核苷环磷酰化反应中的立体化学

我们曾经报道了腺嘌呤核苷3′,5′-环磷酸酯和3′,5′-环磷酰胺对肿瘤细胞的DNA和RNA的合成有明显的抑制作用^[1]。研究这类化合物对进一步了解c-AMP在生物系统中的作用机制以及它们与蛋白激酶和磷酸二酯酶的作用情况有一定价值^[2]。因此在合成一系列腺嘌呤核苷3′,5′-环磷酸酯和3′,5′-环磷酰胺的基础上^[1～3],我们用2′-保护核苷与三价磷试剂反应,经过一步环磷酰化反应合成了核苷3′,5′-环亚磷酸衍生物,后者经氧化和脱保护即可得到核苷3′,5′-环磷酸衍生物。本文将报道核苷环磷酰化反应中的立体化学问题。     

核酸碱基互变异构体的结构、稳定性及其物理化学性质

核酸碱基是DNA及RNA分子的重要组成部分, 在基因遗传信息的传递方面起着主导作用. 核酸碱基存在多种互变异构体, 它们在DNA及RNA分子中主要以最稳定的异构体形式存在, 但是在气相或凝聚相中也有少量的其他异构体形式存在. 核酸碱基的稀有互变异构体往往能够引起碱基对的错配对, 这可能会导致DNA及RNA分子形成不规则的结构, 并进一步导致DNA或RNA双螺旋的自发突变. 因此, 对核酸碱基的互变异构体进行系统的研究, 有助于人们深入认识DNA和RNA分子的结构和性质. 国际上有很多研究小组已经通过实验和理论对核酸碱基互变异构体的结构、相对能量及其性质进行了研究. 本文对文献中有关核酸碱基互变异构体的实验和理论研究进行了综述. 在对前人研究进行归纳总结的基础上, 我们利用密度泛函计算对核酸碱基的互变异构体进行了排序, 得到的最优异构体结构参数和相对能量与实验值相比较为一致. 此外, 因为核酸碱基的物理化学性质可以为生物、化学、材料等方面的研究提供重要的基础性信息, 因此我们还对它们的电子亲和能、电离能、质子亲和能等研究进行了总结.     

基于核酸适体的电化学生物传感器*

核酸适体是一类体外筛选的、可与目标分子高效、高特异亲合的RNA或DNA寡核苷酸片段,与常规识别分子（如抗体等）相比,核酸适体作为一类新型识别分子具有明显特色和优势,已被广泛应用于生物传感等分子识别和应用研究领域。本文就基于核酸适体的电化学生物传感器（标记型和非标记型）的近期进展作简要评述,包括适体简介、标记型（“信号衰减”型、“信号增强”型、酶标记型和纳米粒子标记型）和非标记型电化学适体生物传感器等内容。     

脱氧核糖核酸和核糖核酸与铽离子的荧光反应及其在分析中的应用

本文提出了一种简单灵敏的测定DNA和RNA的方法。实验结果表明RNA可直接与铽离子发生荧光反应,但DNA只有在发生变性之后才能发生同样的反应。酸变性的最佳pH范围在2.2～2.8之间,通过实验确定了荧光反应的最佳条件。在最佳条件下测定DNA和RNA的线性范围在0.5～20.0μg/ml,标准偏差在5％以內。小牛胸腺DNA和鱼精子DNA在酸变性时其对铽离子荧光增强效应提高数十倍,而RNA变性前后对铽离子荧光增强效应变化不大,这本身反映了DNA与RNA二级结构的不同。利用DNA和RNA在酸变性时对Tb(Ⅱ)荧光增强变化的不同可在一定量的DNA存在下测定RNA或在一定量的RNA存在下利用差减法测DNA。     

用于基因和药物传递的多肽及聚多肽材料

多肽和聚多肽作为一类新型的生物医用材料,由于其具有良好的生物活性、生物可降解性以及生物相容性而备受瞩目.将具有特殊生理功能的多肽作为基因或药物载体、或用于药物修饰等,可以提高基因转染效率,增强药物的靶向治疗效果,降低药物的毒副作用.本文综述了近年来多肽及聚多肽材料在这些生物医学领域的应用及进展,对部分活性肽的作用机制和...     

化学核酸酶及其作用机理

化学核酸酶是一类人工设计、合成的DNA 或RNA 定位断裂工具, 由核酸识别结合系统和化学断裂系统组成。它们能够在任何位点断裂单链、双链DNA 或RNA , 不受限制性内切酶的天然专一性限制。本文除介绍了一些新的化学核酸酶体系外, 着重对它们的作用方式及作用机理进行了讨论。     

DNA酶:筛选、生物传感及展望

综述了脱氧核糖核酸酶(DNA酶)的起源及分离富集策略,对比了DNA酶与核糖核酸酶(RNA酶)及蛋白酶的相似点和不同之处,并重点讨论了产物捕获和对冲抵消等策略对筛选获得DNA酶的独到之处;同时系统回顾了近年来分离出的可特异感应各种金属离子或生物样本(包括细菌、细胞等),从而能在特定位点切割RNA底物的DNA酶探针;阐述了DNA酶领域现存的挑战,总结和展望了新思路和新方向.     

两种氧氮杂环戊二烯类化合物的紫外和荧光光谱的溶剂效应

前文报道了1,4-二(5-苯基-1,3,4-嚼二唑基-2)苯(PDPDP)紫外、荧光光谱的溶剂效应.为了与PDPDP比较,又合成了1-(5-苯基-1,3,4-噁二唑基-2)-4-(5′-苯基-1′,3′-噁唑基-2′)苯(PDPOP)和1-(5-苯基-1,3,4-噁二唑基-2)-4-(2′-苯基-1′,3′噁唑基-5′)苯(PDPO′P).     

抗肿瘤新药磷酸氟达拉宾的合成

磷酸氟达拉宾（7,fludarabine phosphate）由德国Bedex Labs开发,1991年首先在美国上市,用于治疗其它疗法无效的慢性淋巴细胞性白血病。本品摄入后经去磷酸作用生成代谢物9-β-氟阿糖腺苷（F-Ara-A）,后者在细胞内经磷酸化变成具抗肿瘤活性的F-Ara-ATP。本品的抗肿瘤机制涉及参入肿瘤细胞DNA或RNA链,使链停止延伸,以达到抑制DNA或RNA合成的目的。     

脱氧核酶传感器研究进展

脱氧核酶是利用体外分子进化技术获得的一类具有高效催化活性和结构识别能力的单链DNA片段。本文主要介绍了两类脱氧核酶,即RNA切割型脱氧核酶和G-四聚体型脱氧核酶在生物传感体系中的发展和应用。     

Intercalating nucleic acids (INAs) containing insertions of 6H-indolo[2,3-b]quinoxaline

6H-Indolo[2,3-b]quinoxaline was studied as a covalently bound heteroaromatic intercalator. Six monomers were synthesized and incorporated into DNA oligonucleotides. Through a study of linker length dependence it was concluded that the linker between the oligo and the intercalator must consist of at least five C atoms in order to stabilize a DNA duplex. An intercalator with a 2′-deoxy-d-riboside linker to the oligo could also stabilize a DNA/RNA duplex, while (S)-4-(6-methylindolo[2,3-b]quinoxalin-3-ylmethoxy)-butane-1,2-diol was able to stabilize both DNA/DNA, DNA/RNA and a DNA/LNA duplex. Mismatch studies revealed a huge sensitivity to the C-C mismatch at the 5′-site of the intercalator.     

Syntheses of 2′-C-amidoalkyl and 2′-C-cyanoalkyl containing oligodeoxyribonucleotides and assessment of their hybridisation affinity for complementary DNA and RNA

Oligodeoxynucleotides containing 2′-C-branched nucleosides with an amide or nitrile appended to either a one or two carbon alkyl chain have been synthesised. The phosphoramidites of the 2′-C-modified nucleosides were prepared and incorporated into the oligonucleotides using automated DNA synthesis. The duplex stability with complementary RNA and DNA was measured by UV melting experiments, in order to assess whether the amide/nitrile function could induce any duplex stability without the presence of the 2′-oxygen. The duplex stabilities of the oligonucleotides containing the 2′-C-modifications were decreased in the absence of the 2′-oxygen.     

Design, synthesis, and evaluation of a novel bridged nucleic acid, 2′,5′-BNA, with S-type sugar conformation fixed by N-O linkage

We designed a novel 2′-O,5′-N bridged nucleic acid, 2′,5′-BNA^ON, whose sugar puckering was fixed to S-type conformation by an N-O linkage. A dimer unit formed from 2′,5′-BNA^ON-U and thymidine was synthesized via a coupling reaction between a protected 2′,5′-BNA^ON-U monomer and a thymidine derivative. Introduction of 2′,5′-BNA^ON-U into DNA was carried out using conventional phosphoramidite chemistry with a DNA synthesizer. The hybridization abilities of 2′,5′-BNA^ON-U-modified oligonucleotides against DNA or RNA complement were evaluated.     

A convenient synthesis of the cytidyl 3′-terminal monomer for solid-phase synthesis of RNG oligonucleotides

Replacing the phosphodiester backbone of RNA with positively charged guanidinium linkages has been shown to enable RNA oligomers to overcome electrostatic repulsion and bind double-stranded DNA in a triplex with high affinity. Ribonucleotide monomers with the ability to form guanidinium linkages have been synthesized for the generation of ribooligonucleotides with guanidinium linkages (RNGs) through solid-phase synthesis. We report herein an efficient method for the synthesis of N⁴-benzoyl-2′-O-(tert-butyldimethylsilyl)-5′-N-(4-monomethoxytritylamino)-3′-O-succinyl-5′-deoxycytidine, a new monomer required for the solid-phase synthesis of cytidyl RNG oligonucleotides.     

A route for preparing new neamine derivatives targeting HIV-1 TAR RNA

In the search for molecules possessing antibiotic or antiviral properties and ribonuclease like activity, that is, able to induce the cleavage of bacterial or viral RNA targets, we report a new route for preparing selectively neamine derivatives modified at their 4′- and/or 5-hydroxyl functions. Using trityl protective groups for the amino functions and 4-methoxybenzyl groups for the hydroxyl functions, new neamine derivatives, such as histidine, phenanthroline, flavin, adenine conjugates were efficiently obtained after a single deprotection step under acid conditions. For the first time, 4′-modified neamine derivatives were prepared. Most of the 4′-derivatives showed affinity and selectivity for TAR RNA close to those of the corresponding 5-derivatives. The most potent compound is the 4′-histidine derivative 31 which binds more tightly to TAR RNA compared to its 5-isomer and neamine and recognizes selectively TAR oligonucleotides having a bulge.     

Improved DNA extraction efficiency from low level cell numbers using a silica monolith based micro fluidic device

The evaluation of a micro fluidic system with an integrated silica monolith for performing DNA extraction from limited biological samples has been carried out. Low DNA target concentrations usually require the addition of carrier RNA to ensure desired extraction efficiencies. Here, we demonstrate a micro fluidic extraction system with increasingly efficient extraction performances for biological samples containing <15 ng of total DNA without the need of adding carrier nucleic acids. All extracted DNA showed successful amplification via the polymerase chain reaction demonstrating both the effectiveness of the proposed system at removing potential inhibitors and yielding good quality DNA. The work presented here beneficially identifies reduced sample volumes/concentrations as suitable for processing with respect to downstream analysis by enabling pre-concentration of the biological sample, particularly important when dealing with clinical or forensic specimens.     

Electrochemical detection of DNA 3′-phosphatases based on surface-extended DNA nanotail strategy

Determination of DNA dephosphorylation is of great value due to its vital role in many cellular processes. Here we report a surface-extended DNA nanotail strategy for simple and ultrasensitive detection of DNA 3′-phosphatases by terminal deoxynucleotidyl transferase (TdT) mediated signal amplification. In this work, DNA probes labeled with thiols at their 5′ terminals and phosphoryls at 3′ terminals are immobilized on gold electrode and are used as substrates for DNA 3′-phosphatases, taking T4 polynucleotide kinase phosphatase (T4PNKP) as an example. T4PNKP can catalyze the dephosphorylation reaction of the substrate DNA, followed by the formation of a long DNA strand by TdT on its 3′ terminal hydroxyl, leading to an evident chronocoulometry signal enhancement. The proposal presents a considerable analytical performance with low detection limit and wide linear range, making it promise to be applied in the fields of DNA dephosphorylation related processes, drug discovery, and clinical diagnostics.     

New junction models for alternate-strand triple-helix formation

Background: Oligonucleotide-directed triple-helix (triplex) formation can interfere with gene expression but only long tracts of oligopyrimidine·oligopurine sequences can be targeted. Attempts have been made to recognize short oligopurine sequences alternating on the two strands of double-stranded DNA by the covalent linkage of two triplex-forming oligonucleotides. Here we focus on the rational optimization of such an alternate-strand triplex formation on a DNA duplex containing a 5′-GpT-3′/3′-CpA-5′ or a 5′-TpG-3′/3′-ApC-5′ step by combination of (G,T)- and (G,A)-contain ing oligonucleotides that bind to the oligopurine strands in opposite orientations.Results: The deletion of one nucleotide in the reverse Hoogsteen region of the oligonucleotide provides the best binding at the 5′-GpT-3′/3′-CpA-5′ step, whereas the addition of two cytosines as a linker between the two oligonucleotides is the best strategy to cross a 5′-TpG-3′/3′-ApC-5′ step. Energy minimization and experimental data suggest that these two cytosines are involved in the formation of two novel base quadruplets.Conclusions: These data provide a rational basis for the design of oligonucleotides capable of binding to oligopurine sequences that alternate on the two strands of double-stranded DNA with a 5′-GpT-3′/3′-CpA-5′ or a 5′-TpG-3′/3′-ApC-5' step at the junction.     

Label-free genotyping of single-nucleotide polymorphisms for DNA and RNA targets using a cationic polythiophene derivate

By using the specific primer extension reaction, a new assay for genotyping of single-nucleotide polymorphisms (SNPs) has been demonstrated. The assay relies on the conformational and colorimetric change of water-soluble polythiophene derivative, poly[3-(3′-N,N,N-triethylamino-1′-propyloxy)-4-methyl-2,5-thiophene hydrochloride] (PMNT), upon forming interpolyelectrolyte complex with extended double strand DNA and non-extended single strand DNA. All three kinds of SNP genotypes can be colorimetrically identified with one primer extension reaction in homogeneous solution. Moreover, combining with the specific digestion of RNA strands in the RNA/DNA hybrids, the proposed assay can also be applied to SNP genotyping for RNA templates. The SNP genotyping assay does not require chemical modification of oligonucleotide probes and nucleic acid targets and any separation step. It would be useful for routinely SNP detection in ordinary laboratories.