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

• 批准号: 7979188
• 负责人: F. Dean Toste
• 金额: $ 21.82万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7979188
• 关键词: Base Pairing; 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; Nuclear Magnetic Resonance; Organometallic Compounds; Osmium; Osmium Tetroxide; Platinum; Production; Protocols documentation; Purines; Pyrimidine; Pyrimidines; 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

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）：用于基于电子显微镜的 DNA 测序的碱基选择性重原子标记 项目摘要/摘要 廉价且快速的 DNA 测序技术的开发仍然是广泛科学兴趣的主要挑战。 Halcyon Molecular 的初步工作表明，透射电子显微镜 (TEM) 可用于获得超快速、超低成本的 DNA 序列。由于向检测器的有效电子散射高度依赖于原子序数 (Z)，因此可以用重原子标记单链 DNA (ssDNA)。为了测试这一趋势的极限，我们提出了一种多管齐下的方法来选择性制备金属-DNA 碱基对复合物。我们的努力将是协同的，利用 Toste 集团在有机金属和重原子簇合成方面的经验，以及 Halcyon Molecular 在操纵 DNA 和执行 TEM 方面的能力。对于这项提案，我们重点关注 DNA 碱基的选择性标记以及开发适当的检测方法来评估我们的成功。将研究两种通用的合成方法，以开发不同的标记方案。首先，将探索与选择性与（烷基化试剂）反应或结合（铂二胺复合物）嘌呤碱基的基团相连的三锇（ZOs = 76）、四铱（ZIr = 77）和三金（ZAu = 79）簇。还将探索通过直接金属-金属键与锇原子结合金（ZAu = 79）和汞（ZHg = 80）原子。在这种情况下，标记将在 TEM 光谱中显示为强点。对于互补嘧啶标记，四氧化锇联吡啶将是胸腺嘧啶和胞嘧啶的选择性结合剂。使用联吡啶配体作为功能化的支架，可以掺入额外的锇、铂（ZPt = 78）或铀（ZU = 92）原子。金属原子的线性排列将允许将位置矢量引向相应的碱基。将使用核磁共振 (NMR) 光谱法使用单个 DNA 碱基进行概念验证实验。如果成功，将对单条 DNA 链进行测试并使用 TEM 进行测序。这些方法的成功将使金属原子对DNA进行碱基选择性标记成为可能，并有助于开发超快速、超低成本的DNA测序技术。使用我们的中试规模仪器组装整个人类基因组可以证明 TEM 测序具有高一致性准确性、极长（> 150kb）读取以及在分子沉积和读出中缺乏序列特异性偏差的潜力。随后，使用该技术进行全人类基因组测序的商业可用性（预计每个基因组的一致性准确性和完整性将超过 99.9999%，不到 10 分钟，成本不到 100 美元）将为转化医学带来新的机遇，并通过以下方式促进许多新发现：美国国立卫生研究院的调查人员。 公共健康相关性：拟议项目旨在开发用于 ssDNA 碱基选择性标记的重原子有机金属化合物，用于基于单原子灵敏度透射电子显微镜 (TEM) 的超低成本 DNA 测序技术。