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Re-evaluation of the Widely Applied Force-Frequency Relation for Frequency-Modulation AFM Under Solution

Journal of Shanghai Jiaotong university(Science) 2014年第5期19卷 612-616页

作者：何健锋胡钧孙洁林 CZAJKOWSKY Daniel M School of Biomedical Engineering Shanghai Jiaotong University Laboratory of Physical Biology Shanghai Institute of Applied PhysicsChinese Academy of Sciences Key Laboratory of Systems Biomedicine Shanghai Jiaotong University

Frequency-modulation atomic force microscopy(FM-AFM) is a highly versatile tool for surface *** imaging surfaces, FM-AFM is capable of measuring interactions between the AFM probe and the surface with high sensitivity, which can provide chemical information at sub-nanometer resolution. This is achieved by deconvoluting the frequency shift, which is directly measured in experiments, into the force between the probe and sample. At present, the widely used method to perform this deconvolution has been shown to be accurate under high quality(high-Q) factor vacuum conditions. However, under low quality(low-Q) factor conditions, such as in solution, it is not clear if this method is valid. A previous study apparently verified this relation for experiments in solution by comparing the force calculated by this equation with that obtained in separate experiments using the surface force apparatus(SFA). Here we show that, in solution, a more direct comparison of the force calculated by this relation with that directly measured by the cantilever deflection in AFM reveals significant differences,both qualitative and quantitative. However, we also find that there are complications that hinder this ***, while contact with the surface is clear in the direct measurements(including the SFA data), it is less certain in the FM-AFM case. Hence, it is not clear if the two methods are measuring the same tip-sample distance regimes. Thus, our results suggest that a more thorough verification of this relation is required, as application of this formulation for experiments in solution may not be valid.

关键词： frequency-modulation atomic force microscopy(FM-AFM) chemical mapping frequency-force relationship force spectroscopy

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Novel Experimental Strategy for High Resolution AFM Imaging of Membrane-Associated Bacterial Toxins

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Journal of Shanghai Jiaotong university(Science) 2014年第5期19卷 569-573页

作者：罗梦麟邵志峰沈轶 CZAJKOWSKY Daniel M 孙洁林 School of Biomedical Engineering Shanghai Jiaotong University Key Laboratory of Systems Biomedicine Shanghai Jiaotong University

Bacterial pore-forming toxins(PFTs) are essential virulence factors of many human pathogens. Knowledge of their structure within the membrane is critical for an understanding of their function in pathogenesis and for the development of useful therapy. Atomic force microscopy(AFM) has often been employed to structurally interrogate many membrane proteins, including PFTs, owing to its ability to produce sub-nanometer resolution images of samples under aqueous solution. However, an absolute prerequisite for AFM studies is that the samples are single-layered and closely-packed, which is frequently challenging with PFTs. Here, using the prototypical member of the cholesterol-dependent cytolysin family of PFTs, perfringolysin O(PFO), as a test sample, we have developed a simple, highly robust method that routinely produces clean, closely-packed samples across the entire specimen surface. In this approach, we first use a small Teflon well to prepare the supported lipid bilayer, remove the sample from the well, and then directly apply the proteins to the bilayer. For reasons that are not clear,bilayer preparation in the Teflon well is essential. We anticipate that this simple method will prove widely useful for the preparation of similar samples, and thereby enable AFM imaging of the greatest range of bacterial PFTs to the highest possible resolution.

关键词： atomic force microscopy(AFM) perfringolysin O(PFO) bacterial pore forming toxins(PFTs) supported lipid bilayers

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5’-Cap Selection Methods and Their Application in Full-Length cDNA Library Construction and Transcription Start Site Profiling

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Journal of Shanghai Jiaotong university(Science) 2014年第5期19卷 580-586页

作者：白玲王琪李红梅程酩张宁波李华 School of Biomedical Engineering Shanghai Jiaotong University

With the accomplishment of the genome draft sequences, identification of functional elements in genome has become an urgent task. Full-length cDNAs provide an important resource for gene identification and their precise structural feature determination. It also provides a basis for genomic element definition. As many regulatory elements are around transcription start sites(TSSs), precise localization of TSSs in the genome becomes a critical step for identifying the associated core promoters. Massive parallel snapshot of TSSs at a particular time under a specific experimental condition makes it possible to globally analyze important regulatory elements around TSSs and further construct transcriptional regulatory networks. In this paper, we first reviewed two important full-length cDNA cloning techniques: cap-trapper technique and oligo-capping technique. Then,we introduced deepCAGE, a cap-trapper and deep sequencing-based TSS profiling technique, and its applications in the research of transcriptional regulation.

关键词： cap-trapper oligo-capping deepCAGE transcription start sites(TSSs) deep sequencing

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用于DNA合成测序的可断裂连接单元研究现状

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化学进展 2016年第1期28卷 58-66页

作者：姜玉谭连江殷燕沈玉梅龚兵邵志峰上海应用技术学院化学与环境工程学院上海201418 上海交通大学系统生物医学研究院系统生物医学教育部重点实验室上海200240 上海交通大学生物医学工程学院Bio-ID中心上海200240 布法罗纽约州立大学化学系美国纽约14260

DNA测序技术是遗传基因组相关疾病研究的基础。合成测序是DNA二代测序技术中非常重要的一种。合成测序技术能够有效地实现大规模平行测序,大大提高测序通量的同时也降低成本,目前已得到广泛的应用。在合成测序中,首先需要合成荧光素标... 详细信息

DNA测序技术是遗传基因组相关疾病研究的基础。合成测序是DNA二代测序技术中非常重要的一种。合成测序技术能够有效地实现大规模平行测序,大大提高测序通量的同时也降低成本,目前已得到广泛的应用。在合成测序中,首先需要合成荧光素标记的核苷酸,作为能够参与DNA链延伸的循环可逆终端。已有文献报道的可逆终端结构主要包括单位点修饰(MRT)和双位点修饰(DRT)两种类型。DRT类型可逆终端的最大优势是DNA聚合酶容易识别且合成路线简单,能够较好地应用于DNA合成测序。在此过程中可断裂连接单元将核苷酸和荧光素连接起来,它的性质直接决定了测序的效率、读长等关键指标。本文主要对目前用于DNA合成测序的可断裂连接单元研究现状进行介绍,并对其发展前景进行了展望。

关键词： DNA合成测序循环可逆终端可断裂连接单元

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Direct Resolution of the Pitch of DNA on Positively Charged Lipid Bilayers by Frequency-Modulation AFM

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Journal of Shanghai Jiaotong university(Science) 2014年第5期19卷 565-568页

作者：张金金吕军鸿孙洁林胡钧 CZAJKOWSKY Daniel M 沈轶 Laboratory of Physical Biology Shanghai Institute of Applied PhysicsChinese Academy of Sciences School of Biomedical Engineering Shanghai Jiaotong University Key Laboratory of Systems Biomedicine Shanghai Jiaotong University

High resolution structural studies of DNA and DNA binding proteins by atomic force microscopy(AFM) require well-bound samples on suitably flat substrates. Adsorbing the DNA onto a positively charged supported lipid bilayer has previously been shown to be a potentially effective strategy for structural studies with AFM. Here, using our home-built frequency-modulation AFM(FM-AFM), we show that these bilayer substrates are only maximally effective for high resolution AFM when the samples are short, linear DNA, compared with circular plasmid DNA. We find that, with the former sample, the measured width of the DNA is about 2 nm, the known DNA diameter, and there is a clear height modulation along the length of the DNA with a periodicity of about 3.4 nm, in excellent agreement with the known pitch of the double helix. This sample preparation strategy is expected to enable higher resolution studies of DNA and DNA binding proteins with FM-AFM than that can presently be achieved.

关键词： frequency-modulation atomic force microscopy(FM-AFM) DNA double helix high resolution

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优化的Native PAGE蛋白回收方法在AFM中的应用

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基因组学与应用生物学 2019年第6期38卷 2706-2712页

作者：刘美君沈轶 Daniel M Czajkowsky 上海交通大学生物医学工程学院

原子力显微镜(AFM)可以在生理条件下获得生物大分子的自然结构信息而不需要进行图像处理。虽然对含有大小范围不一的异质样品成像可以得到几纳米的分辨率,但是具有更高的亚纳米级分辨率的图像仅可以从含有单一亚基数目的高度均一的蛋白... 详细信息

原子力显微镜(AFM)可以在生理条件下获得生物大分子的自然结构信息而不需要进行图像处理。虽然对含有大小范围不一的异质样品成像可以得到几纳米的分辨率,但是具有更高的亚纳米级分辨率的图像仅可以从含有单一亚基数目的高度均一的蛋白复合物获得。在重组蛋白的制备中这种蛋白复合物经常不存在,尤其是对于膜蛋白则更具有挑战性。本研究中,研究者以一种熟知的膜成孔蛋白复合物Perfringolysin O(PFO)作为模型系统,研制出一种方法来分离含有单一亚基数目的复合物,并用于随后在液相的AFM成像。具体而言,研究人员通过非变性聚丙烯酰胺凝胶电泳(Native PAGE)分离出该复合物中特定大小的蛋白成分,然后提取时采用一种新方法使结构完整保持。预计这种方法不仅对更多其他蛋白的高分辨率AFM研究有效用,而且在基于单分子方法和生物技术应用的研究上有更普遍的用途。

关键词：原子力显微镜(AFM) Perfringolysin O (PFO) 蛋白回收

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Interplay between the bacterial protein deacetylase CobB and the second messenger c-di-GMP

Interplay between the bacterial protein deacetylase CobB and...

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中国生物工程学会第十三届学术年会暨2019年全国生物技术大会

作者： Zhaowei Xu Hainan Zhang Xingrun Zhang Hewei Jiang Chengxi Liu Fanlin Wu Lili Qian Bingbing Hao Daniel M Czajkowsky Shujuan Guo Zhijing Xu Lijun Bi Shihua Wang Haitao Li Minjia Tan Wei Yan Lei Feng Jingli Hou Sheng-ce Tao Key Laboratory of Systems Biomedicine (Ministry of Education) Shanghai Center for Systems Biomedicine Shanghai Jiao Tong University MOE Key Laboratory of Protein Sciences Center for Structural Biology School of Life Sciences Tsinghua University Department of Basic Medical Sciences School of Medicine Tsinghua University The Chemical Proteomics Center and State Key Laboratory of Drug Research Shanghai Institute of Materia Medica Chinese Academy of Sciences School of Biomedical Engineering Shanghai Jiao Tong University College of Life Sciences Shanghai Normal University National Key Laboratory of Biomacromolecules Key Laboratory of Non-Coding RNA and Key Laboratory of Protein and Peptide Pharmaceuticals Institute of Biophysics Chinese Academy of Sciences School of Stomatology and Medicine Foshan University School of Life Science Fujian Agriculture and Forestry University Instrumental Analysis Center Shanghai Jiao Tong University State Key Laboratory of Oncogenes and Related Genes

As a ubiquitous bacterial secondary messenger, c-di-GMP plays key regulatory roles in processes such as bacterial motility and transcription regulation. CobB is the Sir2 family protein deacetylase that controls energy metabolism, chemotaxis, and DNA supercoiling in many bacteria. Using an Escherichia coli proteome microarray, we found that c-di-GMP strongly binds to CobB. Further, protein deacetylation assays showed that c-di-GMP inhibits the activity of CobB and thereby modulates the biogenesis of acetyl-CoA. Interestingly, we also found that one of the key enzymes directly involved in c-di-GMP production, DgcZ, is a substrate of CobB. Deacetylation of DgcZ by CobB enhances its activity and thus the production of cdi-GMP. Our work establishes a novel negative feedback loop linking c-di-GMP biogenesis and CobB-mediated protein deacetylation.

关键词： c-di-GMP CobB diguanylate cyclase negative feedback loop protein acetylation

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一种应用于低温原子力显微镜的新型生物样品制备方法

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基因组学与应用生物学 2020年第7期39卷 3031-3036页

作者：汤挺沈轶 Daniel M Czajkowsky 上海交通大学生物医学工程学院上海200240

低温原子力显微镜(cryogenic atomic force microscope,cryo-AFM)能够克服常温原子力显微镜(atomic force microscope,AFM)的诸多局限从而可以在生物大分子样品中获得更高分辨率的结构信息。然而,如何获得接近分子天然状态的洁净样品依... 详细信息

低温原子力显微镜(cryogenic atomic force microscope,cryo-AFM)能够克服常温原子力显微镜(atomic force microscope,AFM)的诸多局限从而可以在生物大分子样品中获得更高分辨率的结构信息。然而,如何获得接近分子天然状态的洁净样品依然是cryo-AFM面临的挑战之一,也是制约cryo-AFM进一步应用于更多不同生物分子结构解析的主要因素之一。针对这一问题,本研究提出了一种新的适用于cryo-AFM样品制备的方法,并使用人类补体分子(complement 1q,C1q)作为典型样品进行验证,实验结果表明使用本研究所提出的样品制备方法能够快速有效地制作出适用于低温原子力显微镜成像的洁净样品且同时能保证样品更接近天然结构。本研究预计该样品制备方法不仅适用于cryo-AFM,也将为其它真空或者低温下的生物大分子结构研究技术的样品制备提供借鉴。

关键词：低温原子力显微镜生物样品制备人类补体分子天然结构

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