A possible mode of the specific recognition of nucleic acids by proteins

Seven sets of protein target sites, which occur in several gene promoters, have been analyzed. The results suggest that there is a possible mode of specific recognition of double-helical nucleic acids by proteins, This recognition mode is related to a spe

Interactions of Rare Earth-Amino Acid Complexes with Nucleic Acids

A number of metal-based anticancer drugs are designed to target nucleic acids. Therefore, the elucidation of their interactions with nucleic acids is important for rational design of new anticancer agents with high selectivity and low toxicity, which has been received much attention in this field. Lanthanide complexes have the potential to be therapeutic agents due to their unique magnetic, optical, electronic, and coordinate characteristics. However, lanthanide ions are easy to hydrolysis under physiological pH, which makes it difficult to study rare earth complexes nucleic acids selectivity. Recent studies have shown that natural amino acids can form stable complexes with rare earth ions under near physiological condition and the complexes have high solubility. This review summarizes the current progress in rare earth-amino acid complexes binding to nuclelic acids and their selectivity.

Interactions of Safranin T with nucleic acids

The interactions of Safranin T (ST) with several nucleic acids have been investigated by electrochemical, UV-visible and CD spectroscopic techniques. The form of the nucleic acid-ST complexes is sensitive to the ratio of the two species. Two electrochemically inactive complexes such as, nucleic acid-ST and nucleic acid-2ST, were formed while ST interacts with nucleic acids. Two processes were obtained from spectral experiments: (1) at the high value of R (R is defined as the ratio of the total concentration of ST to that of nucleic acid), ST is groove-binding with stacking, (2) st the low value of R, ST is groove-binding without stacking. Intrinsic binding constants were obtained by spectral methods. The experiments also show that electrostatic binding plays an important role in the interaction of ST with nucleic acids.

In situ infrared spectroelectrochemical studies on adsorption and oxidation of nucleic acids at glassy carbon electrode

The adsorption and oxidation of yeast RNA and herring sperm DNA (HS DNA) at glass carbon (GC) electrode are studied by differential pulse voltammetry (DPV) and in situ FTIR spectroelectrochemistry. Two oxidation peaks of yeast RNA are obtained by DPV, whose peak potentials shift negatively with increasing pH. The peak currents decrease gradually in successive scans and no corresponding reduction peaks occur, thus indicating that the oxidation process of yeast RNA is completely irreversible. The IR bands in the 1200-1800 cm-l range, attributed to the stretching and ring vibrations of nucleic acid bases, show the main spectral changes when the potential is shifted positively, which gives evidence that the oxidation process takes place in the base residues. The oxidation process of HS DNA is similar to that of yeast RNA. The results both from DPV and in situ FTIR spectroelectrochemistry confirm that the guanine and adenine residues can be oxidized at the electrode surface, which is consistent with the oxidation mechanism of nucleic acids proposed previously. (C) 2001 Elsevier Science B.V. All rights reserved.

Interactions of the human telomeric DNA with terbium-amino acid complexes

The human telomeric DNA can form four-stranded structures: the G-rich strand adopts a G-quadruplex conformation stabilized by G-quartets and the C-rich strand may fold into an I-motif based on intercalated C (.) C+ base pairs. There is intense interests in the design and synthesis of compounds which can target telomeric DNA and inhibit the telomerase activity. Here we report the thermodynamic studies of the two newly synthesized terbium-amino acid complexes bound to the human telomeric G-quadruplex and I-motif DNA which were studied by means of UV-Visible, DNA meltings, fluorescence and circular dichroism. These two complexes can bind to the human telomeric DNA and have shown different features on DNA stability, binding stoichiometry, and sequence-dependent fluorescence enhancement. To our knowledge, this is the first report to show terbium-amino acid complexes can interact with the human telomeric DNA.

Identification of peptides that bind with thymidylate synthase RNA using mRNA display technique

Using in vitro selection method to isolate nucleic acids, peptides and proteins has been studied intensively in recent years. In vitro mRNA display is a new and effective technique for peptides selection, and the rationale of this technique is that a synthetic mRNA with puromycin could covalently link with the protein that it encodes, thus an mRNA-protein fusion is formed. This approach has been used in identification of many functional peptides. The peptides binding with thymidylate synthase RNA were isolated using mRNA display technique from a large peptide library (>10(13) different sequences). The selection scheme was constructed, and the experimental conditions, including library synthesis, formation of RNA-peptide fusion and RNA immobilization were optimized. Eight cycles have been processed and the results confirmed that the selected peptides could bind with thymidylate synthase mRNA specifically. Compared the amino acid sequences of the selected peptides with those from the initial random library, the basic and aromatic residues in selected peptides were enriched significantly, suggesting these peptide regions may be important in the peptide-TS mRNA interaction. As a novel in vitro selection approach, mRNA display technique would be developed as a powerful tool for isolation of functional peptides and proteins that could interact with immobilized targets with high affinity and specificity.

核酸四碱基体及四螺旋模型的理论构建

核酸的结构具有多态性,核酸的功能取决于其结构. 了解核酸不同结构的特征有利 于对其功能的充分认识. 本文对核酸四碱基体及其形成的四链结构模型进行了理论研究. 这些工作 有助于从理论上进一步研究核酸四螺旋结构,对相关的实验研究也将起到有益参考作用.

PNA(T).DNA(AT) triplexes with Hoogsteen base pairing are more favorable

Peptide nucleic acids (PNAs) are nucleic acid analogs with the deoxyribose phosphate backbone replaced by pseudo-peptide polymers to which the nucleobases are linked. The achiral, uncharged and rather flexible properties of the peptide backbone permit peptide nucleic acids more potential than oligonucleotides in application to antisence and antigenic reagents. The process of PNA binding to DNA duplex and forming triplex is the first step of PNA interacting with PNA. But there are no PNA.2DNA triplex crystal data up to date and little has been reported on the structure features and the force of the PNA.2DNA triplex. In this work, PNA(T).DNA(AT) triplexes are successfully built and the structures and forces to stabilize the triplex after optimizations and molecule dynamics are systematically examined, which are expected to aid in the application of PNAs as anticense and antigene agents.

Development of a rapid, sensitive and specific diagnostic assay for fish Aquareovirus based on RT-PCR

A rapid, sensitive and highly specific detection method for Aquareovirus based on reverse-transcription polymerase chain reaction (RT-PCR) was developed. Based on multiple sequence alignment of the cloned sequences of a local isolates, the Threadfin reovirus (TFV) and Guppy reovirus (GPV) with Grass carp reovirus (GCRV), a pair of degenerate primers was selected carefully and synthesized. Using this primer combination, only one specific product, approximately 450 bp in length was obtained when RT-PCR was carried out using the genomic double-stranded RNA (dsRNA) of TFV, GPV and GCRV. Similar results were also obtained when Chum salmon reovirus (CSRV) and Striped bass reovirus (SBRV) dsRNA were used as templates. No products were observed when nucleic acids other than the dsRNA of the aquareoviruses described above were used as RT-PCR templates. This technique could detect not only TFV but also GPV and GCRV in low titer virus-infected cell cultured cells. Furthermore, this method has also been shown to be able to diagnose GPV-infected guppy (Poecilia reticulata) that exhibit clinical symptoms as well as GPV-carrier guppy. Collectively, these results showed that the RT-PCR amplification method using specific degenerate primers described below is very useful for rapid and accurate detection of a variety of aquareovirus strains isolated from different host species and origin. (C) 2004 Elsevier B.V. All rights reserved.

A detection method for grass carp hemorrhagic virus (GCHV) based on a reverse transcription polymerase chain reaction

A rapid, sensitive and highly specific detection method for grass carp hemorrhagic virus (GCHV) based on a reverse transcription-polymerase chain reaction (RT-PCR) has been developed. Two pairs of PCR primers were synthesized according to the cloned cDNA sequences of the GCHV-861 strain. For each primer combination, only one specific major product was obtained when amplification was performed by using the genomic dsRNA of GCHV-861 strain. The lengths of their expected products were 320 and 223 bp, respectively. No products were obtained when nucleic acids other than GCHV-861 genomic RNA were used as RT-PCR templates. To assess the sensitivity of the method, dilutions of purified GCHV-861 dsRNA total genome (0.01 pg up to 1000 pg) were amplified and quantities of as little as 0.1 pg of purified dsRNA were detectable when the amplification product was analyzed by 1.5% agarose gel electrophoresis. This technique could detect GCHV-861 not only in infected cell culture fluids, but also in infected grass carp Ctenopharyngodon idellus and rare minnow Gobiocypris rarus with or without hemorrhagic symptoms. The results show that the RT-PCR amplification method is useful for the direct detection of GCHV.

基于核酸特殊结构的分子识别及核酸相关纳米结构的设计应用

核酸为生命的最基本物质之一，是生物体遗传信息的携带者，在生长、遗传、变异等一系列重大生命现象中起决定性的作用。以核酸作为新药设计的靶分子，越来越受到人们的广泛重视。然而，不像其它靶分子如蛋白质、受体等具有特定的结构和功能，核酸结构在很多情况下是同源的，而且联系到很多人体正常的生理功能；能够与核酸结合的药物又往往不具有序列选择性，这就带来明显的毒副作用。因此，寻找和发现一些与疾病相关的核酸的特殊结构，并筛选对其有特异性结合能力的小分子，是以核酸为靶的药物研究的一个重要课题。 近年来，随着纳米科学技术的兴起，以核酸作为纳米体系的结构材料开始受到人们的广泛关注。作为一类特殊的线性高分子，核酸具有化学性质稳定，结构丰富且可控，良好的刚性和柔性，精确识别，高度生物相容性，合成方便等诸多优点，是一类优良的结构材料。目前核酸相关的纳米组装结构和器件研究还处于起步阶段，但是已经呈现出良好的发展前景。 本论文主要针对核酸特殊结构的分子识别及核酸相关功能纳米结构的设计这两方面展开了研究，全文由以下两大部分组成： 第一部分通过光谱学和生物化学等手段，研究了小分子对不同核酸结构的识别作用。借助于竞争平衡透析技术，发现一类恶嗪染料(oxazine dyes)能够与多种结构核酸结合。热变性及光谱实验结果表明，oxazine染料能够诱导杂合体三链核酸poly(rA):2poly(dT)的形成，并强烈地稳定其结构，其中以cresyl violet作用最强，是迄今发现的化合物中最强的。进一步研究发现，此类化合物以嵌插方式与杂合体三链核酸结合。RNase H酶切实验表明，杂合体三链核酸的形成能够强烈地抑制RNase H核酸酶的活性。研究了oxazine-170与三链核酸poly(dA):2poly(dT)及poly(rA):2poly(rU)的相互作用，发现oxazine-170能够强烈稳定三链DNA poly(dA):2poly(dT)的结构，而对相应双链DNA不具稳定作用；对三链RNA poly(rA):2poly(rU)及相应的双链RNA都有一定稳定作用，但作用不强。进一步研究发现，oxazine-170能够以两种结合方式与核酸结合：嵌插方式和外部静电堆积作用。研究了oxazine-170及cresyl violet与单链核酸的相互作用。研究发现oxazine-170能够序列特异性地与单链核酸poly(rA) 结合，CD光谱及AFM研究发现oxazine-170诱导poly(rA)形成新的二级结构。UV光谱、FL光谱及RLS研究发现poly(rA)促使oxazine 170形成H-aggregate，并以poly(rA)为模板自组装。而cresyl violet能够与单链核酸poly(rA)及poly(dA)结合，且采用不同的结合方式： cresyl violet能够与oxazine-170 类似地以poly(rA)为模板自组装；以嵌插方式与poly(dA)结合，并诱导其单链碱基堆积方式的改变。通过以上实验结果，我们进一步揭示了oxazine染料作为肿瘤细胞染色及光动力学治疗试剂的结构基础，对进一步设计、合成更加高效的抗肿瘤药物具有一定的指导意义。 第二部分中，我们尝试设计了几种基于核酸的纳米结构功能体系，并讨论了其相关应用。利用有机小分子coralyne能够诱导聚腺嘌呤序列反平行双链结构的形成，实现了一类新型的小分子诱导的纳米金组装结构。并以(dA)16功能化的金纳米粒子作为新型纳米探针，发展了一种简单的筛选单链核酸聚腺嘌呤序列结合分子的筛选方法。利用核酸限制性内切酶酶切位点回文序列的结构特点，设计了一种以DNA功能化的金纳米粒子组装体为酶切底物的比较通用的核酸限制性内切酶活力检测方法，并进一步用于甲基化酶活性检测及其抑制剂的筛选。基于单链DNA富胞嘧啶i-motif结构在不同pH值条件下的形成与解离，设计了一类质子驱动的DNA分子镊子，与基于链交换反应的DNA分子镊子相比，该体系更加简单，工作效率更高。随后，我们又通过合理设计，得到了两种分别能够结合与释放DNA和蛋白的分子镊子，为其应用做了一些探索。

A switch-on mechanism to activate maize ribosome-inactivating protein for targeting HIV-infected cells

Maize ribosome-inactivating protein (RIP) is a plant toxin that inactivates eukaryotic ribosomes by depurinating a specific adenine residue at the a-sarcin/ricin loop of 28S rRNA. Maize RIP is first produced as a proenzyme with a 25-amino acid internal inactivation region on the protein surface. During germination, proteolytic removal of this internal inactivation region generates the active heterodimeric maize RIP with full N-glycosidase activity. This naturally occurring switch-on mechanism provides an opportunity for targeting the cytotoxin to pathogen-infected cells. Here, we report the addition of HIV-1 protease recognition sequences to the internal inactivation region and the activation of the maize RIP variants by HIV-1 protease in vitro and in HIV-infected cells. Among the variants generated, two were cleaved efficiently by HIV-1 protease. The HIV-1 protease-activated variants showed enhanced N-glycosidase activity in vivo as compared to their un-activated counterparts. They also possessed potent inhibitory effect on p24 antigen production in human T cells infected by two HIV-1 strains. This switch-on strategy for activating the enzymatic activity of maize RIP in target cells provides a platform for combating pathogens with a specific protease.

Capillary electrophoresis of urinary normal and modified nucleosides of cancer patients

This paper gives a capillary electrophoretic method for the separation of 15 urinary normal and modified nucleosides from cancer patients in less than 40 min. A 500 mmx50 mu m uncoated capillary column (437.5 mm to window) was used. The effects of the voltage and the sodium dodecyl sulfate (SDS) concentration in the buffer on the separation were studied. With reproducibilities of migration times better than 1.2% (R.S.D.) and determined concentrations better than 5-25%, depending on the concentrations of nucleosides in the urine, the analytical characteristics of the method were food. Using this developed method, the concentrations of 13 normal and modified nucleosides, extracted on a phenyl boronic acid affinity chromatography column, in 25 urines from patients of 14 kinds of cancer were determined. The levels (nmol/mol creatinine) of modified nucleosides in urines from cancer patients were increased as compared with those in normal urines. (C) 1998 Elsevier Science B.V.

Soil microbial community analysis using denaturing gradient gel electrophoresis

The most biological diversity on this planet is probably harbored in soils. Understanding the diversity and function of the microbiological component of soil poses great challenges that are being overcome by the application of molecular biological approaches. This review covers one of many approaches being used: separation of polymerase chain reaction (PCR) amplicons using denaturing gradient gel electrophoresis (DGGE). Extraction of nucleic acids directly from soils allows the examination of a community without the limitation posed by cultivation. Polymerase chain reaction provides a means to increase the numbers of a target for its detection on gels. Using the rRNA genes as a target for PCR provides phylogenetic information on populations comprising communities. Fingerprints produced by this method have allowed spatial and temporal comparisons of soil communities within and between locations or among treatments. Numerous samples can be compared because of the rapid high throughput nature of this method. Scientists now have the means to begin addressing complex ecological questions about the spatial, temporal, and nutritional interactions faced by microbes in the soil environment.