用于金刚石表面单分子有序固定的DNA折纸制备

近年来兴起的以金刚石氮-空位(NV)色心为量子传感器的微观磁共振技术得到快速发展,已经实现单个生物分子磁共振谱的探测,正在向单分子结构和功能的研究推进.这其中需要解决一个重要的技术问题,即单分子在金刚石表面的有序分散和固定. DNA的自组装为解决这一问题提供了可行途径,本文使用DNA折纸技术,制备了一种60 nm边长的正方形双层DNA折纸作为单分子载体,并与金刚石表面结合.首先采用双层结构提高了DNA折纸的稳定性,其次通过在DNA折纸边缘添加发卡结构减少了DNA折纸结构间的聚团,最终成功将DNA折纸装配到金刚石表面.通过原子力显微镜图像进行表征显示其结构完整、分散均匀.本工作为后续的单分子磁共振技术在单分子生物物理领域的应用推广奠定了样品制备的基础.     

AFM Imaging and Analysis of Electrostatic Double Layer Forces on Single DNA Molecules

Electrical double layer (EDL) forces develop between charged surfaces immersed in an electrolyte solution. Biological material surrounded by its physiological medium constitutes a case where these forces play a major role. Specifically, this work is focused on the study of the EDL force exerted by DNA molecules, a standard reference for the study of single biomolecules of nanometer size. The molecules deposited on plane substrates have been characterized by means of the atomic force microscope operated in the force spectroscopy imaging mode. Force spectroscopy imaging provides images of the topography of the DNA molecules, and of the EDL force spectrum. Due to the size of the molecule being much smaller than that of the tip, both the tip-substrate and tip-molecule interactions need to be considered in the analysis of the experimental results. We solve this problem by linearly superposing the two contributions. EDL force images are presented where DNA molecules are clearly resolved. The lateral resolution of the EDL force is discussed and compared with that of the topography. The method also allows the estimation of the DNA surface charge density, thereby obtaining reasonable values.     

Oxaliplatin and Its Enantiomer Induce Different Condensation Dynamics of Single DNA Molecules

The interactions of DNA with oxaliplatin (Pt(R,R-DACH)) or its enantiomer (Pt(S,S-DACH)) were investigated using magnetic tweezers and atomic force microscope. In the process of DNA condensation induced by Pt-DACH, only diadducts and micro-loops are formed at low Pt-DACH concentrations, while at high Pt-DACH concentrations, besides the diadducts and micro-loops, long-range cross-links are also formed. The diadduct formation rate of Pt(R,R-DACH) is higher than that of Pt(S,S-DACH). However, the proportions of micro-loops and long-range cross-links for Pt(S,S-DACH) are higher than those for Pt(R,R-DACH). We propose a model to explain these differences between the effect of Pt(R,R-DACH) and that of Pt(S,S-DACH) on DNA condensation. The study has strong implications for the understanding of the effect of chirality on the interaction between Pt-DACH and DNA and the kinetics of DNA condensation induced by platinum complexes.     

High-Speed DNA Sequencing: An Approach Based Upon Fluorescence Detection of Single Molecules

Abstract

We are developing a laser based technique for the rapid sequencing of large fragments (～40 kb) of DNA based upon the detection of single, fluorescently tagged nucleotides cleaved from a single DNA fragment. We have demonstrated significant progress on several of the important steps of this technique. The projected rate of sequencing is several hundred bases per second which is orders of magnitude faster than existing methods. Once developed, this technology could be utilized by investigators for rapid sequencing of genetic material from virtually any source.     

Adenovirus type 2 DNA replication. II. Termini of DNA replication.

Complete, mature adenovirus type 2 DNA molecules were isolated from virus-infected HeLa cells, pulse-labeled at 20 h postinfection in [3H]thymidine pulses shorter than the time necessary for one round of viral DNA replication. After digestion with the restriction endonucleases Eco RI, Hpa I, and Hind III, a temporal order of synthesis of different regions of the viral genome was established from the relative specific radioactivities in the restriction enzyme fragments. A comparison with the physical order of these fragments revealed the existence of two termini of DNA replication towards both the molecular right and left ends, respectively, of the viral chromosome.     

Requirement of Watson-Crick hydrogen bonding for DNA synthesis by yeast DNA polymerase eta

Classical high-fidelity DNA polymerases discriminate between the correct and incorrect nucleotides by using geometric constraints imposed by the tight fit of the active site with the incipient base pair. Consequently, Watson-Crick (W-C) hydrogen bonding between the bases is not required for the efficiency and accuracy of DNA synthesis by these polymerases. DNA polymerase eta (Poleta) is a low-fidelity enzyme able to replicate through DNA lesions. Using difluorotoluene, a nonpolar isosteric analog of thymine unable to form W-C hydrogen bonds with adenine, we found that the efficiency and accuracy of nucleotide incorporation by Poleta are severely impaired. From these observations, we suggest that W-C hydrogen bonding is required for DNA synthesis by Poleta; in this regard, Poleta differs strikingly from classical high-fidelity DNA polymerases.     

Quantum-chemical investigation of tautomerization ways of Watson-Crick DNA base pair guanine-cytosine

A novel physico-chemical mechanism of the Watson-Crick DNA base pair Gua.Cyt tautomerization Gua.Cyt*<---->Gua.Cyt<---->Gua*.Cyt (mutagenic tautomers of bases are marked by asterisks) have been revealed and realized in a pathway of single proton transfer through two mutual isoenergetic transition states with Gibbs free energy of activation 30.4 and 30.6 kcal/mol and they are ion pairs stabilized by three (N2H...N3, N1H...N4- and O6+H...N4-) and five (N2H...O2, N1H...O2, N1H...N3, O6+H...N4- and 06+H...N4-) H-bonds accordingly. Stable base pairs Gua-Cyt* and Gua*.Cyt which dissociate comparably easy into monomers have acceptable relative Gibbs energies--12.9 and 14.3 kcal/mol--for the explanation of the nature of the spontaneous transitions of DNA replication. Results are obtained at the MP2/6-311++G(2df,pd)//B3LYP/6-31 1++G(d,p) level of theory in vacuum approach.     

PCR产物末端性质的分析研究

利用PCR、UT-PCR、克隆及测序等技术,对强直性肌营养不良基因（MT-PK）3′-非翻译区分别用Taq,Taq＋Pwo DNA聚合酶进行了扩增、克隆和测序,研究了PCR产物末端组成情况,并比较了上述两种DNA聚合酶对PCR产物末端的影响.结果在用Taq DNA聚合酶扩增的PCR产物主要得到3′端突出1个A（占67.3％,35/52）;在Taq＋Pwo DNA聚合酶扩增的PCR产物末端中得到3′端+A的仅占17.4％,而－1的占34.8％,与前者显著不同.表明PCR扩增产物的末端是复杂多样的.     

Stability of DNA duplexes with Watson-Crick base pairs: a predicted model

The conformational stability (difference between the free energies of the folded and unfolded states, DeltaG degrees ) of a DNA duplex is considered as a function of component energy terms, hydrophobic, base stacking, hydrogen bonding, van der Waals, and electrostatic, and a trinucleotide-level helix stiffness parameter measured in terms of its Young's modulus. Hydrophobic and base stacking energy components were determined with the use of the crystal structure data of 30 DNA duplexes judicially selected within a resolution of 1.5 A, and hydrogen bonding, van der Waals and electrostatic terms were determined through an extensive review of experimental and theoretical studies. The stiffness indices for the trinucleotides were the ones realized by M. M. Gromiha [(2000) J. Biol. Phys. 26, 43-50] using the crystal structure data of 70 DNA duplexes. The unfolded state was treated in the classical way to determine its stability. Thermodynamically determined DeltaG degrees values for 111 DNA duplexes, with the number of base pairs ranging from 4 to 16, were selected in two sets, and the regression equation formed with one set was used to predict the stabilities of the other set, taking the energy components and the stiffness parameter to be independent variables. The computed energy terms indicate that the base stacking and hydrogen bonding forces are the dominant and the hydrophobic and electrostatic forces the weak partners in imparting stability to the duplexes. This model predicts DeltaG degrees values for DNA duplexes examined with a level of accuracy similar to that used for predictions made by the widely used nearest-neighbor models. The uniqueness of this model is that it combines the crystal and thermodynamic data for interpretation of conformational stability.     

Productive and Nonproductive Complexes of Ku and DNA-Dependent Protein Kinase at DNA Termini

DNA-dependent protein kinase (DNA-PK) is the only eukaryotic protein kinase known to be specifically activated by double-stranded DNA (dsDNA) termini, accounting for its importance in repair of dsDNA breaks and its role in physiologic processes involving dsDNA breaks, such as V(D)J recombination. In this study we conducted kinase and binding analyses using DNA-PK on DNA termini of various lengths in the presence and absence of Ku. We confirmed our previous observations that DNA-PK can bind DNA termini in the absence of Ku, and we determined rate constants for binding. However, in the presence of Ku, DNA-PK can assume either a productive or a nonproductive configuration, depending on the length of the DNA terminus. For dsDNA greater than 26 bp, the productive mode is achieved and Ku increases the affinity of the DNA-PK for the Ku:DNA complex. The change in affinity is achieved by increases in both the kinetic association rate and reduction in the kinetic dissociation rate. For dsDNA smaller than 26 bp, the nonproductive mode, in which DNA-PK is bound to Ku:DNA but is inactive as a kinase, is assumed. Both the productive and nonproductive configurations are likely to be of physiologic importance, depending on the distance of the dsDNA break site to other protein complexes, such as nucleosomes.     

Nanomanipulation of Single RNA Molecules by Optical Tweezers

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be increasingly important. As RNA function often depends on its ability to adopt alternative structures, it is difficult to predict RNA three-dimensional structures directly from sequence. Single-molecule approaches show potentials to solve the problem of RNA structural polymorphism by monitoring molecular structures one molecule at a time. This work presents a method to precisely manipulate the folding and structure of single RNA molecules using optical tweezers. First, methods to synthesize molecules suitable for single-molecule mechanical work are described. Next, various calibration procedures to ensure the proper operations of the optical tweezers are discussed. Next, various experiments are explained. To demonstrate the utility of the technique, results of mechanically unfolding RNA hairpins and a single RNA kissing complex are used as evidence. In these examples, the nanomanipulation technique was used to study folding of each structural domain, including secondary and tertiary, independently. Lastly, the limitations and future applications of the method are discussed.     

Segregation of Partly Melted DNA Molecules

Segregation of partly melted DNA molecules is a convenient and efficient method to isolate DNA fragments associated with CpG islands. The method stands on the observation that the electrophoretic mobility of partly melted DNA fragments in a denaturing gradient gel is low and that they persist in the gel so long as the remaining helical part is sufficiently resistant to strand dissociation and dissociates slowly. Such features are observed in DNA fragments derived from CpG islands. These DNA fragments are preferentially retained in a denaturing gradient gel after prolonged electric field exposure, permitting the enrichment of DNA fragments derived from CpG islands. The principle and practical application of this method are reviewed.     

Internal Dynamics of Supercoiled DNA Molecules

The intramolecular diffusive motion within supercoiled DNA molecules is of central importance for a wide array of gene regulation processes. It has recently been shown, using fluorescence correlation spectroscopy, that plasmid DNA exhibits unexpected acceleration of its internal diffusive motion upon supercoiling to intermediate density. Here, we present an independent study that shows a similar acceleration for fully supercoiled plasmid DNA. We have developed a method that allows fluorescent labeling of a 200-bp region, as well as efficient supercoiling by Escherichia coli gyrase. Compared to plain circular or linear DNA, the submicrosecond motion within the supercoiled molecules appears faster by up to an order of magnitude. The mean-square displacement as a function of time reveals an additional intermediate regime with a lowered scaling exponent compared to that of circular DNA. Although this unexpected behavior is not fully understood, it could be explained by conformational constraints of the DNA strand within the supercoiled topology in combination with an increased apparent persistence length.     

Fano Resonance of Nanocrescent for the Detection of Single Molecules and Single Nanoparticles

This paper reports a theoretical study on the Fano resonance of a 3D nanocrescent and its application in single molecular detection. The resonance wavelength changes with the crescent radius, gap width and thickness. The Fano resonance is attributed to the interference between the quadrupolar mode supported by the horizontal crescent and the quadrupolar mode supported by the nanotip oscillating along the height direction. The Fano resonance is highly sensitive to a nanoparticle trapped by the nanocrescent. The wavelength shift is larger than 0.5 nm when a single protein nanoparticle with radius only of 1.25 nm is trapped. For a protein with radius of 0.3 nm, the wavelength shift is still larger than 0.03 nm, over the detection limit (10^?5 nm) by 3 orders in the magnitude, which indicates that the nanocrescent can be used to detect small molecule with several atoms.     

A Highly Accurate Pixel-Based FRAP Model Based on Spectral-Domain Numerical Methods

We introduce a new, to our knowledge, numerical model based on spectral methods for analysis of fluorescence recovery after photobleaching data. The model covers pure diffusion and diffusion and binding (reaction-diffusion) with immobile binding sites, as well as arbitrary bleach region shapes. Fitting of the model is supported using both conventional recovery-curve-based estimation and pixel-based estimation, in which all individual pixels in the data are utilized. The model explicitly accounts for multiple bleach frames, diffusion (and binding) during bleaching, and bleaching during imaging. To our knowledge, no other fluorescence recovery after photobleaching framework incorporates all these model features and estimation methods. We thoroughly validate the model by comparison to stochastic simulations of particle dynamics and find it to be highly accurate. We perform simulation studies to compare recovery-curve-based estimation and pixel-based estimation in realistic settings and show that pixel-based estimation is the better method for parameter estimation as well as for distinguishing pure diffusion from diffusion and binding. We show that accounting for multiple bleach frames is important and that the effect of neglecting this is qualitatively different for the two estimation methods. We perform a simple experimental validation showing that pixel-based estimation provides better agreement with literature values than recovery-curve-based estimation and that accounting for multiple bleach frames improves the result. Further, the software developed in this work is freely available online.     

DNA 分子简谐特性的研究

考虑周期驱动力下的ＤＮＡ分子系统的福克普朗克方程。通过数值计算，研究期望值〈ｘ（ｔ）〉的简谐性。根据随机力强度对简谐特性的研究，我们得到温度有益于癌症治疗的结论。     

Mutagenic role of Watson-Crick protons in alkylated DNA bases: A theoretical study

Loss of Watson-Crick protons following DNA base alkylation has been proposed as a key event which confers mutation-inducing properties on to alkylated DNA bases. In this theoretical study, the promutagenic O⁶-guanine and O⁴-thymine sites are clearly distinguished from the nonmutagenic N⁷-guanine site on the basis of calculated values of mechanistic indicators for Watson-Crick proton acidity following alkylation at these respective sites. The degree of acidity predicted for these protons for each type of alkylated base accords well with the presence or absence of mutagenicity observed experimentally in each case.