Controlled intracellular localization and enhanced antisense effect of oligonucleotides by chemical conjugation

Oligonucleotides can be covalently linked to peptides composed of any sequence of amino acids by solid phase fragment condensation. The peptides incorporated into the conjugates include nuclear localizing signals (NLS), nuclear export signals (NES), membrane fusion domain of some viral proteins and some designed peptides with amphipathic character. Evaluation of biological properties of DNA-peptide conjugates indicated that (a) the conjugates could bind to target RNA and dsDNA with increased affinity, (b) the conjugates were more resistant to cellular nuclease degradation, (c) the conjugate-RNA hybrids could activate RNase H as effectively as native oligonucleotides, (d) the conjugates with fusion peptides showed largely enhanced cellular uptake, (e) the conjugates with NLS could be predominantly delivered into the cell nucleus, (f) the conjugates with NES could be localized in the cytoplasm. As a result, antisense oligonucleotides conjugated with NLS could inhibit human telomerase in human leukemia cells much more strongly than phosphorothioate oligonucleotides.     

Identification of novel ligands for the Z-DNA binding protein by structure-based virtual screening

We describe the first discovery of small molecules that bind to the Z-DNA binding domain of human ADAR1 (Adenosine Deaminase Acting on RNA 1) by structure-based virtual screening of chemical database. These molecules bind to Z-DNA binding domain to inhibit the interaction with the Z-DNA. Many viruses have Z-DNA binding proteins, which are structurally similar to Z-DNA binding domain of human ADAR1, and the ability of Z-DNA binding protein to bind the Z-DNA is essential for their pathogenicity. Therefore, the molecules identified in this study may serve as novel leads for the design of agents that inhibit biological functions of those pathogenic viruses.     

Site-specific cleavage of RNA by a metal-free artificial nuclease attached to antisense oligonucleotides

RNA cleaving tris(2-aminobenzimidazoles) have been attached to DNA oligonucleotides via disulfide or amide bonds. The resulting conjugates are effective organocatalytic nucleases showing substrate and site selectivity as well as saturation kinetics. The benzimidazole conjugates also degrade enantiomeric RNA. This observation rules out contamination effects as an alternative explanation of RNA degradation. The pH dependency shows that the catalyst is most active in the deprotonated state. Typical half-lifes of RNA substrates are in the range of 12-17 h. Thus, conjugates of tris(2-aminobenzimidazoles) can compete with the majority of metal-dependent artificial nucleases.     

In Vitro Selection of Specific Ligand-binding Nucleic Acids

In vitro selection is a method that allows the simultaneous screening of very large numbers of nucleic acid molecules for a wide range of properties from binding characteristics to catalytic properties; moreover, the isolation of the very rare functional molecules becomes possible. Binding sites between proteins and nucleic acids, for example, have been evaluated by this methodology in order to gain information about protein/nucleic acid interactions. Structure and function of catalytic RNA (“ribozymes”) has been studied by in vitro selection and has led to new ribozymes with improved catalytic function. Substrate specificity of catalytic RNA has been changed and has led to a ribozyme that cleaves DNA. Other applications include the isolation of nucleic acids that bind specifically to small organic molecules and of RNA molecules that form triple helices with double-stranded DNA. In this article we discuss the background, design, and results of in vitro genetic experiments, which bridge biochemical/molecular biological and organic chemical approaches to molecular recognition.     

Templated Formation of Discrete RNA and DNA:RNA Hybrid G‐Quadruplexes and Their Interactions with Targeting Ligands

G‐rich RNA and DNA oligonucleotides derived from the human telomeric sequence were assembled onto addressable cyclopeptide platforms through oxime ligations and copper‐catalyzed azide‐alkyne cycloaddition (CuAAc) reactions. The resulting conjugates were able to fold into highly stable RNA and DNA:RNA hybrid G‐quadruplex (G4) architectures as demonstrated by UV, circular dichroism (CD), and NMR spectroscopic analysis. Whereas rationally designed parallel RNA and DNA:RNA hybrid G4 topologies could be obtained, we could not force the formation of an antiparallel RNA G4 structure, thus supporting the idea that this topology is strongly disfavored. The binding affinities of four representative G4 ligands toward the discrete RNA and DNA:RNA hybrid G4 topologies were compared to the one obtained with the corresponding DNA G4 structure. Surface plasmon resonance (SPR) binding analysis suggests that the accessibility to G4 recognition elements is different among the three structures and supports the idea that G4 ligands might be shaped to achieve structure selectivity in a biological context.     

Design of Bioinorganic Materials at the Interface of Coordination and Biosupramolecular Chemistry

Protein assemblies have recently become known as potential molecular scaffolds for applications in materials science and bio‐nanotechnology. Efforts to design protein assemblies for construction of protein‐based hybrid materials with metal ions, metal complexes, nanomaterials and proteins now represent a growing field with a common aim of providing novel functions and mimicking natural functions. However, the important roles of protein assemblies in coordination and biosupramolecular chemistry have not been systematically investigated and characterized. In this personal account, we focus on our recent progress in rational design of protein assemblies using bioinorganic chemistry for (1) exploration of unnatural reactions, (2) construction of functional protein architectures, and (3) in vivo applications.     

Recognition of chromosomal DNA by PNAs

The recognition of cellular nucleic acids by synthetic oligonucleotides is a versatile strategy for regulating biological processes. The vast majority of published studies have focused on antisense oligonucleotides that target mRNA, but it is also possible to design antigene oligonucleotides that are complementary to chromosomal DNA. Antigene oligomers could be used to inhibit the expression of any gene or analyze promoter structure and the mechanisms governing gene regulation. Other potential applications of antigene oligomers include activation of expression of chosen genes or the introduction of mutations to correct genetic disease. Peptide nucleic acid (PNA) is a nonionic DNA/RNA mimic that possesses outstanding potential for recognition of duplex DNA. Here we describe properties of PNAs and the challenges for their development as robust antigene agents.     

Toward the synthesis of artificial proteins: the discovery of an amphiphilic helical peptoid assembly

While nature exploits folded biopolymers to achieve molecular recognition and catalysis, comparable abiological heteropolymer systems have been difficult to create. We synthesized and identified abiological peptoid heteroploymers capable of binding a dye. Using combinatorial synthesis, we constructed a library of 3400 amphiphilic 15-mer peptoids on an ultra-high-capacity beaded support. Individual macrobeads, each containing a single peptoid sequence, were arrayed into plates, cleaved, and screened in aqueous solution to locate dye binding heteropolymer assemblies. Resynthesis and characterization demonstrated the formation of defined helical assemblies as judged by size-exclusion chromatography, circular dichroism, and analytical ultracentrifugation. Inspired by nature's process of sequence variation and natural selection, we identified rare abiological sequence-specific heteropolymers that begin to mimic the structure and functional properties of their biological counterparts.     

Recent advances in biocompatible supramolecular assemblies for biomolecular detection and delivery

Inspired by sophisticated biological structures and their physiological processes,supramolecular chemistry has been developed for understanding and mimicking the behaviors of natural species. Through spontaneous self-assembly of functional building blocks,we are able to control the structures and regulate the functions of resulting supramolecular assemblies.Up to now,numerous functional supramolecular assemblies have been constructed and successfully employed as molecular devices, machines and biological diagnostic platforms.This review will focus on molecular structures of functional molecular building blocks and their assembled superstructures for biological detection and delivery.     

肽与寡核苷酸缀合物的合成及应用研究进展

寡核苷酸可作为基因表达的抑制剂和潜在的治疗药物,但许多类型的寡核苷酸为聚阴离子化合物,难以跨过细胞膜,而许多生物活性肽具有跨膜与核定位能力,通过合成的方法可以将这两种具有重要功能的生物高聚物以共价键连接在一起,从而实现药物的有效载运．本文综述了肽寡核苷酸缀合物的合成方法及其应用．     

Polyphenol‐Mediated Assembly of Proteins for Engineering Functional Materials

Functional materials composed of proteins have attracted much interest owing to the inherent and diverse functionality of proteins. However, establishing general techniques for assembling proteins into nanomaterials is challenging owing to the complex physicochemical nature and potential denaturation of proteins. Here, a simple, versatile strategy is introduced to fabricate functional protein assemblies through the interfacial assembly of proteins and polyphenols (e.g., tannic acid) on various substrates (organic, inorganic, and biological). The dominant interactions (hydrogen‐bonding, hydrophobic, and ionic) between the proteins and tannic acid were elucidated; most proteins undergo multiple noncovalent stabilizing interactions with polyphenols, which can be used to engineer responsiveness into the assemblies. The proteins retain their structure and function within the assemblies, thereby enabling their use in various applications (e.g., catalysis, fluorescence imaging, and cell targeting).     

Peptide Nanomaterials Designed from Natural Supramolecular Systems

Natural supramolecular assemblies exhibit unique structural and functional properties that have been optimized over the course of evolution. Inspired by these natural systems, various bio‐nanomaterials have been developed using peptides, proteins, and nucleic acids as components. Peptides are attractive building blocks because they enable the important domains of natural protein assemblies to be isolated and optimized while retaining the original structures and functions. Furthermore, the peptide subunits can be conjugated with exogenous molecules such as peptides, proteins, nucleic acids, and metal nanoparticles to generate advanced functions. In this personal account, we summarize recent progress in the construction of peptide‐based nanomaterial designed from natural supramolecular systems, including (1) artificial viral capsids, (2) self‐assembled nanofibers, and (3) protein‐binding motifs. The peptides inspired by nature should provide new design principles for bio‐nanomaterials.     

Polyphenol-Mediated Assembly of Proteins for Engineering Functional Materials

Functional materials composed of proteins have attracted much interest owing to the inherent and diverse functionality of proteins. However, establishing general techniques for assembling proteins into nanomaterials is challenging owing to the complex physicochemical nature and potential denaturation of proteins. Here, a simple, versatile strategy is introduced to fabricate functional protein assemblies through the interfacial assembly of proteins and polyphenols (e.g., tannic acid) on various substrates (organic, inorganic, and biological). The dominant interactions (hydrogen-bonding, hydrophobic, and ionic) between the proteins and tannic acid were elucidated; most proteins undergo multiple noncovalent stabilizing interactions with polyphenols, which can be used to engineer responsiveness into the assemblies. The proteins retain their structure and function within the assemblies, thereby enabling their use in various applications (e.g., catalysis, fluorescence imaging, and cell targeting).     

基于冠醚衍生物的超分子聚合物

自组装现象是生命科学最本质的内容之一,生物体系可以精确地利用非共价键相互作用形成高度有序的功能组装体.受到大自然的启发,近年来利用分子自组装构筑包括超分子聚合物在内的有序聚集体是超分子科学的研究热点.此类组装体不仅在拓扑学上具有重要的意义,而且可以用来制备动态的超分子功能材料.冠醚作为第一代超分子主体化合物,由于其结构...     

Physics of engineered protein hydrogels

Artificially engineered proteins and synthetic polypeptides have attracted widespread interest as building blocks for polymer hydrogels. The biophysical properties of the proteins, such as molecular recognition abilities, folded chain structures, and sequence-dependent thermodynamic behavior, enable advances in functional, responsive, and tunable gels. This review discusses the design of polymer hydrogels that incorporate protein domains, highlighting new challenges in polymer physics that are presented by this emerging class of materials. Five types of engineered protein hydrogels are discussed: (a) physically associating protein polymer gels, (b) amorphous artificially engineered protein networks, (c) engineered proteins with crystalline domains, (d) stretchable protein tertiary structures in gels, and (e) protein gels with biological recognition properties. The physics of the protein component and the physical properties of the resulting hydrogels are summarized, illustrating how advances in understanding these systems are leading to exciting novel biofunctional hydrogels. © 2013 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys, 2013     

Nucleic acid-based fluorescent probes and their analytical potential

It is well known that nucleic acids play an essential role in living organisms because they store and transmit genetic information and use that information to direct the synthesis of proteins. However, less is known about the ability of nucleic acids to bind specific ligands and the application of oligonucleotides as molecular probes or biosensors. Oligonucleotide probes are single-stranded nucleic acid fragments that can be tailored to have high specificity and affinity for different targets including nucleic acids, proteins, small molecules, and ions. One can divide oligonucleotide-based probes into two main categories: hybridization probes that are based on the formation of complementary base-pairs, and aptamer probes that exploit selective recognition of nonnucleic acid analytes and may be compared with immunosensors. Design and construction of hybridization and aptamer probes are similar. Typically, oligonucleotide (DNA, RNA) with predefined base sequence and length is modified by covalent attachment of reporter groups (one or more fluorophores in fluorescence-based probes). The fluorescent labels act as transducers that transform biorecognition (hybridization, ligand binding) into a fluorescence signal. Fluorescent labels have several advantages, for example high sensitivity and multiple transduction approaches (fluorescence quenching or enhancement, fluorescence anisotropy, fluorescence lifetime, fluorescence resonance energy transfer (FRET), and excimer-monomer light switching). These multiple signaling options combined with the design flexibility of the recognition element (DNA, RNA, PNA, LNA) and various labeling strategies contribute to development of numerous selective and sensitive bioassays. This review covers fundamentals of the design and engineering of oligonucleotide probes, describes typical construction approaches, and discusses examples of probes used both in hybridization studies and in aptamer-based assays.     

A phosphoramidate substrate analog is a competitive inhibitor of the Tetrahymena group I ribozyme

BACKGROUND: Phosphoramidate oligonucleotide analogs containing N3'-P5' linkages share many structural properties with natural nucleic acids and can be recognized by some RNA-binding proteins. Therefore, if the N-P bond is resistant to nucleolytic cleavage, these analogs may be effective substrate analog inhibitors of certain enzymes that hydrolyze RNA. We have explored the ability of the Tetrahymena group I intron ribozyme to bind and cleave DNA and RNA phosphoramidate analogs. RESULTS: The Tetrahymena group I ribozyme efficiently binds to phosphoramidate oligonucleotides but is unable to cleave the N3'-P5' bond. Although it adopts an A-form helical structure, the deoxyribo-phosphoramidate analog, like DNA, does not dock efficiently into the ribozyme catalytic core. In contrast, the ribo-phosphoramidate analog docks similarly to the native RNA substrate, and behaves as a competitive inhibitor of the group I intron 5' splicing reaction. CONCLUSIONS: Ribo-N3'-P5' phosphoramidate oligonucleotides are useful tools for structural and functional studies of ribozymes as well as protein-RNA interactions.     

核酸适体靶向的膜蛋白识别与功能调控研究进展

膜蛋白在细胞生命活动中发挥着重要作用, 研究并调控细胞膜蛋白的结构和功能有助于阐明生命活动的基本规律, 为新型药物研发和高效疾病诊治提供研究基础. 核酸适体是一类特殊的寡核苷酸序列, 因具有特异性识别靶标的能力而被广泛用于生物传感领域. 将核酸适体与DNA纳米技术相结合, 利用DNA分子可程序化设计、 可功能化修饰等优势, 发展核酸适体靶向的膜蛋白识别与功能调控方法可为研究膜蛋白相互作用提供有力工具. 本文介绍了基于核酸适体靶向识别的DNA纳米技术在膜蛋白识别及细胞功能调控中的研究进展, 并对核酸适体靶向的膜蛋白识别及功能调控领域面临的挑战进行了分析, 对其应用前景进行了展望.