Base-selective heavy atom labels for electron microscopy-based DNA sequencing

用于基于电子显微镜的 DNA 测序的碱基选择性重原子标记

基本信息

批准号：
8134491
负责人：
F. Dean Toste
金额：
$ 21.76万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-09-01 至 2013-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8134491
关键词：
Base Pairing Beryllium Binding Biological Assay Church Collaborations Complex Consensus Cytosine DNA DNA Sequence Data Deposition Development Diamines Dose Electron Microscopy Electrons Elements Engineering Fostering Genome Gold Guanosine Human Human Genome Individual Label Ligands Measures Mediating Mercury Metals Methods Molecular NMR Spectroscopy Organometallic Compounds Osmium Osmium Tetroxide Platinum Production Protocols documentation Purines Pyrimidine Reaction Reading Reagent Relative (related person)Research Personnel Scheme Single-Stranded DNA Speed Spottings Technology Testing Thymine Transmission Electron Microscopy United States National Institutes of Health Uranium Work abstracting base cost density design detector experience genome sequencing improved instrument interest lens medical schools programs public health relevance purine research study scaffold success translational medicine trend vector

项目摘要

DESCRIPTION (provided by applicant): Base-selective heavy atom labels for electron microscopy-based DNA sequencing Project Summary/Abstract The development of inexpensive and rapid DNA sequencing technology remains a major challenge of broad scientific interest. Preliminary work at Halcyon Molecular has shown that transmission electron microscopy (TEM) can be used to obtain ultra-fast ultra-low-cost DNA sequences. Since efficient electron scattering to a detector is highly dependent on atomic number (Z), it is possible to label single stranded DNA (ssDNA) with heavy atoms. To test the limits of this trend, we propose a multipronged approach to selectively prepared metal-DNA base pair complexes. Our effort will be synergistic, taking advantage of the experience of the Toste group in organometallic and heavy atom cluster synthesis, and the capabilities of Halcyon Molecular in manipulating DNA and performing TEM. For this proposal, we are focusing on the selective labeling of DNA bases and the development of an appropriate assay to evaluate our success. Two general synthetic methods will be investigated in order to develop distinct labeling protocols. First, triosmium (ZOs = 76), tetrairidium (ZIr = 77) and trigold (ZAu = 79) clusters tethered to a group that selectively react with (alkylating reagents) or bind (platinum diamine complexes) purine bases will be explored. Incorporation of gold (ZAu = 79) and mercury (ZHg = 80) atoms through direct metal-metal bonds to the osmium atoms will also be explored. In this case, the labels would appear as intense spots in the TEM spectra. For the complimentary pyrimidine label, osmium tetraoxide bipyridine will be the selective binding agent thymine and cytosine. Using the bipyridine ligand as a scaffold for functionalization, additional osmium, platinum (ZPt = 78) or uranium (ZU = 92) atoms may be incorporated. A linear arrangement of metal atoms would allow a positional vector to be drawn towards the corresponding base. Proof-of-concept experiments will be performed using nuclear magnetic resonance (NMR) spectroscopy using individual DNA bases. If successful, testing will be performed on single DNA strands and sequenced using TEM. The success of these methods will enable the base-selective labeling of DNA with metal atoms and help develop ultra-fast ultra-low-cost DNA sequencing technology. The assembly of a whole human genome with our pilot-scale instrument can demonstrate TEM sequencing's potential for high consensus accuracy, extremely long (>150kb) reads, and lack of sequence specific bias in molecule deposition and readout. The subsequent, commercial availability of whole human genome sequencing using this technology (with expected >99.9999% consensus accuracy and completeness in <10 minutes/genome, at a cost of <$100) will enable new opportunities in translational medicine and foster many new discoveries by NIH investigators. PUBLIC HEALTH RELEVANCE: The proposed program aims to develop heavy atom organometallic compounds for ssDNA base-selective labeling for use in ultra-low-cost DNA sequencing technology based on single- atom sensitivity transmission electron microscopy (TEM).

描述（由申请人提供）：基于电子显微镜的DNA测序项目摘要/摘要廉价和快速DNA测序技术的发展仍然是广泛科学兴趣的主要挑战。 Halcyon分子的初步工作表明，可以使用透射电子显微镜（TEM）来获得超快速的超低成本DNA序列。由于有效的电子散射到检测器上高度依赖于原子数（z），因此可以将单链DNA（ssDNA）标记为重原子。为了测试这种趋势的极限，我们提出了一种多收益的方法来选择性准备好的金属DNA碱基对配合物。我们的努力将是协同作用的，利用了托斯特组在有机金属和重原子簇合成中的经验，以及Halcyon Molecular在操纵DNA和执行TEM方面的能力。对于此提案，我们专注于DNA碱基的选择性标签和开发适当的测定法以评估我们的成功。将研究两种一般的合成方法，以制定不同的标记协议。首先，将探索三杆（ZOS = 76），四翼族（ZIR = 77）和Trigold（Zau = 79）簇簇为束缚在选择性地与（烷基化试剂）或结合（铂二氨酸复合物）的组中的组系在一起。还将探索将金（ZAU = 79）和汞（ZHG = 80）通过直接金属金属键与osmium原子的掺入。在这种情况下，标签将在TEM光谱中显示为强烈的斑点。对于免费的嘧啶标记，四氧化甲氧烷双吡啶将是选择性结合剂胸骨和胞嘧啶。可以使用双吡啶配体作为功能化的支架，额外的oSmium，platinum（ZPT = 78）或铀（ZU = 92）原子可以合并。金属原子的线性排列将允许将位置矢量伸向相应的碱基。概念验证实验将使用核磁共振（NMR）光谱使用单个DNA碱基进行。如果成功，将对单个DNA链进行测试，并使用TEM测序。这些方法的成功将使DNA用金属原子对基础选择性标记，并有助于开发超快速的超低成本DNA测序技术。整个人类基因组与我们的试验尺度仪器的组装可以证明TEM测序具有高共识精度的潜力，非常长（> 150KB）的读取，并且缺乏分子沉积和读数的序列特定偏差。随后使用该技术的整个人类基因组测序的商业可用性（预期> 99.9999％的共识准确性和<10分钟/基因组的共识准确性和完整性为<10分钟/基因组，费用<$ 100）将为转化医学提供新的机会，并促进NIH研究人员的许多新发现。公共卫生相关性：拟议的计划旨在开发用于SSDNA基碱选择标签的重原子有机金属化合物，用于基于单原子灵敏度传输电子显微镜（TEM）的超低成本DNA测序技术。