Self-assembly of single-stranded RNA on carbon nanotube: Polyadenylic acid to form a duplex structure

All messenger-RNA (mRNA) molecules in eukaryotic cells have a polyadenylic acid [poly (rA)] tail at the 3'-end and human poly (rA) polymerase (PAP) has been considered as a tumor-specific target. A ligand that is capable of recognizing and binding to the poly(M) tail of mRNA might interfere with the full processing of mRNA by PAP and can be a potential therapeutic agent. We report here for the first time that single-walled carbon nanotubes (SWNTs) can cause single-stranded poly (M) to self-structure and form a duplex structure, which is studied by UV melting, atomic force microscopy, circular dichroism spectroscopy, and NMR spectrometry.

7-Amino actinomycin D bound to single-stranded hairpin DNA enhanced by loop sequence-dependent luminescent Eu3+ and Tb3+ binding

Lanthanide Eu3+ and Tb3+ ions have been widely used in luminescent resonance energy transfer (LRET) for bioassays to study metal binding microenvironments. We report here that Eu3+ or Tb3+ can increase the binding affinity of antitumor antibiotic drug agent, 7-amino actinomycin D (7AACTD), binding to 5'-GT/TG-5' or 5'-GA/AG-5' mismatched stem region of the single-stranded hairpin DNA. Further studies indicate that the effect of Eu3+ or Tb3+ on 7AACTD binding is related to DNA loop sequence. Our results will provide new insights into how metal ions can enhance antitumor agents binding to their targets.

Structural diversity of lanthanide-amino acid complexes under near physiological pH conditions and their recognition of single-stranded DNA

We reported here four structures of lanthanide-amino acid complexes obtained under near physiological pH conditions and their individual formula can be described as [Tb-2(DL-Cys)(4)(H2O)(8)]Cl-2 (1), [Eu-4(mu(3)-OH)(4)(L-Asp)(2)(L-HAsp)(3)(H2O)(7)] Cl center dot 11.5H(2)O (2), [Eu-8-(L-HVal) (16)(H2O)(32)]Cl-24 center dot 12.5H(2)O (3), and [Tb-2(DL-HVal)(4)(H2O)(8)]Cl-6 center dot 2H(2)O (4). These complexes showed diverse structures and have shown potential application in DNA detection. We studied the interactions of the complexes with five single-stranded DNA and found different fluorescence enhancement, binding affinity and binding stoichiometry when the complexes are bound to DNA.

DNAzyme-based Colorimetric Sensing of Lead (Pb2+) Using Unmodified Gold Nanoparticle Probes

Novel functional oligonucleotides, especially DNAzymes with RNA-cleavage activity, have been intensively studied due to their potential applications in therapeutics and sensors. Taking advantage of the high specificity of 17E DNAzyme for Pb2+, highly sensitive and selective fluorescent, electrochemical and colorimetric sensors have been developed for Pb2+. In this work, we report a simple, sensitive and label-free 17E DNAzyme-based sensor for Pb2+ detection using unmodified gold nanoparticles (GNPs) based on the fact that unfolded single-stranded DNA could be adsorbed on the citrate protected GNPs while double-stranded DNA could not. By our method the substrate cleavage by the 17E DNAzyme in the presence of Pb2+ could be monitored by color change of GNPs, thereby Pb2+ detection was realized.

The topological configuration and conformational analysis of mRNA in translation

The theoretical model construction of mRNA hairpin structure and single-stranded structure as well as the simulation studies on RNA structure determined by the X-ray crystal diffraction and nuclear magnetic resonance revealed that in translation, after mRNA being unfolded into single-stranded structure, its topological configuration was closely correlative with the original hairpin structure. The conformational features of single-stranded mRNA appeared as helical regions alternating with curly regions to different extents, which might exert the influence on the folding of nascent polypeptide by various regulating effects including different translational rates.

Origin and evolution of the RIG-I like RNA helicase gene family

Background: The DExD/H domain containing RNA helicases such as retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are key cytosolic pattern recognition receptors (PRRs) for detecting nucleotide pathogen associated molecular patterns (PAMPs) of invading viruses. The RIG-I and MDA5 proteins differentially recognise conserved PAMPs in double stranded or single stranded viral RNA molecules, leading to activation of the interferon system in vertebrates. They share three core protein domains including a RNA helicase domain near the C terminus (HELICc), one or more caspase activation and recruitment domains (CARDs) and an ATP dependent DExD/H domain. The RIG-I/MDA5 directed interferon response is negatively regulated by laboratory of genetics and physiology 2 (LGP2) and is believed to be controlled by the mitochondria antiviral signalling protein (MAVS), a CARD containing protein associated with mitochondria. Results: The DExD/H containing RNA helicases including RIG-I, MDA5 and LGP2 were analysed in silico in a wide spectrum of invertebrate and vertebrate genomes. The gene synteny of MDA5 and LGP2 is well conserved among vertebrates whilst conservation of the gene synteny of RIG-I is less apparent. Invertebrate homologues had a closer phylogenetic relationship with the vertebrate RIG-Is than the MDA5/LGP2 molecules, suggesting the RIG-I homologues may have emerged earlier in evolution, possibly prior to the appearance of vertebrates. Our data suggest that the RIG-I like helicases possibly originated from three distinct genes coding for the core domains including the HELICc, CARD and ATP dependent DExD/H domains through gene fusion and gene/domain duplication. Furthermore, presence of domains similar to a prokaryotic DNA restriction enzyme III domain (Res III), and a zinc finger domain of transcription factor (TF) IIS have been detected by bioinformatic analysis. Conclusion: The RIG-I/MDA5 viral surveillance system is conserved in vertebrates. The RIG-I like helicase family appears to have evolved from a common ancestor that originated from genes encoding different core functional domains. Diversification of core functional domains might be fundamental to their functional divergence in terms of recognition of different viral PAMPs.

RecA-mediated, targeted mutagenesis in zebrafish

We have evaluated the efficacy of RecA, a prokaryotic protein involved with homologous recombination, to direct site-specific mutagenesis in zebrafish embryos. For this we coinjected a vector containing a mutated enhanced green fluorescent protein (EGFP) gene plus 236-nucleotide corrective single-stranded DNAs coated with RecA into I-cell zebrafish embryos. Twenty-hours after fertilization, about 5% to 20% of injected embryos showed EGFP expression in I or more cells when RecA-coated corrective DNAs were used, but not when RecA was omitted. Mutated EGFP genes with 1-bp insertions or deletions were inefficiently activated, whereas those with 7-bp insertions were activated about 4-fold more efficiently. RecA-coated template strand had a higher efficiency than its complementary strand in activation of EGFP expression. Prior irradiation of the embryos with UV light enhanced RecA-mediated restoration of gene activity, suggesting that the effects we observed were augmented by one or more factors of zebrafish DNA repair systems.

固氮树木丛枝菌根真菌(AMF)遗传多样性的研究

丛枝菌根是自然生态系统中分布最广的内生菌根，在促进植物生存与生长、植被恢复以及生物多样性保护等方面有着非常重要的作用。 随着现代分子生物学技术的不断发展，丛枝菌根真菌研究得到空前发展。大量DNA分析新技术在丛枝菌根真菌的分子遗传、分类鉴定、种间及种内亲缘关系、菌株持久性等方面得到应用，与传统菌根研究方法相比，表现出巨大的优越性。 本项研究利用分子生物学技术和研究方法对中国吉林长白山地区非豆科固氮植物以及东北地区固氮树木的丛枝菌根真菌DNA分子多态性及其与宿主植物之间的相互关系等进行初步研究，旨在利用分子生态学理论和研究方法揭示丛枝菌根真菌多样性及其与宿主植物之间相互适应和协同进化的一般规律，为更好地保护和利用这一重要的微生物资源提供理论依据。 通过比较与筛选，建立起丛枝菌根真菌痕量DNA快速、简便、高效的提取纯化方法——改良CTAB法。经PCR检测，所得DNA满足进一步研究的要求。 根据丛枝菌根真菌18s rRNA 小亚基核基因片段的特点，利用“科”特异性引物进行半巢式标记PCR (Labelled Primers-PCR，LP-PCR) 及单链构象多态性（Single-Stranded Conformation Polymorphism，SSCP）分析技术研究了长白山赤杨在属水平上表现出的多样性。另外，利用巢式PCR-RFLP技术，分别对来源于长白山不同海拔的四种赤杨菌根样品的AMF侵染情况及其系统进化进行了研究。利用AMF特异性PCR技术对我国东北地区四种非豆科树木和5种豆科树木菌根侵染情况和系统发育规律进行了研究 研究结果显示：赤杨根内AMF存在丰富的基因多样性。AMF的侵染有从宿主混乱性向宿主专一性发展的趋势。 长白山地区赤杨属植物至少有东北赤杨、西伯利亚赤杨和色赤杨三个树种在其“属”的水平上与共生的球囊霉科（Glomaceae）至少一个“种” 的丛枝菌根真菌，即根内球囊霉（Glomus intraradix），在“种”的水平上表现出不相关于宿主海拔高度的某种相互选择性。 东北赤杨AMF菌的宿主专一性水平最强，球囊霉属已成为东北赤杨的优势侵染类群；对于其余三种赤杨，AMF则出现宿主混乱现象。宿主因素比海拔因素对AMF侵染特异性的影响更为重要。 豆科与非豆科样本的混乱性都比较强，在特定植物和AMF属之间无特异侵染规律，相对来说，非豆科树木比豆科树木对于AMF的选择性要更强一些，更倾向于和球囊霉属与无梗孢囊霉属的AMF构建共生体。

固氮树木丛枝菌根真菌（AMF）遗传多样性的研究

丛枝菌根是自然生态系统中分布最广的内生菌根，在促进植物生存与生长、植被恢复以及生物多样性保护等方面有着非常重要的作用。 随着现代分子生物学技术的不断发展，丛枝菌根真菌研究得到空前发展。大量DNA分析新技术在丛枝菌根真菌的分子遗传、分类鉴定、种间及种内亲缘关系、菌株持久性等方面得到应用，与传统菌根研究方法相比，表现出巨大的优越性。 本项研究利用分子生物学技术和研究方法对中国吉林长白山地区非豆科固氮植物以及东北地区固氮树木的丛枝菌根真菌DNA分子多态性及其与宿主植物之间的相互关系等进行初步研究，旨在利用分子生态学理论和研究方法揭示丛枝菌根真菌多样性及其与宿主植物之间相互适应和协同进化的一般规律，为更好地保护和利用这一重要的微生物资源提供理论依据。 通过比较与筛选，建立起丛枝菌根真菌痕量DNA快速、简便、高效的提取纯化方法——改良CTAB法。经PCR检测，所得DNA满足进一步研究的要求。 根据丛枝菌根真菌18s rRNA 小亚基核基因片段的特点，利用“科”特异性引物进行半巢式标记PCR (Labelled Primers-PCR，LP-PCR) 及单链构象多态性（Single-Stranded Conformation Polymorphism，SSCP）分析技术研究了长白山赤杨在属水平上表现出的多样性。另外，利用巢式PCR-RFLP技术，分别对来源于长白山不同海拔的四种赤杨菌根样品的AMF侵染情况及其系统进化进行了研究。利用AMF特异性PCR技术对我国东北地区四种非豆科树木和5种豆科树木菌根侵染情况和系统发育规律进行了研究 研究结果显示：赤杨根内AMF存在丰富的基因多样性。AMF的侵染有从宿主混乱性向宿主专一性发展的趋势。 长白山地区赤杨属植物至少有东北赤杨、西伯利亚赤杨和色赤杨三个树种在其“属”的水平上与共生的球囊霉科（Glomaceae）至少一个“种” 的丛枝菌根真菌，即根内球囊霉（Glomus intraradix），在“种”的水平上表现出不相关于宿主海拔高度的某种相互选择性。 东北赤杨AMF菌的宿主专一性水平最强，球囊霉属已成为东北赤杨的优势侵染类群；对于其余三种赤杨，AMF则出现宿主混乱现象。宿主因素比海拔因素对AMF侵染特异性的影响更为重要。 豆科与非豆科样本的混乱性都比较强，在特定植物和AMF属之间无特异侵染规律，相对来说，非豆科树木比豆科树木对于AMF的选择性要更强一些，更倾向于和球囊霉属与无梗孢囊霉属的AMF构建共生体.

长白山赤杨从枝菌根真菌DNA分子多态性的研究

丛枝菌根是自然生态系统中分布最广的内生菌根，在促进植物生存与生长、植被恢复以及生物多样性保护等方面有着非常重要的作用。随着现代分子生物学技术的发展，丛枝菌根真菌的研究得到空前发展。大量DNA分析新技术在丛枝菌根真菌的分子遗传、分类鉴定、种间及种内亲缘关系、菌株持久性等方面得到应用，与传统菌根研究方法相比，表现出巨大的优越性。但相比国际而言，国内针对菌根真菌分子水平上的研究发展较为缓慢。本项研究对中国吉林长白山东北赤杨、西伯利亚赤杨、色赤杨丛枝菌根真菌DNA分子多态性进行初步研究，试图揭示其一般规律，为更好地利用这一资源提供理论依据。通过比较与筛选，得到丛枝菌根真菌痕量DNA快速、简便的提取纯化方法—改良CTAB法。经PCR检测，所得DNA满足进一步研究的要求。根据丛枝菌根真菌185 rRNA小亚基核基因片段的特点，利用“科”特异性引物进行半巢式标记PCR（Labelled Primers-PCR，LP-PCR）扩增，再经单链构象多态性（Single-Stranded Conformation Polymorphism，SSCP）分析来检测其DNA分子在“种”水平上表现出的多态性。研究结果显示：丛枝菌根真菌在“种”的水平上并未随各宿主的变化表现出丰富的多样性；长白山地区赤杨属植物至少有东北赤杨、西伯利亚赤杨和色赤杨三个树种在自身“属”的水平上与共生的球囊霉科（Glomaceae）至少一个“种”的丛枝菌根真菌，即根内球囊霉（Glomus intradix），在“种”的水平上表现出不相关于宿主海拔高度的某种相互选择性。

Amplified electrochemical aptasensor taking AuNPs based sandwich sensing platform as a model

Here, we report a sensitive amplified electrochemical impedimetric aptasensor for thrombin, a kind of serine protease that plays important role in thrombosis and haemostasis. For improving detection sensitivity, a sandwich sensing platform is fabricated, in which the thiolated aptamers are firstly immobilized on a gold substrate to capture the thrombin molecules, and then the aptamer functionalized Au nanoparticles (AuNPs) are used to amplify the impedimetric signals.

Atomic force microscopic study of low temperature induced disassembly of RecA-dsDNA filaments

The assembly and disassembly of RecA-DNA nucleoprotein filaments on double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) are important steps for homologous recombination and DNA repair. The assembly and disassembly of the nucleoprotein filaments are sensitive to the reaction conditions. In this work, we investigated different morphologies of the formed nucleoprotein filaments at low temperature under different solution conditions by atomic force microscopy (AFM). We found that low temperature and long keeping time could induce the incomplete disassembly of the formed nucleoprotein filaments.

Real-Time Study of Genomic DNA Structural Changes upon Interaction with Small Molecules Using Dual-Polarization Interferometry

The structural changes of genomic DNA upon interaction with small molecules have been studied in real time using dual-polarization interferometry (DPI). Native or thermally denatured DNA was immobilized on the silicon oxynitride surface via a preadsorbed poly(ethylenimine) (PEI) layer. The mass loading was similar for both types of DNA, however, native DNA formed a looser and thicker layer due to its rigidity, unlike the more flexible denatured DNA, which mixed with PEI to form a denser and thinner layer. Ethidium bromide (EtBr), a classical intercalator, induced the large thickness decrease and density increase of native DNA (double-stranded), but a slight increase in both the thickness and density of denatured DNA (partial single-stranded).

Influence of Polyelectrolyte on DNA-RecA Nucleoprotein Filaments: Poly-L-Lysine Used as a Model

RecA of Escherichia coli and its active nucleoprotein filaments with DNA are important for the genomic integrity and the genetic diversity. The formation of the DNA-RecA nucleoprotein filaments is a complex multiple-step process and can be affected by many factors. In this work, the effects of poly-L-lysine (PLL) on the DNA-RecA nucleoprotein filaments are investigated in vitro by agarose gel electrophoresis and atomic force microscopy (AFM). The observed morphologies vary with the concentration, the length, and the addition order of PLL. These distinctions provide information for the conformation change of DNA and the binding sites of RecA protein in the formation process of nucleoprotein filaments.