PNA synthesis using a novel Boc/acyl protecting group strategy.

The synthesis of novel Boc/acyl protected monomers for the synthesis of peptide nucleic acid (PNA) is described. The oligomerization protocol using these new monomers has been optimized with regard to coupling reagents. The use of base-labile acyl protecting groups at the exocyclic amines of the heterocyclic bases (isobutyryl for guanine and benzoyl for adenine and cytosine) and a PAM-linked solid support offers an attractive alternative to the present procedures used in PNA synthesis. This strategy has been applied for the synthesis of a test 17mer PNA on both control pore glass (CPG) and a polystyrene MBHA support and was used in the preparation of PNA-DNA chimeras.     

Foldamers Derived From Nucleoside β-Amino Acids: Pna Or Dna? Can We Have Both In One Place?

The synthesis of a modified thymidine (nucleoside β-amino acid) monomer and preliminary investigations into the solid phase peptide synthesis of PNA/DNA chimeras containing a neutral, internucleoside amide linkage are described.     

Facile synthesis of peptide nucleic acids and peptide nucleic acid‐peptide conjugates on an automated peptide synthesizer

Peptide nucleic acids (PNAs) are DNA mimics with a neutral peptide backbone instead of the negatively charged sugar phosphates. PNAs exhibit several attractive features such as high chemical and thermal stability, resistance to enzymatic degradation, and stable binding to their RNA or DNA targets in a sequence‐specific manner. Therefore, they are widely used in molecular diagnosis of antisense‐targeted therapeutic drugs or probes and in pharmaceutical applications. However, the main hindrance to the effective use of PNAs is their poor uptake by cells as well as the difficult and laborious chemical synthesis. In order to achieve an efficient delivery of PNAs into cells, there are already many published reports of peptides being used for transport across the cell membrane. In this protocol, we describe the automated as well as cost‐effective semi‐automated synthesis of PNAs and PNA‐peptide constructs on an automated peptide synthesizer. The facile synthesis of PNAs will be helpful in generating PNA libraries usable, e.g. for high‐throughput screening in biomolecular studies. Efficient synthetic schemes, the automated procedure, the reduced consumption of costly reagents, and the high purity of the products are attractive features of the reported procedure. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

功能核酸DNA水凝胶的制备与组装

功能核酸DNA水凝胶是一种以DNA为构建单元通过化学反应或物理缠结自组装而成的新型柔性材料,其构建单元中包含1种或多种能够形成功能核酸的特定序列。功能核酸是通过碱基修饰和DNA分子之间的相互作用力组合的一类特定核酸结构,包括核酸适配体、DNA核酶、G-四联体(G-quadruplex,G4)和i-motif结构等。传统上,高浓度的长DNA链是制备DNA水凝胶的必要条件,而核酸扩增方法的引入为DNA水凝胶的组装方式提供了新的可能。因此,对常用于制备DNA水凝胶的多种功能核酸以及核酸的提取、合成和扩增手段进行了详细的介绍。在此基础上,综述了通过化学或物理交联方式组装功能核酸DNA水凝胶的制备方法。最后,提出了DNA纳米材料的组装所面临的挑战和潜在的发展方向,以期为开发高效组装的功能核酸DNA水凝胶提供参考。     

Synthèses ďacides nucléiques et de protéines au cours de ľinitiation de bourgeons adventifs sur des explantats de Cichorium intybus cultivés in vitro

Synthesis of nucleic acids and proteins during initiation of vegetative buds from explants of Cichorium intybus cultivated in vitro .
Explants of 6 mm diameter and 2 mm thickness from roots of Cichorium intybus L. (var. WitIoof cv. "Zoom"), grown in vitro in the presence of kinetin (l0_–6 M ), produced only buds. By comparing the histological events and the biochemical modifications which occurred during the neoformation of buds, it is possible to distinguish two distinct phases. The first phase starts immediately after the excision of the explants and continues for 30 h, when the mitotic index is at its highest. This corresponds to a phase of cellular activation, which is characterized by early synthesis of RNA, permitting the synthesis of proteins necessary for duplication of DNA and cell divisions. The second period starts after 30 h and ends after approximately 72 h of culture, at which time the first bud meristematic nodules were detected. This is a preparatory phase for organogenesis and above all related to synthesis of RNA and proteins.     

Effects of Fusarium moniliforme culture extracts and fumonisin B1 on DNA,RNA and protein synthesis by baby hamster kidney cells

Baby hamster kidney cells (BHK-21) were exposed to culture filtrates of 4 Fusarium moniliforme isolates containing varying levels of fumonisin B1 (FMB1) and the effects upon RNA, DNA and protein synthesis were monitored. Cells were also grown on medium amended with FMB1 only for comparison. After 24 h incubation FMB1 (100 μg/100 ml medium) reduced protein synthesis by 4% and by 18% after 48 h. Culture filtrates containing the highest levels of FMB1 also caused the greatest inhibition in protein synthesis after 24 h but after 48 h protein synthesis levels were the same as controls even though the FMB1 level was 360 μg/100 ml. Only FMB1 reduced DNA synthesis, by 8% after 24 h but after 48 h DNA levels had increased by 40 % over controls. The culture filtrates containing the highest levels of FMB1 (360 μg/100 ml) reduced DNA synthesis more than 50% after 24 h and 48 h. Culture filtrates containing lesser amounts of FMB1 in some instances stimulated DNA synthesis and inhibited it in others. There was also no correlation in the level of FMB1 with the inhibition of RNA synthesis by BHK cells. It appears that metabolites other than fumonisin produced by F. moniliforme in culture can affect and both stimulate and inhibit RNA, DNA and protein synthesis by BHK cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

A rapid coupling protocol for the synthesis of peptide nucleic acids

With the current interest in anti-sense and anti-gene technologies, an efficient, fast and less toxic synthesis protocol would be advantageous for the oligomerisation of Peptide Nucleic Acids (PNA). Most of the methods currently in use for the t-Boc synthesis of PNA's use TFA/m-cresol, pyridine, piperidine and capping reagents. In this work, a rapid synthesis protocol has been adapted from an earlier published peptide synthesis method allowing a reduction in cycle time from around 30 min down to 16 min. By utilising quantitative deprotection with 100% TFA, a coupling time of 10 min and a four-fold excess of monomer, this synthesis protocol has been used to synthesise a number of PNA's incorporating all four nucleotides of varying sequence, up to 17 residues in length.     

A rapid coupling protocol for the synthesis of peptide nucleic acids

Summary With the current interest in anti-sense and anti-gene technologies, an efficient, fast and less toxic synthesis protocol would be advantageous for the oligomerisation of Peptide Nucleic Acids (PNA). Most of the methods currently in use for thet-Boc synthesis of PNA's use TFA/m-cresol, pyridine, piperidine and capping reagents. In this work, a rapid synthesis protocol has been adapted from an earlier published peptide synthesis method allowing a reduction in cycle time from around 30 min down to 16 min. By utilising quantitative deprotection with 100% TFA, a coupling time of 10 min and a four-fold excess of monomer, this synthesis protocol has been used to synthesise a number of PNA's incorporating all four nucleotides of varying sequence, up to 17 residues in length.     

Mode of action of a 5-nitrofuran drug (SQ 18506) against Trypanosoma cruzi.

In order to determine the mode of action of SQ18506 (a 5-nitrofuran drug) against Trypanosoma cruzi, we investigated the action of the drug in critical biochemical areas: energy metabolism, membranes and their functioning, macromolecular synthesis. In these areas SQ18506, at physiological concentrations, only caused substantial effects on macromolecular synthesis, and in particular on the incorporation of radioacitve precursors into DNA and RNA. For this reason the mode of action of SQ18506 was considered to be inhibition of nucleic acid synthesis.     

Conformationally Restricted Chiral Peptide Nucleic Acids Derived from Azetidines

Abstract

The initial experiments towards the chemical synthesis of conformationally rigid peptide nucleic acid analogues with azetidine moieties have been described.     

绿豆子叶衰老期间核酸代谢的变化

萌发绿豆的子叶自然衰老期间,核酸含量降低,RNA降低的幅度比DNA大。电泳分析结果表明,子叶衰老期间细胞核主带DNA明显降低;而迁移慢的卫星带DNA变化不大。在RNA各组分中,18S rRNA从衰老前期就开始降低;25S rRNA和4～5S小分子RNA到衰老后期才缓慢下降。DNase和RNase活性在子叶整个衰老期间都明显升高,是导致核酸含量下降的主要原因。~3H-核苷掺入试验表明,核酸的合成速率在子叶衰老前期有所上升,到衰老后期又降低。poly(A)~ -mRNA含量在子叶开始衰老时明显上升。     

Synthesis of a new disulfide Fmoc monomer for creating biologically susceptible linkages in peptide nucleic acid oligomers

Peptide nucleic acids (PNA) are one of many synthetic mimics of DNA and RNA that have found applications as biological probes, as nano-scaffold components, and in diagnostics. In an effort to use PNA as constructs for cellular delivery we investigated the possibility of installing a biologically susceptible disulfide bond in the backbone of a PNA oligomer. Here we report the synthesis of a new abasic Fmoc monomer containing a disulfide bond that can be incorporated into a PNA oligomer (DS-PNA) using standard solid phase peptide synthesis. The disulfide bond survives cleavage from the resin and DS-PNA forms duplexes with complementary PNA oligomers. Initial studies aimed at determining if the disulfide bond is cleavable to reducing agents while in a duplex are explored using UV thermal analysis and HPLC.     

RNA—RNA原位杂交实验条件探讨

将Vigilin和qroal(Ⅰ)cDNA亚克隆到pGEM3Z和pGEM4Z载体,体外转录合成35s标记的cRNA探针。经RNA凝胶电泳,Southern Northern,杂交检查探针长度,杂交特性和特异性,通过系列实验探讨了RNA-RNA原位杂交实验中固定、杂交前处理、杂交温度,探针量、探针长度,洗脱严格性和RNA酶处理等对杂交结果的影响,建立了简化的RNA-RNA原位杂交方法。     

β-PNA: peptide nucleic acid (PNA) with a chiral center at the β-position of the PNA backbone

Peptide nucleic acid (PNA) monomers with a methyl group at the β-position have been synthesized. The modified monomers were incorporated into PNA oligomers using Fmoc chemistry for solid-phase synthesis. Thermal denaturation and circular dichroism (CD) studies have shown that PNA containing the S-form monomers was well suited to form a hybrid duplex with DNA, whose stability was comparable to that of unmodified PNA–DNA duplex, whereas PNA containing the R-form monomers was not.     

多肽核酸

通过计算机模型设计出一种以肽骨架取代ＤＮＡ中脱氧核糖磷酸酯骨架的ＤＮＡ类似物．实验已证明其与互补寡核苷酸的结合具有高度的特异性．综述了其合成、杂交性及在分子生物学等方面的应用．     

肽核酸——一种新型的反义物质

肽核酸是以肽为骨架的一种新型ＤＮＡ模拟物。已经证明肽核酸具有与ＤＮＡ和ＲＮＡ结合的高度亲合性、良好的稳定性及能方便地固相合成等特性。在反义技术和基因治疗中有着很好的前景。本文综述了肽核酸的生物化学特性及其在反义技术方面的应用。     

Stabilization of duplex DNA and RNA by dangling ends studied by free energy simulations

Single unpaired nucleotides at the end of double‐stranded nucleic acids, termed dangling ends, can contribute to duplex stability. Umbrella sampling free energy simulations of dangling cytosine and guanine nucleotides at the end of duplex and single stranded RNA and DNA molecules have been used to investigate the molecular origin of dangling end effects. In unrestraint simulations, the dangling end nucleotides stayed close to placements observed in experimental structures. Calculated free energy contributions associated with the presence of dangling nucleotides were in reasonable agreement with experiment predicting the general trend of a more stabilizing effect of purine vs. pyrimidine dangling ends. In addition, the calculations indicate a more significant stabilizing effect of dangling ends at the 5′‐end vs. 3′‐end in case of DNA and the opposite trend in case of RNA. Both electrostatic and van der Waals interactions contribute to the duplex stabilizing effect of dangling end nucleotides. The free energy simulation scheme could also be used to design dangling end nucleotides that result in enhanced duplex stabilization. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 418–427, 2014.     

Experimental Evidence That GNA and TNA Were Not Sequential Polymers in the Prebiotic Evolution of RNA

Systematic investigation into the chemical etiology of ribose has led to the discovery of glycerol nucleic acid (GNA) and threose nucleic acid (TNA) as possible progenitor candidates of RNA in the origins of life. Coupled with their chemical simplicity, polymers for both systems are capable of forming stable Watson-Crick antiparallel duplex structures with themselves and RNA, thereby providing a mechanism for the transfer of genetic information between successive genetic systems. Investigation into whether both polymers arose independently or descended from a common evolutionary pathway would provide additional constraints on models that describe the emergence of a hypothetical RNA world. Here we show by thermal denaturation that complementary GNA and TNA mixed sequence polymers are unable, even after prolonged incubation times, to adopt stable helical structures by intersystem cross-pairing. This experimental observation suggests that GNA and TNA, whose structures derive from one another, were not consecutive polymers in the same evolutionary pathway to RNA. Reviewing Editor: Dr. Niles Lehman     

Qualitative and Quantitative Studies on DNA and RNA Synthesis in Loxodes striatus Nuclei*

SYNOPSIS. In this study the characteristics of the synthesis of DNA and RNA in the nuclei of Loxodes were investigated. Loxodes striatus is a primitive ciliate with 2 pairs of structurally differentiated diploid nuclei, the macro- and micronuclei. The macronuclei are differentiated morphologically into a clearly recognizable central core and an outer zone. To determine DNA and RNA synthesis, individual organisms were analyzed by autoradiography after incubating groups of cells with a ³H-labeled precursor ([³H]thymidine for DNA and [³H]uridine for RNA). The following observations were made: (A) All portions of macro- and micronuclei appeared to contain DNA as judged by the localizations of incorporated [³H]thymidine. (B) The macro- and micronuclei did not synthesize DNA at the same time; moreover, the duration of DNA synthesis in the former was much longer than of the latter nucleus. (C) Replication of DNA in the inner core and outer zone of the macronucleus occurred at separate times with little if any overlap. (D) All of the detectable [³H]uridine incorporation was found in the macronucleus and none in the micronucleus. Within the macro-nucleus the central core was more heavily labeled. (E) The quantitative differences in the label of the different components of the nuclear complex were investigated. (F) Contrary to the previously reported information our results suggest that DNA synthesis can occur in adult macronuclei. The possible explanation of these results is discussed in the context of the nuclear evolution of ciliates and of recent information on nuclear differentiation.     

功能核酸概念的内涵与外延

对功能核酸概念的分析需要建立在对功能核酸研究的基础上,从内涵和外延两个方面来进行探析。从内涵来看,它是对具有特殊结构、执行特定生物功能的核酸分子的统称;从外延来看,它包括适体、核酸核酶、核糖开关、发光核酸、修饰核酸、功能核酸裁剪、核酸自组装、功能核酸纳米材料、核酸纳米酶、核酸药物、核酸补充剂以及DNA存储技术等。目前功能核酸已成功地应用于生物传感、生物成像、生物医学等诸多领域。对功能核酸这一概念进行了探讨,并尝试对其范畴、特点进行归纳总结,以期梳理和完善功能核酸的基本概念,促进该领域的进一步发展。