-like chain model. Thiol-terminated Poly(N-isopropylacrylamide) was used as an example study and its behavior change near its LCST was investigated. The temperature dependence of the plateau force at the low-extension region agreed well with the prediction by the pearl-necklace model developed by Prof. F. Tanaka. Two types of dynamic SMFS methods were also reviewed. The first noise-analysis SMFS may need future instrumental improvement. The second forced-oscillation SMFS measurements have resulted in a lot of interesting insights. As an example, the relaxation time of the polystyrene chain in N,N-dimethylformamide was measured and its dependence on the degree of polymerization was investigated. The Kirkwood model reproduced well the experimental results, while the necessity for a new concept of effective viscosity is proposed. Keywords: Single Polymer Chain / Atomic Force Microscopy / Single Molecule Force Spectroscopy / Poly(N-isopropylacrylamide) / Relaxation Time / Kirkwood Model |
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