SSB蛋白与ssDNA相互作用的动力学和可视化研究

研究大肠杆菌单链结合蛋白(single-stranded DNA-binding protein,SSB)与单链DNA(single-stranded DNA,ssDNA)的相互作用对于了解其在DNA复制、重组和修复中的作用是非常重要的。通过表面等离子共振技术(surface plasmon resonance,SPR)得到了在有、无镁离子的情况下,SSB与ssDNA两者的平衡解离常数(equilibrium dissociation constant,KD)分别为9.67×10-7M和4.79×10-7M,阐明了镁离子对于两者作用形式的影响。利用原子力显微镜技术分别观察SSB蛋白、ssDNA和SSB-ssDNA复合物的成像,为下一步研究SSB在DNA代谢中作用模式的单分子可视化奠定了基础。     

随机单链DNA文库SELEX筛选寡核苷酸适配子方法的建立

指数富集配基的系统进化(SELEX)技术是一种新的组合化学技术.体外构建了一个长度为81 nt、含有35个随机序列的单链DNA(ssDNA)文库,优化了ssDNA文库扩增为双链DNA (dsDNA)文库的PCR反应条件.通过对比不对称PCR和生物素-链亲和素磁珠分离方法制备ssDNA文库的效果,确定了以生物素-链亲和素磁珠分离方法制备ssDNA.由于脱氧核糖核酸的疏水性导致ssDNA文库与硝酸纤维素滤膜的结合背景过高,因此选择以微孔板为介质,分离与靶蛋白结合的适配子.经过9轮循环筛选,随机ssDNA文库与丙型肝炎病毒(HCV)核心蛋白(C蛋白)的结合率从0.5%上升到32.5%.     

荧光偏振技术研究Bloom解旋酶催化核心与双链DNA的相互作用

Bloom 综合症(BLM)解旋酶是RecQ家族DNA解旋酶中的一个重要成员,参与了DNA复制、修复、转录、重组以及端粒的维持等细胞代谢过程,在维持染色体的稳定性中具有重要的作用．BLM解旋酶的突变可导致Bloom综合症,患者遗传不稳定易患多种类型癌症．本研究运用荧光偏振技术研究BLM解旋酶催化核心(BLM642-1290)与双链DNA(dsDNA)的相互作用,分析其相关特征参数,了解BLM642-1290解旋酶与dsDNA的结合和解链特性．结果表明,BLM642-1290解旋酶与dsDNA的结合和解链和dsDNA3’末端的单链DNA(ssDNA)长度有关;解旋酶优先结合于dsDNA底物的ssDNA末端,且每分子解旋酶可结合9.6 nt的ssDNA;dsDNA3’末端ssDNA的长度为9.6 nt时,解旋酶的解链效率达到最大且不再随其长度而变化．另外,BLM642-1290解旋酶也能够结合和解链钝末端dsDNA,但其结合亲和力和解链效率低于有3’末端ssDNA的dsDNA．推测BLM642-1290解旋酶在与dsDNA底物结合和解链时是单体形式,可能以尺蠖的形式解开dsDNA．这些结果可为进一步研究BLM解旋酶的功能特征提供理论基础．     

大肠杆菌RecQ解旋酶的生物学活性分析

RecQ家族解旋酶是DNA解旋酶中高度保守的一个重要家族,在维持染色体的稳定性中起着重要的作用.人类RecQ家族解旋酶突变会导致几种与癌症有关的疾病.本研究旨在诱导大肠杆菌RecQ解旋酶体外表达,并应用生物化学和生物物理学技术研究大肠杆菌RecQ解旋酶的生物学活性.体外诱导表达获得纯度达90%以上并具有高活性的大肠杆菌重组RecQ解旋酶,其可溶性好;经生物学活性分析显示具有DNA结合活性、ATP依赖的DNA解链活性、DNA依赖的ATP酶活性.较之双链DNA(dsDNA),大肠杆菌RecQ解旋酶更容易与单链DNA(ssDNA)结合(P0.01),但与长度不同的dsDNA的结合特性有差异(P0.01)而与ssDNA没有差异(P0.05);大肠杆菌RecQ解旋酶对3种dsDNA的解链速率不同(P0.05);大肠杆菌RecQ解旋酶的ATP酶活性与辅助因子ssDNA长度也呈正相关(P0.01).这些研究结果将有助于阐明大肠杆菌RecQ解旋酶的分子作用机制,并为研究RecQ解旋酶家族其它成员的结构与功能提供帮助。     

大肠杆菌RecQ解旋酶的表达纯化和生物学活性研究

RecQ家族解旋酶是DNA解旋酶中高度保守的一个重要家族,在维持染色体的稳定性中起着重要的作用.人类RecQ家族解旋酶突变会导致几种与癌症有关的疾病.本研究旨在诱导大肠杆菌RecQ解旋酶体外表达,并应用生物化学和生物物理学技术研究大肠杆菌RecQ解旋酶的生物学活性. 体外诱导表达获得纯度达90% 以上并具有高活性的大肠杆菌重组RecQ解旋酶,其可溶性好;经生物学活性分析显示具有DNA结合活性、ATP依赖的DNA解链活性、DNA依赖的ATP酶活性. 较之双链DNA（dsDNA）,大肠杆菌RecQ解旋酶更容易与单链DNA(ssDNA)结合( P<0.01 ),但与长度不同的dsDNA的结合特性有差异(P<0.01)而与ssDNA没有差异(P>0.05);大肠杆菌RecQ解旋酶对3种dsDNA的解链速率不同(P<0.05);大肠杆菌RecQ解旋酶的ATP酶活性与辅助因子ssDNA长度也呈正相关(P<0.01). 这些研究结果将有助于阐明大肠杆菌RecQ解旋酶的分子作用机制,并为研究RecQ解旋酶家族其它成员的结构与功能提供帮助.     

腾冲嗜热菌单链 DNA 结合蛋白 SSB2 和 SSB3 新的单链 DNA 结合特性

摘要：【目的】揭示腾冲嗜热菌中两个单链DNA结合蛋白SSB2和SSB3的全新的底物结合功能及其不同的体内表达模式。【方法】利用腾冲嗜热菌复制起始位点附近的长度较短的单链DNA为底物,采用非变性聚丙烯酰胺凝胶电泳及western blot方法,研究SSB2和SSB3体外单链DNA结合特征和体内表达模式。【结果】SSB2 与35nt的复制起始区单链DNA（ssDNA）结合, 形成单个SSB2-DNA复合物;当与59 nt ssDNA结合时,可以随着蛋白浓度的递增形成一个或两个SSB2-DNA复合物;而与70n     

荧光偏振技术研究Bloom解旋酶催化核心与双链DNA的相互作用

Bloom 综合症(BLM)解旋酶是RecQ家族DNA解旋酶中的一个重要成员,参与了DNA复制、修复、转录、重组以及端粒的维持等细胞代谢过程,在维持染色体的稳定性中具有重要的作用．BLM解旋酶的突变可导致Bloom综合症,患者遗传不稳定易患多种类型癌症．本研究运用荧光偏振技术研究BLM解旋酶催化核心(BLM^642～1290)与双链DNA(dsDNA)的相互作用,分析其相关特征参数,了解BLM^642～1290解旋酶与dsDNA的结合和解链特性．结果表明：BLM^642～1290解旋酶与dsDNA的结合和解链与dsDNA 3′端的单链DNA(ssDNA)长度有关;解旋酶优先结合于dsDNA底物的ssDNA末端,且每分子解旋酶可结合9.6 nt的ssDNA;dsDNA 3′端ssDNA的长度为9.6 nt时,解旋酶的解链效率达到最大且不再随其长度而变化．另外,BLM^642～1290解旋酶也能够结合和解链钝末端dsDNA,但其结合亲和力和解链效率低于有3′端ssDNA的dsDNA．推测BLM^642～1290解旋酶在与dsDNA底物结合和解链时是单体形式,可能以尺蠖的形式解开dsDNA．这些结果可为进一步研究BLM解旋酶的功能特征提供理论基础．     

用单链DNA转化植物细胞

Rodenburg等最近报道,如果用电激法将DNA直接导入烟草原生质体,那么单链DNA(ssDNA)的稳定的转化频率将比双链DNA(dsDNA)高3～10倍.他们认为,ssDNA能比dsDNA高效地进入植物原生质体的核或整合到植物的基因组中. 但是,剑桥大学的I.J.Furner等分析了导入碧冬茄叶片原生质体中的ssDNA的暂时表达,认为直接导入的ssDNA在植物染色体外变成双链DNA,然后整合到核基因组中.他们发现,在有37%聚乙二醇6000存在时,通过孵育细胞而导人ssDNA和dsDNA,在暂时的和稳定的测定中它们的转化率相当.     

一种有效的DNA序列测定方法

比较了单链和双链DNA(ss和dsDNA)模板和两种不同的聚丙烯酰胺凝胶电泳对DNA序列测定的影响。结果表明:在相同的条件下,ssDNA模板明显优于dsDNA模板;缓冲液梯度聚丙烯酰胺凝胶电泳测定DNA序列的长度在40cm长的胶上可达350nt。而一般的线性聚丙烯酰胺凝胶电泳才可测150nt。对于基因组序列分析,采用单链DNA模板和缓冲液梯度聚丙烯酰胺凝胶电泳是一种有效的测定方法。     

核黄素光敏损伤DNA的凝胶电泳

以琼脂糖凝胶电泳研究了波长为427~457nm可见光照射下核黄素(VB₂)光敏诱导的质粒DNA损伤,结果发现DNA光敏损伤主要与照光量、核黄素和DNA浓度比、溶液中的氧气等有关.在无氧条件下光敏损伤更为显著,证明VB₂光敏损伤DNA主要是通过VB₂激发三重态与DNA碱基组分间发生电荷转移导致其产生氧化性损伤实现的.表明核黄素光敏损伤DNA时最佳浓度比为bp∶VB₂ = 3.5∶1.对照结果表明,单链DNA(ssDNA)比双链DNA(dsDNA)的碱基更易被核黄素敏化发生损伤,这可能是由于ssDNA中碱基暴露的原因.     

Rectangular coordination polymer nanoplates: large-scale, rapid synthesis and their application as a fluorescent sensing platform for DNA detection

In this paper, we report on the large-scale, rapid synthesis of uniform rectangular coordination polymer nanoplates (RCPNs) assembled from Cu(II) and 4,4'-bipyridine for the first time. We further demonstrate that such RCPNs can be used as a very effective fluorescent sensing platform for multiple DNA detection with a detection limit as low as 30 pM and a high selectivity down to single-base mismatch. The DNA detection is accomplished by the following two steps: (1) RCPN binds dye-labeled single-stranded DNA (ssDNA) probe, which brings dye and RCPN into close proximity, leading to fluorescence quenching; (2) Specific hybridization of the probe with its target generates a double-stranded DNA (dsDNA) which detaches from RCPN, leading to fluorescence recovery. It suggests that this sensing system can well discriminate complementary and mismatched DNA sequences. The exact mechanism of fluorescence quenching involved is elucidated experimentally and its use in a human blood serum system is also demonstrated successfully.     

Electrostatic-assembly-driven formation of supramolecular rhombus microparticles and their application for fluorescent nucleic acid detection

In this paper, we report on the large-scale formation of supramolecular rhombus microparticles (SRMs) driven by electrostatic assembly, carried out by direct mixing of an aqueous HAuCl(4) solution and an ethanol solution of 4,4'-bipyridine at room temperature. We further demonstrate their use as an effective fluorescent sensing platform for nucleic acid detection with a high selectivity down to single-base mismatch. The general concept used in this approach is based on adsorption of the fluorescently labeled single-stranded DNA (ssDNA) probe by SRM, which is accompanied by substantial fluorescence quenching. In the following assay, specific hybridization with its target to form double-stranded DNA (dsDNA) results in desorption of ssDNA from SRM surface and subsequent fluorescence recovery.     

Characterizing and controlling the motion of ssDNA in a solid-state nanopore

Sequencing DNA in a synthetic solid-state nanopore is potentially a low-cost and high-throughput method. Essential to the nanopore-based DNA sequencing method is the ability to control the motion of a single-stranded DNA (ssDNA) molecule at single-base resolution. Experimental studies showed that the average translocation speed of DNA driven by a biasing electric field can be affected by ionic concentration, solvent viscosity, or temperature. Even though it is possible to slow down the average translocation speed, instantaneous motion of DNA is too diffusive to allow each DNA base to stay in front of a sensor site for its measurement. Using extensive all-atom molecular dynamics simulations, we study the diffusion constant, friction coefficient, electrophoretic mobility, and effective charge of ssDNA in a solid-state nanopore. Simulation results show that the spatial fluctuation of ssDNA in 1 ns is comparable to the spacing between neighboring nucleotides in ssDNA, which makes the sensing of a DNA base very difficult. We demonstrate that the recently proposed DNA transistor could potentially solve this problem by electrically trapping ssDNA inside the DNA transistor and ratcheting ssDNA base-by-base in a biasing electric field. When increasing the biasing electric field, we observed that the translocation of ssDNA changes from ratcheting to steady-sliding. The simulated translocation of ssDNA in the DNA transistor was theoretically characterized using Fokker-Planck analysis.     

Label-free optical detection of single-base mismatches by the combination of nuclease and gold nanoparticles

We report here an optical approach that enables highly selective and colorimetric single-base mismatch detection without the need of target modification, precise temperature control or stringent washes. The method is based on the finding that nucleoside monophosphates (dNMPs), which are digested elements of DNA, can better stabilize unmodified gold nanoparticles (AuNPs) than single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) with the same base-composition and concentration. The method combines the exceptional mismatch discrimination capability of the structure-selective nucleases with the attractive optical property of AuNPs. Taking S1 nuclease as one example, the perfectly matched 16-base synthetic DNA target was distinctively differentiated from those with single-base mutation located at any position of the 16-base synthetic target. Single-base mutations present in targets with varied length up to 80-base, located either in the middle or near to the end of the targets, were all effectively detected. In order to prove that the method can be potentially used for real clinic samples, the single-base mismatch detections with two HBV genomic DNA samples were conducted. To further prove the generality of this method and potentially overcome the limitation on the detectable lengths of the targets of the S1 nuclease-based method, we also demonstrated the use of a duplex-specific nuclease (DSN) for color reversed single-base mismatch detection. The main limitation of the demonstrated methods is that it is limited to detect mutations in purified ssDNA targets. However, the method coupled with various convenient ssDNA generation and purification techniques, has the potential to be used for the future development of detector-free testing kits in single nucleotide polymorphism screenings for disease diagnostics and treatments.     

A graphene-based platform for single nucleotide polymorphism (SNP) genotyping

A facile, rapid, stable and sensitive approach for fluorescent detection of single nucleotide polymorphism (SNP) is designed based on DNA ligase reaction and π-stacking between the graphene and the nucleotide bases. In the presence of perfectly matched DNA, DNA ligase can catalyze the linkage of fluorescein amidite-labeled single-stranded DNA (ssDNA) and a phosphorylated ssDNA, and thus the formation of a stable duplex in high yield. However, the catalytic reaction cannot effectively carry out with one-base mismatched DNA target. In this case, we add graphene to the system in order to produce different quenching signals due to its different adsorption affinity for ssDNA and double-stranded DNA. Taking advantage of the unique surface property of graphene and the high discriminability of DNA ligase, the proposed protocol exhibits good performance in SNP genotyping. The results indicate that it is possible to accurately determine SNP with frequency as low as 2.6% within 40 min. Furthermore, the presented flexible strategy facilitates the development of other biosensing applications in the future.     

Identification of a new motif required for the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I (Klenow fragment): the RRRY motif is necessary for the binding of single-stranded DNA substrate and the template strand of the mismatched duplex

The Klenow fragment of Escherichia coli DNA polymerase I houses catalytic centers for both polymerase and 3'-5' exonuclease activities that are separated by about 35 A. Upon the incorporation of a mismatched nucleotide, the primer terminus is transferred from the polymerase site to an exonuclease site designed for excision of the mismatched nucleotides. The structural comparison of the binary complexes of DNA polymerases in the polymerase and the exonuclease modes, together with a molecular modeling of the template strand overhang in Klenow fragment, indicated its binding in the region spanning residues 821-824. Since these residues are conserved in the "A" family DNA polymerases, we have designated this region as the RRRY motif. The alanine substitution of individual amino acid residues of this motif did not change the polymerase activity; however, the 3'-5' exonuclease activity was reduced 2-29-fold, depending upon the site of mutation. The R821A and R822A/Y824A mutant enzymes showed maximum cleavage defect with single-stranded DNA, mainly due to a large decrease in the ssDNA binding affinity of these enzymes. Mismatch removal by these enzymes was only moderately affected. However, data from the exonuclease-polymerase balance assays with mismatched template-primer suggest that the mutant enzymes are defective in switching mismatched primer from the polymerase to the exonuclease site. Thus, the RRRY motif provides a binding track for substrate ssDNA and for nonsubstrate single-stranded template overhang, in a polarity-dependent manner. This binding then facilitates cleavage of the substrate at the exonuclease site.     

HU binds and folds single-stranded DNA

The nucleoid-associated protein HU plays an important role in bacterial nucleoid organization and is involved in numerous processes including transposition, recombination and DNA repair. We show here that HU binds specifically DNA containing mismatched region longer than 3 bp as well as DNA bulges. HU binds single-stranded DNA (ssDNA) in a binding mode that is reminiscent but different from earlier reported specific HU interactions with double-helical DNA lesions. An HU dimer requires 24 nt of ssDNA for initial binding, and 12 nt of ssDNA for each additional dimer binding. In the presence of equimolar amounts of HU dimer and DNA, the ssDNA molecule forms an U-loop (hairpin-like) around the protein, providing contacts with both sides of the HU body. This mode differs from the binding of the single-strand-binding protein (SSB) to ssDNA: in sharp contrast to SSB, HU binds ssDNA non-cooperatively and does not destabilize double-helical DNA. Furthermore HU has a strong preference for poly(dG), while binding to poly(dA) is the weakest. HU binding to ssDNA is probably important for its capacity to cover and protect bacterial DNA both intact and carrying lesions.     

A chemically synthesized peptoid-based drag-tag enhances free-solution DNA sequencing by capillary electrophoresis

We report a capillary-based DNA sequencing read length of 100 bases in 16 min using end-labeled free-solution conjugate electrophoresis (FSCE) with a monodisperse poly-N-substituted glycine (polypeptoid) as a synthetic drag-tag. FSCE enabled rapid separation of single-stranded (ss) DNA sequencing fragments with single-base resolution without the need for a viscous DNA separation matrix. Protein-based drag-tags previously used for FSCE sequencing, for example, streptavidin, are heterogeneous in molar mass (polydisperse); the resultant band-broadening can make it difficult to obtain the single-base resolution necessary for DNA sequencing. In this study, we synthesized and HPLC-purified a 70mer poly-N-(methoxyethyl)glycine (NMEG) drag-tag with a molar mass of - 11 kDa. The NMEG monomers that comprise this peptoid drag-tag are interesting for bioanalytical applications, because the methoxyethyl side chain's chemical structure is reminiscent of the basic monomer unit of polyethylene glycol, a highly biocompatible commercially available polymer, which, however, is not available in monodisperse preparation at an - 11 kDa molar mass. This is the first report of ssDNA separation and of four-color, base-by-base DNA sequencing by FSCE through the use of a chemically synthesized drag-tag. These results show that high-molar mass, chemically synthesized drag-tags based on the polyNMEG structure, if obtained in monodisperse preparation, would serve as ideal drag-tags and could help FSCE reach the commercially relevant read lengths of 100 bases or more.     

2'-anthraquinone-conjugated oligonucleotide as an electrochemical probe for DNA mismatch

We previously prepared the oligonucleotides (ODNs) conjugated to an anthraquinone (AQ) group via one carbon linker at the 2'-sugar position. When these modified ODNs bind to cDNA sequences, the AQ moiety can be intercalated into the predetermined base-pair pocket of a duplex DNA. In this paper, 2'-AQ-modified ODNs are shown to be an excellent electrochemical probe to clarify the effect of a mismatch base on the charge transfer (CT) though DNA. Two types of DNA-modified electrodes were constructed by assembly of disulfide-terminated 2'-AQ-ODN duplexes onto gold electrodes. One type of electrodes (system I) contains fully matched base pairs or a single-base mismatch in duplex DNA between the redox center and the electrode. The other (system II) consists of the mismatch but at the outside of the redox center. The modified electrodes were analyzed by cyclic voltammetry to estimate the CT rate through duplex DNA. In system I, the CT rate was found to be approximately 50 s (-1) for the fully matched AQ-ODN duplexes, while the CT rates of the mismatched DNA were considerably slower than that of the fully matched DNA. In system II, the AQ-ODN duplexes showed almost similar CT rates ( approximately 50 s (-1)) for the fully matched DNA and for the mismatched DNAs. The detection of a single-base mismatch was then performed by chronocoulometry (CC). All the DNA duplexes containing a mismatch base in system I gave the reduced electrochemical responses when compared to the fully matched DNA. In particular, the mismatched DNAs including G--A mismatch can be differentiated from fully matched DNA without using any electrochemical catalyst. We further tested the usefulness of single-stranded (ss) AQ-ODN immobilized on a gold electrode in the electrochemical detection of a single-base mismatch through hybridization assay. The ss-AQ-ODN electrodes were immersed in target-containing buffer at room temperature, and the CC measurements were carried out to see the changes in the integrated charge. Within 60 min, the mismatched DNA was clearly distinguishable by the CC differences from the fully matched target. Thus, the electrochemical hybridization assay provides an easy and convenient detection for DNA mutation that does not require any extra reagents, catalyst, target labeling, and washing steps.     

The function of the secondary DNA-binding site of RecA protein during DNA strand exchange.

RecA protein features two distinct DNA-binding sites. During DNA strand exchange, the primary site binds to single-stranded DNA (ssDNA), forming the helical RecA nucleoprotein filament. The weaker secondary site binds double-stranded DNA (dsDNA) during the homology search process. Here we demonstrate that this site has a second important function. It binds the ssDNA strand that is displaced from homologous duplex DNA during DNA strand exchange, stabilizing the initial heteroduplex DNA product. Although the high affinity of the secondary site for ssDNA is essential for DNA strand exchange, it renders DNA strand exchange sensitive to an excess of ssDNA which competes with dsDNA for binding. We further demonstrate that single-stranded DNA-binding protein can sequester ssDNA, preventing its binding to the secondary site and thereby assisting at two levels: it averts the inhibition caused by an excess of ssDNA and prevents the reversal of DNA strand exchange by removing the displaced strand from the secondary site.