DNA and histone protein are important in the formation of nucleosomal arrays, which are the first packaging level of DNA into a more compact chromatin structure. To characterize the interactions of DNA and histone pro...DNA and histone protein are important in the formation of nucleosomal arrays, which are the first packaging level of DNA into a more compact chromatin structure. To characterize the interactions of DNA and histone proteins, we reconstitute nucleosomes using lambda DNA and whole histone proteins by dialysis and perform direct atomic force microscopy （AFM） imaging. Compared with non-specific DNA and histone binding, nucleosomes are formed within the assembled “beads-on-a-string” nucleosomal array by dialysis. These observations facilitate the establishment of the molecular mechanisms of nucleosome and demonstrate the capability of AFM for protein-DNA interaction analysis.展开更多
Nano-manipulation of single atoms and molecules is a critical technique in nanoscience and nanotech- nology. This review paper will focus on the recent development of the manipulation of single DNA molecules based on ...Nano-manipulation of single atoms and molecules is a critical technique in nanoscience and nanotech- nology. This review paper will focus on the recent development of the manipulation of single DNA molecules based on atomic force microscopy (AFM). Precise manipulation has been realized including varied manipulating modes such as “cutting”, “pushing”, “folding”, “kneading”, “picking up”, “dipping”, etc. The cutting accuracy is dominated by the size of the AFM tip, which is usually 10nm or less. Single DNA fragments can be cut and picked up and then amplified by single molecule PCR. Thus positioning isolation and sequencing can be performed.展开更多
Anticoagulation factor I (ACF I) from the venom of Agki^strodon acutus is a binding protein to activated coagulation factor X (FXa) and possesses marked anticoagulant activity. Single ACF I molecule has been succe...Anticoagulation factor I (ACF I) from the venom of Agki^strodon acutus is a binding protein to activated coagulation factor X (FXa) and possesses marked anticoagulant activity. Single ACF I molecule has been successfully imaged in air by tapping mode atomic force microscopy (AFM) with high resolution using glutaraldehyde as a coupling agent. The physical adsorption and covalent binding of ACF I onto the mica show very different surface topographies. The former exhibits the characteristic strand like structure with much less reproducibility, the latter displays a elliptic granular structure with better reproducibility, which suggests that the stability of ACF I molecules on the mica is enhanced by covalent bonding in the presence of glutaraldehyde. A small scale AFM amplitude mode image clearly shows that the covalently bonded ACF I molecule by glutaraldehyde has olive shape structure with an average size of 7 4 nm×3 6 nm×3 1 nm, which is very similar to the size determined from the crystal structure of ACF I.展开更多
Elucidating the underlying mechanisms of cell physiology is currently an important research topic in life sciences. Atomic force microscopy methods can be used to investigate these molecular mechanisms. In this study,...Elucidating the underlying mechanisms of cell physiology is currently an important research topic in life sciences. Atomic force microscopy methods can be used to investigate these molecular mechanisms. In this study, single-molecule force spectroscopy was used to explore the specific recognition between the CD20 antigen and anti-CD20 antibody Rituximab on B lymphoma cells under near-physiological conditions. The CD20-Rituximab specific binding force was measured through tip functionalization. Distribution of CD20 on the B lymphoma cells was visualized three-dimensionally. In addition, the relationship between the intramolecular force and the molecular extension of the CD20-Rituximab complex was analyzed under an external force. These results facilitate further investigation of the mechanism of Rituximab’s anti-cancer effect.展开更多
DNA molecules were stretched on silanized mica surface with the molecular combing technique, and detected with fluorescence microscopy and atomic force microscopy. Meantime, DNA molecules were stretched with a modifie...DNA molecules were stretched on silanized mica surface with the molecular combing technique, and detected with fluorescence microscopy and atomic force microscopy. Meantime, DNA molecules were stretched with a modified dynamic molecular combing technique and studied with atomic force microscopy. The results indicate that, compared with the dynamic molecular combing technique, the modified dynamic molecular combing technique has advantages of less-sample demand and less contamination to sample; as compared with the molecular combing technique, it has better aligning effect and reproducibility. Combination of this kind of DNA molecular manipulating technique with the single DNA molecule detecting technique by atomic force microscopy and fluorescence microscopy will play an important role in the basic research of molecular dynamics and the application of gene research.展开更多
In this paper, a new approach is demonstrated to measure the compression elasticity of single biomolecule in small force regime (<0.5 nN) using vibrating mode scanning polarization force microscopy (VSPFM). With th...In this paper, a new approach is demonstrated to measure the compression elasticity of single biomolecule in small force regime (<0.5 nN) using vibrating mode scanning polarization force microscopy (VSPFM). With this method we investigate the compression elasticity of a single DNA molecule in the radial direction (perpendicular to DNA strands). The radial deformation of DNA molecules deposited on mica surface is shown to be able to reach about 50% un der external load, and this remarkable deformation is re- versible. In addition, the compression spring constant of DNA molecules is estimated to be about 0.6 nN/nm according to the height-force curves.展开更多
Atomic force micriscope (AFM)-based dip-pen nanolithography (DPN) is an emerging approach for con-structing nanostructures on material surfaces such as gold, silicon and silicon oxide. Although DPN is a powerful tech-...Atomic force micriscope (AFM)-based dip-pen nanolithography (DPN) is an emerging approach for con-structing nanostructures on material surfaces such as gold, silicon and silicon oxide. Although DPN is a powerful tech-nique, it has not shown its ability of direct-writing and pat-terning of nanostructures on surfaces of soft materials, for example biomacromolecules. Direct depositing on soft sur-faces becomes possible with the introduction of a com-bined-dynamic mode DPN rather than mostly used contact mode DPN or tapping mode DPN. In this report, the com-bined dynamic mode DPN is used for direct depositing pro-tein ink on DNA molecules at the nanometer scale.展开更多
A method for real time observation of photo-cleavage of stretched λDNA at single molecular level by a fluorescent microscope coupled with CCD is developed. DNA molecules stained with YOYO-1 are stretched by the mo-le...A method for real time observation of photo-cleavage of stretched λDNA at single molecular level by a fluorescent microscope coupled with CCD is developed. DNA molecules stained with YOYO-1 are stretched by the mo-lecular combing technique and fixed on a modified slide. Then the process of photocleavage and relaxation of DNA under radiation of blue light is observed. We speculate that the conformation change of stretched DNA and the effect of water are likely to facilitate the effect of YOYO photocleav-age DNA molecules. The photocleavage effect of YOYO for stretched DNA may be useful to study DNA elasticity, cancer research as well as the interaction between DNA and dyes.展开更多
The rupture force of the streptavidin-biotin complex was investigated using atomic force microscopy (AFM). The most frequently observed rupture force (MFOF), which is essential for the evaluation of the potential land...The rupture force of the streptavidin-biotin complex was investigated using atomic force microscopy (AFM). The most frequently observed rupture force (MFOF), which is essential for the evaluation of the potential landscape, was evaluated by processing 22,500 force curves using two methods. One method is a conventional method, which is usually built in commercial AFM systems, i.e., difference between the baseline value and the minimum force value in the force curve. The other is a detection of rupture events based on a fuzzy logic algorithm to detect the rupture event from analyzing the shape of the force curves. Our statistical analysis revealed that the conventional method exhibited a significant artifact, which is the increase in the population of small forces comparable to thermal noise of cantilevers, resulting in a smaller MFOF. Based on this finding, we discuss the choice of a method and its effecton the illustrated potential landscapes of ligand-receptor complexes.展开更多
A modified "dynamic molecular combing"(DMC) technique used for stretching double-stranded DNA is reported. DNA molecules were stretched on the silanized mica surface by this technique, its speed being precis...A modified "dynamic molecular combing"(DMC) technique used for stretching double-stranded DNA is reported. DNA molecules were stretched on the silanized mica surface by this technique, its speed being precisely controlled with a computer. This approach combined the precise DNA stretching method with high resolution AFM imaging at nanometer scale, thus making it useful for DNA alignment manipulation and subsequent gene research.展开更多
该文利用一种基于原子力显微镜(AFM)抬高模式(Lift mode)的"逐线反馈纳米操纵"技术成功地进行了DNA单分子水平上的切割、拾取及连接酶分子的传递,系统地完成了DNA的分子手术。在切割和拾取过程中,以PBR322/Pst I DNA为研究对...该文利用一种基于原子力显微镜(AFM)抬高模式(Lift mode)的"逐线反馈纳米操纵"技术成功地进行了DNA单分子水平上的切割、拾取及连接酶分子的传递,系统地完成了DNA的分子手术。在切割和拾取过程中,以PBR322/Pst I DNA为研究对象,进行了单分子水平上的切割,实验发现由于DNA分子本身弹性,切割过程极易出现切割端变粗的现象。在分子传递过程中,分别以T4 DNA连接酶和小牛胸腺组蛋白分子为研究对象,实现针尖和基底表面之间的分子传递。同时通过控制针尖运动成功获得连接酶分子点阵排列及小牛胸腺组蛋白方块状纳米结构。结果表明利用此操纵方法可以获得很高的精确度,切割DNA时的空间精确度小于5 nm。系列单分子水平上的分子手术的整合为实现单分子水平上生化反应,甚至构造智能机器提供了可能。展开更多
链霉亲和素(Streptavidin,STV)和生物素(Biotin)之间的非共价相互作用很强,已被运用到包括分子生物学、免疫学以及生物技术领域。STV与Biotin间优异的结合能力与其结构密切相关,晶体解析结果证明STV是一个四聚体蛋白。原子力显微镜技术(...链霉亲和素(Streptavidin,STV)和生物素(Biotin)之间的非共价相互作用很强,已被运用到包括分子生物学、免疫学以及生物技术领域。STV与Biotin间优异的结合能力与其结构密切相关,晶体解析结果证明STV是一个四聚体蛋白。原子力显微镜技术(Atomic Force Microscopy,AFM)可以在液体环境下获得生物单分子的形貌信息,对结构和功能的研究具有非常重要的作用。然而,由于STV分子小而柔软,获得高分辨的AFM图像极其困难。本研究利用DNA折纸可寻址的特点,将Biotin定位修饰在折纸上,选择性地将STV固定在折纸设定的位置上,实现了对单个STV分子的高分辨成像,观察到了单个STV呈"沙漏形"结构,与其晶体结构符合性较好。同时,我们注意到STV形貌易受成像力的影响,可能与STV分子的柔性相关。这种基于DNA折纸的高分辨成像方法,简单、方便,为研究生物分子形貌和功能提供了新思路。展开更多
基于单链DNA(single-stranded DNA,ss DNA)和单链DNA键合蛋白质(ss DNA binding protein SSBP)的高亲和力,在十六烷基硫醇修饰的金基底(HDT/Au)上构建了SSBP的分形结构。利用高分辨原子力显微镜表征了SSBP的枝状结构,在不同的组装时间下...基于单链DNA(single-stranded DNA,ss DNA)和单链DNA键合蛋白质(ss DNA binding protein SSBP)的高亲和力,在十六烷基硫醇修饰的金基底(HDT/Au)上构建了SSBP的分形结构。利用高分辨原子力显微镜表征了SSBP的枝状结构,在不同的组装时间下,SSBP在HDT/Au基底形成不同结构的可控形貌,形成的自组装结构具有高度有序性,可以有效地阻止生物分子的相互交叉或重叠。利用这种二维自组装方法形成的蛋白质纤维结构的变化遵循有限扩散凝聚(Diffusionlimited aggregation,DLA)过程。SSBP和ss DNA之间的高亲和力是整个自组装过程的驱动力,由DNA引导蛋白质构筑的分形结构提供了一种构建形貌可控的蛋白质纳米纤维的新方法。展开更多
We construct a system of magnetic tweezers and apply it to study the interaction between histones and DNA. The condensation of DNA by purified histones at low ionic strengths is directly monitored by recording the len...We construct a system of magnetic tweezers and apply it to study the interaction between histones and DNA. The condensation of DNA by purified histones at low ionic strengths is directly monitored by recording the length of the DNA as a function of elapsed time. It is found that DNA condensates in a dynamic manner. The binding of hist, ones to DNA is energetically favoured, but the ten,sion applied on DNA tends to unravel the DNA-histone complex, The competition between the two processes determiners the rate of the DNA condensation.展开更多
Investigation on the folding mode of a single polymer chain in its crystal is significant to the understanding of the mechanism of the fundamental crystallization as well as the engineering of new polymer crystal-base...Investigation on the folding mode of a single polymer chain in its crystal is significant to the understanding of the mechanism of the fundamental crystallization as well as the engineering of new polymer crystal-based materials. Herein, we use the combined techniques of atomic force microscopy （AFM） imaging and force spectroscopy to pull a single polyethylene oxide （PEO） chain out of its spiral crystal in amyl acetate. From these data, the folding mode of polymer chains in the spiral crystal has been reconstructed. We find that the stems tilt in the typical flat area, leading to the decrease in the apparent lamellar height. While in the area of screw dislocation, the lamellar height gradually increases in the range of several nanometers. These results indicate that the combined techniques present a novel tool to directly unravel the chain folding mode of spiral crystals at single-molecule level.展开更多
基金ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China （No.11274374）, the National Basic Research Program of China （No.2009CB930704）, and the Basic Scientific Research Foundation of China Agricultural University （No.2012QJ026）.
文摘DNA and histone protein are important in the formation of nucleosomal arrays, which are the first packaging level of DNA into a more compact chromatin structure. To characterize the interactions of DNA and histone proteins, we reconstitute nucleosomes using lambda DNA and whole histone proteins by dialysis and perform direct atomic force microscopy （AFM） imaging. Compared with non-specific DNA and histone binding, nucleosomes are formed within the assembled “beads-on-a-string” nucleosomal array by dialysis. These observations facilitate the establishment of the molecular mechanisms of nucleosome and demonstrate the capability of AFM for protein-DNA interaction analysis.
文摘Nano-manipulation of single atoms and molecules is a critical technique in nanoscience and nanotech- nology. This review paper will focus on the recent development of the manipulation of single DNA molecules based on atomic force microscopy (AFM). Precise manipulation has been realized including varied manipulating modes such as “cutting”, “pushing”, “folding”, “kneading”, “picking up”, “dipping”, etc. The cutting accuracy is dominated by the size of the AFM tip, which is usually 10nm or less. Single DNA fragments can be cut and picked up and then amplified by single molecule PCR. Thus positioning isolation and sequencing can be performed.
文摘Anticoagulation factor I (ACF I) from the venom of Agki^strodon acutus is a binding protein to activated coagulation factor X (FXa) and possesses marked anticoagulant activity. Single ACF I molecule has been successfully imaged in air by tapping mode atomic force microscopy (AFM) with high resolution using glutaraldehyde as a coupling agent. The physical adsorption and covalent binding of ACF I onto the mica show very different surface topographies. The former exhibits the characteristic strand like structure with much less reproducibility, the latter displays a elliptic granular structure with better reproducibility, which suggests that the stability of ACF I molecules on the mica is enhanced by covalent bonding in the presence of glutaraldehyde. A small scale AFM amplitude mode image clearly shows that the covalently bonded ACF I molecule by glutaraldehyde has olive shape structure with an average size of 7 4 nm×3 6 nm×3 1 nm, which is very similar to the size determined from the crystal structure of ACF I.
基金supported by the National Natural Science Foundation of China (Grant Nos. 60904095 and 60635040)the National High Technology Research and Development Program of China (Grant No. 2009AA03Z316)the CAS FEA International Partnership Program for Creative Research Teams and the State Key Laboratory of Drug Research.
文摘Elucidating the underlying mechanisms of cell physiology is currently an important research topic in life sciences. Atomic force microscopy methods can be used to investigate these molecular mechanisms. In this study, single-molecule force spectroscopy was used to explore the specific recognition between the CD20 antigen and anti-CD20 antibody Rituximab on B lymphoma cells under near-physiological conditions. The CD20-Rituximab specific binding force was measured through tip functionalization. Distribution of CD20 on the B lymphoma cells was visualized three-dimensionally. In addition, the relationship between the intramolecular force and the molecular extension of the CD20-Rituximab complex was analyzed under an external force. These results facilitate further investigation of the mechanism of Rituximab’s anti-cancer effect.
文摘DNA molecules were stretched on silanized mica surface with the molecular combing technique, and detected with fluorescence microscopy and atomic force microscopy. Meantime, DNA molecules were stretched with a modified dynamic molecular combing technique and studied with atomic force microscopy. The results indicate that, compared with the dynamic molecular combing technique, the modified dynamic molecular combing technique has advantages of less-sample demand and less contamination to sample; as compared with the molecular combing technique, it has better aligning effect and reproducibility. Combination of this kind of DNA molecular manipulating technique with the single DNA molecule detecting technique by atomic force microscopy and fluorescence microscopy will play an important role in the basic research of molecular dynamics and the application of gene research.
基金This work was supported by the National Natural Science Foundation of China(Grant Nos.10304011 and 10335070)Chinese Academy of Sciences+1 种基金Shanghai Science Committee Ningbo University.
文摘In this paper, a new approach is demonstrated to measure the compression elasticity of single biomolecule in small force regime (<0.5 nN) using vibrating mode scanning polarization force microscopy (VSPFM). With this method we investigate the compression elasticity of a single DNA molecule in the radial direction (perpendicular to DNA strands). The radial deformation of DNA molecules deposited on mica surface is shown to be able to reach about 50% un der external load, and this remarkable deformation is re- versible. In addition, the compression spring constant of DNA molecules is estimated to be about 0.6 nN/nm according to the height-force curves.
文摘Atomic force micriscope (AFM)-based dip-pen nanolithography (DPN) is an emerging approach for con-structing nanostructures on material surfaces such as gold, silicon and silicon oxide. Although DPN is a powerful tech-nique, it has not shown its ability of direct-writing and pat-terning of nanostructures on surfaces of soft materials, for example biomacromolecules. Direct depositing on soft sur-faces becomes possible with the introduction of a com-bined-dynamic mode DPN rather than mostly used contact mode DPN or tapping mode DPN. In this report, the com-bined dynamic mode DPN is used for direct depositing pro-tein ink on DNA molecules at the nanometer scale.
基金supposed by the National Natural Science Foundation of China(Grant No.19890385)the fund from the Chinese Academy of Sciences(Grant Nos.STZ-00-07,KJCX-06 and KSCXI-06)the Science and Technology Commission of Shanghai(Grant No.00XD1444029).
文摘A method for real time observation of photo-cleavage of stretched λDNA at single molecular level by a fluorescent microscope coupled with CCD is developed. DNA molecules stained with YOYO-1 are stretched by the mo-lecular combing technique and fixed on a modified slide. Then the process of photocleavage and relaxation of DNA under radiation of blue light is observed. We speculate that the conformation change of stretched DNA and the effect of water are likely to facilitate the effect of YOYO photocleav-age DNA molecules. The photocleavage effect of YOYO for stretched DNA may be useful to study DNA elasticity, cancer research as well as the interaction between DNA and dyes.
文摘The rupture force of the streptavidin-biotin complex was investigated using atomic force microscopy (AFM). The most frequently observed rupture force (MFOF), which is essential for the evaluation of the potential landscape, was evaluated by processing 22,500 force curves using two methods. One method is a conventional method, which is usually built in commercial AFM systems, i.e., difference between the baseline value and the minimum force value in the force curve. The other is a detection of rupture events based on a fuzzy logic algorithm to detect the rupture event from analyzing the shape of the force curves. Our statistical analysis revealed that the conventional method exhibited a significant artifact, which is the increase in the population of small forces comparable to thermal noise of cantilevers, resulting in a smaller MFOF. Based on this finding, we discuss the choice of a method and its effecton the illustrated potential landscapes of ligand-receptor complexes.
文摘A modified "dynamic molecular combing"(DMC) technique used for stretching double-stranded DNA is reported. DNA molecules were stretched on the silanized mica surface by this technique, its speed being precisely controlled with a computer. This approach combined the precise DNA stretching method with high resolution AFM imaging at nanometer scale, thus making it useful for DNA alignment manipulation and subsequent gene research.
文摘链霉亲和素(Streptavidin,STV)和生物素(Biotin)之间的非共价相互作用很强,已被运用到包括分子生物学、免疫学以及生物技术领域。STV与Biotin间优异的结合能力与其结构密切相关,晶体解析结果证明STV是一个四聚体蛋白。原子力显微镜技术(Atomic Force Microscopy,AFM)可以在液体环境下获得生物单分子的形貌信息,对结构和功能的研究具有非常重要的作用。然而,由于STV分子小而柔软,获得高分辨的AFM图像极其困难。本研究利用DNA折纸可寻址的特点,将Biotin定位修饰在折纸上,选择性地将STV固定在折纸设定的位置上,实现了对单个STV分子的高分辨成像,观察到了单个STV呈"沙漏形"结构,与其晶体结构符合性较好。同时,我们注意到STV形貌易受成像力的影响,可能与STV分子的柔性相关。这种基于DNA折纸的高分辨成像方法,简单、方便,为研究生物分子形貌和功能提供了新思路。
文摘基于单链DNA(single-stranded DNA,ss DNA)和单链DNA键合蛋白质(ss DNA binding protein SSBP)的高亲和力,在十六烷基硫醇修饰的金基底(HDT/Au)上构建了SSBP的分形结构。利用高分辨原子力显微镜表征了SSBP的枝状结构,在不同的组装时间下,SSBP在HDT/Au基底形成不同结构的可控形貌,形成的自组装结构具有高度有序性,可以有效地阻止生物分子的相互交叉或重叠。利用这种二维自组装方法形成的蛋白质纤维结构的变化遵循有限扩散凝聚(Diffusionlimited aggregation,DLA)过程。SSBP和ss DNA之间的高亲和力是整个自组装过程的驱动力,由DNA引导蛋白质构筑的分形结构提供了一种构建形貌可控的蛋白质纳米纤维的新方法。
基金Supported by the National Natural Science Foundation of China under Grant No 10334100. The authors acknowledge the help of Professor .J. Yan of the National University of Singapore in setting up the instrument.
文摘We construct a system of magnetic tweezers and apply it to study the interaction between histones and DNA. The condensation of DNA by purified histones at low ionic strengths is directly monitored by recording the length of the DNA as a function of elapsed time. It is found that DNA condensates in a dynamic manner. The binding of hist, ones to DNA is energetically favoured, but the ten,sion applied on DNA tends to unravel the DNA-histone complex, The competition between the two processes determiners the rate of the DNA condensation.
基金financially supported by the National Natural Science Foundation of China(Nos.20974039,21221063 and 91127031)the National Basic Research Program(2013CB834503)the Program for New Century Excellent Talents in Universities(NCET-11-0205)
文摘Investigation on the folding mode of a single polymer chain in its crystal is significant to the understanding of the mechanism of the fundamental crystallization as well as the engineering of new polymer crystal-based materials. Herein, we use the combined techniques of atomic force microscopy （AFM） imaging and force spectroscopy to pull a single polyethylene oxide （PEO） chain out of its spiral crystal in amyl acetate. From these data, the folding mode of polymer chains in the spiral crystal has been reconstructed. We find that the stems tilt in the typical flat area, leading to the decrease in the apparent lamellar height. While in the area of screw dislocation, the lamellar height gradually increases in the range of several nanometers. These results indicate that the combined techniques present a novel tool to directly unravel the chain folding mode of spiral crystals at single-molecule level.
