Sequence-specific cleavage of RNA by designed ribozymes

Abstract

Ribozymes that distinguish a single base change in RNA were synthesized and used to specifically cleave c-Ha-ras messenger RNA. Using phosphorothioate containing oligonucleotide substrates, we have shown that Mg²⁺ binds to the pro-R oxygen of the phosphate and that the RNA cleavage reaction occurs via an in-line mechanism. Oligoribonucleotides containing a modified nucleoside are described.     

Discrimination between 5-hydroxymethylcytosine and 5-methylcytosine by a chemically designed peptide

An artificial phosphopeptide recognized the difference between methylated and hydroxymethylated cytosines in DNA. The Sp1 zinc finger peptide substituted by phosphotyrosine effectively discriminated between 5-methylcytosine, 5-hydroxymethylcytosine ((hm)C) and unmethylated cytosine. The DNA recognition properties of the peptide differ from those of other chemicals that detect (hm)C.     

一种新型设计肽作为药物载体的潜在应用研究

目前有大量关于全程电荷匹配设计肽的应用的研究,但是关于半程电荷匹配设计肽作为药物的应用研究却未见报道.本文对新型设计的半程电荷匹配肽P9（AC-Pro-Ser-Phe-Asn-Phe-Lys-Phe-Glu-Pro-NH2）的研究发现,P9可以成功地将作为疏水药物模型的芘稳定悬浮于水溶液中.大豆卵磷脂微囊被用以模拟细胞膜.荧光数据显示芘可以借助P9的包埋作用以晶体状态存在于水溶液中.并且当包埋体加入到脂质体微囊体系中时,芘可以从P9的包埋体系中以分子形态释放到膜的脂质层内.实验发现当包埋于芘上的P9较多,包埋壁较厚,芘的释放速度相对变缓.我们可以利用这一特性,通过调节加入的P9的浓度来控制包埋于芘上P9的厚度,从而控制芘的释放速率.研究表明,具有半程电荷匹配性质的P9肽可以作为运载疏水药物的载体.     

Application research of a novel designed peptide as a potential carrier

While a great deal of research has focused on the application of full-sequence ionic complementary peptide, detection of the capability of half-sequence ionic complementary peptide such as drug carriers, is rarely reported. This paper presents that the half-sequence ionic complementary peptide P9 (AC-Pro- Ser-Phe-Asn-Phe-Lys-Phe-Glu-Pro-NH2) can successfully stabilize a model hydrophobic drug pyrene in the aqueous solution. Soybean lecithin vesicles were used to mimic plasma membranes. Fluorescence data show that the pyrene is presented in the crystalline form when stabilized by P9 solution, and molecularly migrated from its peptide encapsulations into the membrane bilayers when the suspension is mixed with lipidosome vesicles. Slower release was observed when thicker coating was applied onto pyrene, which could be to control the wall thickness coating the cargo, and consequently the release rate. The result indicated that P9, with half-sequence ionic complement, may serve as a hydrophobic compounds carrier. Supported by the National ‘985 Project’ of Ministry of Education of China     

Responsive hydrogels from the intramolecular folding and self-assembly of a designed peptide

A general peptide design is presented that links the pH-dependent intramolecular folding of beta-hairpin peptides to their propensity to self-assemble, affording hydrogels rich in beta-sheet. Chemical responsiveness has been specifically engineered into the material by linking intramolecular folding to changes in solution pH, and mechanical responsiveness, by linking hydrogelation to self-assembly. Circular dichroic and infrared spectroscopies show that at low pH individual peptides are unstructured, affording a low-viscosity aqueous solution. Under basic conditions, intramolecular folding takes place, affording amphiphilic beta-hairpins that intermolecularly self-assemble. Rheology shows that the resulting hydrogel is rigid but is shear-thinning. However, quick mechanical strength recovery after cessation of shear is observed due to the inherent self-assembled nature of the scaffold. Characterization of the gelation process, from the molecular level up through the macroscopic properties of the material, suggests that by linking the intramolecular folding of small designed peptides to their ability to self-assemble, responsive materials can be prepared. Cryo-transmission electron and laser scanning confocal microscopies reveal a water-filled porous scaffold on both the nano- and microscale. The environmental responsiveness, morphology, and peptidic nature make this hydrogel a possible material candidate for biomedical and engineering technology.     

Catalysis of acyl group transfer by a double-displacement mechanism: the cleavage of aryl esters catalyzed by calixcrown-Ba2+ complexes

The scope of the barium salt of p-tert-butylcalix[4]arene-crown-5 as a transacylation catalyst has been defined by evaluating its efficiency in the methanolysis of a series of aryl acetates at 25.0 degrees C in MeCN/MeOH 9:1 (v/v) under slightly basic conditions. In this system a phenolic hydroxyl is the acyl-receiving and -releasing unit in a double-displacement mechanism. The complexed barium ion acts both as a nucleophile carrier and a built-in Lewis acid in providing electrophilic assistance to the ester carbonyl both in the acylation and deacylation step (nucleophilic-electrophilic catalysis). Turnover capability is ensured by the acylated intermediate reacting with the solvent more rapidly than the original ester, but a serious drawback derives from the incursion of back-acylation of the liberated phenol. A gradual shift from rate-determining deacylation (p-nitrophenyl acetate) to rate-determining acylation (phenyl acetate) is observed along the investigated series. It is shown that the scope of the catalyst is restricted to acetate esters whose reactivity lies in the range approximately defined by the phenyl acetate-p-nitrophenyl acetate pair, with a maximum efficiency for p-chlorophenyl acetate. Moreover, the catalyst effectively promotes ester interchange between phenols, showing that its activity is not limited to solvolysis reactions. The very high sensitivity of the rate of acylation of the catalyst to leaving group basicity has been interpreted as due to rate-determining decomposition of the tetrahedral intermediate, which is believed to arise from the presumably low basicity of the metal ion stabilized nucleophile. The turnover frequency was in the range of 3.8 x 10(-4) min(-1) for phenyl acetate to 7.4 x 10(-3) min(-1) for p-nitrophenyl acetate ([ArOAc]0=4.0 mM]). A first attempt to enhance the rate of acylation of the catalyst through intramolecular general acid catalysis is also described.     

Kinetics of amide and peptide cleavage by alkaline hydrogen peroxide

The hydroperoxide anion cleaves unactivated amides and peptides although it is completely unreactive toward ethyl esters. The cleavage by HO2(-) proceeds faster than by OH(-) and involves additional routes with general acid assistance by H2O2 and general base assistance by OH(-) and HO2(-). Cleavage of polypeptides occurs at the N-terminal peptide bond. [reaction: see text]     

Cytocompatible encapsulation of individual Chlorella cells within titanium dioxide shells by a designed catalytic peptide

The individual encapsulation of living cells has a great impact on the areas of single cell-based sensors and devices as well as fundamental studies in single cell-based biology. In this work, living Chlorella cells were encapsulated individually with abiological, functionalizable TiO(2), by a designed catalytic peptide that was inspired by biosilicification of diatoms in nature. The bioinspired cytocompatible reaction conditions allowed the encapsulated Chlorella cells to maintain their viability and original shapes. After formation of the TiO(2) shells, the shells were postfunctionalized by using catechol chemistry. Our work suggests a bioinspired approach to the interfacing of individual living cells with abiological materials in a controlled manner.     

A new class of DNA quadruplexes formed by oligodeoxyribonucleotides containing a 3'-3' or 5'-5' inversion of polarity site

Unprecedented DNA quadruplex structures containing a 3'-3' or 5'-5' inversion of polarity site in the G-tract are presented; the quadruplexes are characterized by different elements of symmetry and glycosidic angle conformations.     

Light-activated hydrogel formation via the triggered folding and self-assembly of a designed peptide

Photopolymerization can be used to construct materials with precise temporal and spatial resolution. Applications such as tissue engineering, drug delivery, the fabrication of microfluidic devices and the preparation of high-density cell arrays employ hydrogel materials that are often prepared by this technique. Current photopolymerization strategies used to prepare hydrogels employ photoinitiators, many of which are cytotoxic and require large macromolecular precursors that need to be functionalized with moieties capable of undergoing radical cross-linking reactions. We have developed a simple light-activated hydrogelation system that employs a designed peptide whose ability to self-assemble into hydrogel material is dependent on its intramolecular folded conformational state. An iterative design strategy afforded MAX7CNB, a photocaged peptide that, when dissolved in aqueous medium, remains unfolded and unable to self-assemble; a 2 wt % solution of freely soluble unfolded peptide is stable to ambient light and has the viscosity of water. Irradiation of the solution (260 < lambda < 360 nm) releases the photocage and triggers peptide folding to produce amphiphilic beta-hairpins that self-assemble into viscoelastic hydrogel material. Circular dichroic (CD) spectroscopy supports this folding and self-assembly mechanism, and oscillatory rheology shows that the resulting hydrogel is mechanically rigid (G' = 1000 Pa). Laser scanning confocal microscopy imaging of NIH 3T3 fibroblasts seeded onto the gel indicates that the gel surface is noncytotoxic, conducive to cell adhesion, and allows cell migration. Lastly, thymidine incorporation assays show that cells seeded onto decaged hydrogel proliferate at a rate equivalent to cells seeded onto a tissue culture-treated polystyrene control surface.     

3'-immobilized probes with 2'-caps: synthesis of oligonucleotides with 2'-N-methyl-2'-(anthraquinone carboxamido)uridine residues

[reaction: see text] A synthesis for oligodeoxynucleotides with a 3'-terminal 2'-N-methyl-2'-acylamido-2'-deoxyuridine residue was developed. Unlike their unmethylated counterparts, these oligodeoxynucleotides can be stably immobilized on aldehyde-displaying glass surfaces to provide DNA microarrays. An anthraquinone carboxamido group as a 2'-substituent doubled the capture efficiency of an immobilized tetradecamer.     

Tuning the stacking properties of C3-symmetrical molecules by modifying a dipeptide motif

C3-symmetrical molecules are described which consists of a 1,3,5-benzenetricarboxamide core extended with dipeptide fragments bearing peripheral mesogenic groups. Small structural modifications in the dipeptide fragment have been performed to demonstrate their influence on the stability of the stacks and on the order within the self-assemblies formed. Seven C3-symmetrical discs have been investigated, all with different combinations of glycine, L- and/or D-phenylalanine in the dipeptide fragments. Characterization of these discotics in the neat state using differential scanning calorimetry (DSC), X-ray diffraction (XRD) and polarized optical microscopy (POM) and in solution with circular dichroism (CD), UV-visible spectroscopy, low-concentration proton nuclear magnetic resonance and IR spectroscopy reveals that there is a clear trend in the stack stability, going from the glycine-phenylalanine motifs to the phenylalanine-phenylalanine ones. The combination of a larger hydrophobic core, more confinement of space and the possibility of additional pi-pi interactions leads to more stable stacks. Surprisingly, the weakest stacks consist of discotics of which the center is extended with L-phenylalanyl-glycines and not of discotics of which the center is extended with the glycyl-L-phenylalanine sequences. Furthermore, the XRD investigations show that it is difficult to form well-ordered self-assemblies in the neat state. And, CD measurements point out that some of the discs have a very complex energy landscape in solution. These observations suggest that small differences in the balance between the secondary interactions originating from the benzenetricarboxamide core and the dipeptide fragments, have a strong influence on the order within the stack. From these results it can be concluded that subtle modifications in the peptide fragments of the discs cause significant changes in the stacking properties, stressing the importance of understanding the self-assembly mechanism of each discotic in order to clarify its self-assembly behavior.     

Identification of a potent peptide deformylase inhibitor from a rationally designed combinatorial library

Peptide deformylase catalyzes the removal of the N-terminal formyl group from nascent polypeptides during prokaryotic protein synthesis and maturation and is essential for bacterial survival. Its apparent absence from mammalian organisms makes it an attractive target for designing novel antibacterial agents. Based on the substrate specificity of peptide deformylase from Escherichia coli, a focused library of peptide thiols was synthesized on TentaGel resin using a disulfide linkage. Screening of the library against the purified deformylase was carried out in solution phase after the inhibitors were released from the resin with a reducing agent. A potent deformylase inhibitor was obtained from a 750-member library and was further optimized through rational modification into a low nanomolar inhibitor (KI = 15 nM against E. coli deformylase).     

Advanced approaches for the characterization of a de novo designed antiparallel coiled coil peptide

We report here an advanced approach for the characterization of the folding pattern of a de novo designed antiparallel coiled coil peptide by high-resolution methods. Incorporation of two fluorescence labels at the C- and N-terminus of the peptide chain as well as modification of two hydrophobic core positions by Phe/[15N,13C]Leu enable the study of the folding characteristics and of distinct amino acid side chain interactions by fluorescence resonance energy transfer (FRET) and NMR spectroscopy. Results of both experiments reveal the antiparallel alignment of the helices and thus prove the design concept. This finding is also supported by molecular dynamics simulations. Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) in combination with NMR experiments was used for verification of the oligomerization equilibria of the coiled coil peptide.