细胞特异性核酸适配体筛选新策略的研究进展

核酸适配体(aptamer)是在体外采用指数富集的配基系统进化技术(systematic evolution of ligands by exponential enrichment, SELEX)从人工合成的随机寡核苷酸文库中筛选得到的一段寡核苷酸序列(RNA或DNA),能折叠成特定的三维空间结构同靶物质进行高特异性与高亲和性的结合。近年来,以全细胞为靶标筛选(cell-SELEX)获得的核酸适配体在疾病相关的领域有很大的应用潜力,尤其在细胞分子的识别、生物标志物的发现等方面,但cell-SELEX的过程复杂、难度大及获得的核酸适配体性能不佳等问题仍然制约着细胞特异性核酸适配体的进一步发展,如何高效地筛选获得核酸适配体是其应用的关键。本文总结了目前在cell-SELEX技术基础上发展起来的新方法、新策略及核酸适配体在肿瘤研究中的应用,望为相关科研人员的研究提供参考。     

A Polymeric Support for Chemical and Enzymatic Nucleic Acid Synthesis (1)

Abstract

A non-swellable, highly porous support material -CPG 3000 - was used in building up covalently bound nucleic acids by combined chemical and enzymatic methods. Bases are optimal accessible for hybridization and enzymatic reactions because they are not involved in the linking procedure.     

Design of a New Type of Compact Chemical Heater for Isothermal Nucleic Acid Amplification

Previous chemical heater designs for isothermal nucleic acid amplification have been based on solid-liquid phase transition, but using this approach, developers have identified design challenges en route to developing a low-cost, disposable device. Here, we demonstrate the feasibility of a new heater configuration suitable for isothermal amplification in which one reactant of an exothermic reaction is a liquid-gas phase-change material, thereby eliminating the need for a separate phase-change compartment. This design offers potentially enhanced performance and energy density compared to other chemical and electric heaters.     

Nucleic Acid Amplification Strategies for DNA Microarray-Based Pathogen Detection

DNA microarray-based screening and diagnostic technologies have long promised comprehensive testing capabilities. However, the potential of these powerful tools has been limited by front-end target-specific nucleic acid amplification. Despite the sensitivity and specificity associated with PCR amplification, the inherent bias and limited throughput of this approach constrain the principal benefits of downstream microarray-based applications, especially for pathogen detection. To begin addressing alternative approaches, we investigated four front-end amplification strategies: random primed, isothermal Klenow fragment-based, 29 DNA polymerase-based, and multiplex PCR. The utility of each amplification strategy was assessed by hybridizing amplicons to microarrays consisting of 70-mer oligonucleotide probes specific for enterohemorrhagic Escherichia coli O157:H7 and by quantitating their sensitivities for the detection of O157:H7 in laboratory and environmental samples. Although nearly identical levels of hybridization specificity were achieved for each method, multiplex PCR was at least 3 orders of magnitude more sensitive than any individual random amplification approach. However, the use of Klenow-plus-Klenow and 29 polymerase-plus-Klenow tandem random amplification strategies provided better sensitivities than multiplex PCR. In addition, amplification biases among the five genetic loci tested were 2- to 20-fold for the random approaches, in contrast to >4 orders of magnitude for multiplex PCR. The same random amplification strategies were also able to detect all five diagnostic targets in a spiked environmental water sample that contained a 63-fold excess of contaminating DNA. The results presented here underscore the feasibility of using random amplification approaches and begin to systematically address the versatility of these approaches for unbiased pathogen detection from environmental sources.     

Automated Chemical Synthesis of PNA and PNA-DNA Chimera on a Nucleic Acid Synthesizer

Abstract

Automated chemical synthesis of PNA and PNA-DNA chimera on a DNA synthesizer using the monomethoxytrityl/acyl protecting group strategy is described.     

Method for the Synthesis of Uric Acid Derivatives

Abstract

A general procedure to obtain tetra-substituted uric acid by stepwise N-alkylation is described. 2,6-Dichloropurine (1) was condensed with 1-propanol by Mitsunobu reaction to give 9-propyl congener (2). Treatment of 2 with ammonia gave adenine derivative (4a), which was converted to the 8-oxoadenine (5b) in 3 steps. Methylation of 5b proceeded site-specifically to give 6-amino-2-chloro-7,8-dihydro-7-methyl-9-propylpurin-8-one (6) as a sole product. Compound 6 was successively treated with NaNO₂ and iodomethane to give 2-chloro-1,6,7,8-tetrahydro-1,7-dimethyl-9-propylpurin-6,8-dione (9) accompanied by the O ⁶-methyl product (8) in 75% and 6.9%, respectively. After nucleophilic substitution of 9 with NaOAc, the product (11) was reacted with iodomethane to give the uric acid (12) and the 2-methoxy product (13) in 46% and 15.5%, respectively. However, the reaction of 11 with the benzylating agents gave only O-benzyl products (14a,b).     

Synthesis of Peptide Nucleic Acid Monomers

Abstract

The chemical synthesis of peptide nucleic acid (PNA) monomers is described using Fmoc (backbone), anisoyl (cytosine, adenine), 4-tert-butylbenzoyl (cytosine) and isobutyryl/diphenylcarbamoyl (guanine) protecting group combinations. For the guanine monomer the alkylation was realized both in a Mitsunobu [DIAD, triphenylphosphine or (4-dimethylaminophenyl)diphenylphosphine, tert-butyl glycolate] and in a low-temperature, sodium-hydride mediated alkylation (tert-butyl bromoacetate) to give the N⁹ -substituted derivative.     

Synthesis of Dihydroquinoline-Based Derivatives of Fluorescent Rhodamine Dyes for Nucleic Acid Analysis by a Real-Time Polymerase Chain Reaction

Russian Journal of Bioorganic Chemistry - An optimized synthesis scheme for the dihydroquinoline-based derivatives of xanthene fluorescent dye has been developed. For the first time, their use as...     

Towards a Chemical Etiology of Nucleic Acid Structure

To Leslie E. Orgel, arbiter elegantiarum of contemporary origin-of-life science and far-sighted pioneer of non-enzymic molecular replication, on the occasion of his 70th birthday, with cordial congratulations and best wishes.In the sequel of some general remarks on a chemical etiology of nucleic-acid structure, the paper presents a reproduction of the sequence of slides which were shown in the author's lecture Pyranosyl-RNA at the 8. ISSOL Conference in Orléans. Each slide figure is accompanied by a short explanatory comment.     

Liquid Phase Synthesis of Peptide Nucleic Acid (or Polyamide Nucleic Acid) Dimers

Abstract

The liquid phase synthesis of “polyamide nucleic acid” (PNA) dimers containing the purine nucleic acid bases adenine and guanine has been achieved in good yields. This strategy was elaborated in order to circumvent difficult direct coupling of protected PNA monomers. This method can be applied to the liquid phase synthesis of short protected polyPNAs fragments, which can then selectively be deprotected.     

Nucleic Acid Synthesis in the Chloroplasts of Acetabularia mediterranea

Radioautographic and radiochemical techniques were used to establish the presence of replicating DNA in the chloroplasts of Acetabularia mediterranea. These techniques also demonstrated that these chloroplasts synthesize RNA. It was found that label from thymine was also incorporated into DNA and RNA in these chloroplasts.

With the establishment of protein and nucleic acid synthesis in Acetabularia chloroplasts, it is clear that these chloroplasts carry out those metabolic processes which are most characteristic of autonomous cells.

     

An Improved Screening Method for Inhibitors of Nucleic Acid Synthesis

A screening method for the antibiotics capable of inhibiting synthesis of nucleic acid in bacterial cells or mammalian tumor cells was investigated. The DNA and RNA syntheses in Bacillus subtilis 168 thymine^?, indole^? were studied by the assay of incorporations of ³H-thymine and ¹⁴C-uracil into the cells, respectively. With known antibiotics against nucleic acid synthesis, the adequacy of the method was examined, and the result proved that this method is more sensitive and specific than the conventional assay methods.

It was found as a new fact that cellocidin is a potent and specific inhibitor to the thymine incorporation into DNA.

By an almost similar procedure, an inhibitory effect of several antibiotics on the incorporations of ³H-thymidine and ¹⁴C-uridme into Ehrlich ascites carcinoma cells was also studied.     

Nucleic Acid Components and Their Analogues: New Synthesis of Bicyclic Thiopyrimidine Nucleosides

Abstract

A novel synthesis of condensed bicyclic thiopyrimidine glycosides utilising 1H-cyclopentapyrimidine-2(3H)-thiones and α-bromoglucose or α-bromogalactose tetraacetate as starting components is described.     

乌索酸与齐墩果酸衍生物的合成进展

乌索酸和齐墩果酸在多种植物中均有分布.二者互为同分异构体,均属于五环三萜类化合物,由于其结构多样性而具有广泛的生物活性,包括抗肿瘤、抗氧化、抗艾滋病、抗溃疡、抗炎、免疫调节等功能.有关这两种活性物质的衍生化研究一直为科研人员所关注,本文主要综述了近10年来二者衍生物合成方面的新进展,为寻找活性先导化合物、合理设计药物分子提供依据.     

Localization of Nucleic Acid Synthesis in Root Meristems

Adenine-8-C1¹⁴ was supplied to roots of Vicia faba and Alliumascalonicum and its incorporation into DNA was studied fromautoradiographs of hydrolysed sections. These roots have a quiescentcentre to the meristem where the cells do not synthesize DNAand probably, therefore, play no part in the construction ofthe root. The boundary between the quiescent centre and thecentral cap initials is clearly denned and this suggests thatthere is as little cell interchange between the histogens asthere is in roots with visibly discrete histogens.     

Enzymatic Synthesis of Cinnamic Acid Derivatives

Using Novozym 435 as catalyst, the syntheses of ethyl ferulate (EF) from ferulic acid (4-hydroxy 3-methoxy cinnamic acid) and ethanol, and octyl methoxycinnamate (OMC) from p-methoxycinnamic acid and 2-ethyl hexanol were successfully carried out in this study. A conversion of 87% was obtained within 2 days at 75 °C for the synthesis of EF. For the synthesis of OMC at 80 °C, 90% conversion can be obtained within 1 day. The use of solvent and high reaction temperature resulted in better conversion for the synthesis of cinnamic acid derivatives. Some cinnamic acid esters could also be obtained with higher conversion and shorter reaction times in comparison to other methods reported in the literature. The enzyme can be reused several times before significant activity loss was observed. Revisions requested 10 January 2006; Revisions received 17 January 2006     

Synthesis and Immunomodulating Activity of New Amino Acid Derivatives of Glycyrrhizic Acid and Its Methyl Ester

New amino acid derivatives of glycyrrhizic acid and its methyl ester were selectively synthesized using active N-succinimide esters. The compounds with residues of glycine ethyl ester and alanine methyl and butyl esters increased the level of agglutinins and hemolysins in blood serum of mice two- to threefold in comparison with the control upon parenteral administration at a dose of 2 mg/kg for 14 days.