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

描述(申请人提供)：碱基选择性重原子标记用于基于电子显微镜的DNA测序项目摘要/摘要发展廉价和快速的DNA测序技术仍然是引起广泛科学兴趣的重大挑战。Halcyon分子公司的初步工作表明，透射电子显微镜(TEM)可以用来获得超快、超低成本的DNA序列。由于探测器的有效电子散射高度依赖于原子序数(Z)，因此有可能用重原子标记单链DNA(SsDNA)。为了测试这一趋势的局限性，我们提出了一种多管齐下的方法来选择性地制备金属-DNA碱基对复合体。我们的努力将是协同的，利用Toste小组在有机金属和重原子簇合方面的经验，以及Halcyon分子在操纵DNA和进行透射电子显微镜方面的能力。对于这项提议，我们将重点放在选择性标记DNA碱基上，并开发一种适当的分析来评估我们的成功。为了开发不同的标记方案，将研究两种通用的合成方法。首先，将探索与(烷基化试剂)或结合(铂二胺络合物)嘌呤碱选择性反应的三铯(zOS=76)、四锶(zir=77)和三联体(zau=79)簇合物。还将探索通过直接金属-金属键将金(ZAU=79)和汞(ZHg=80)原子结合到Os原子中。在这种情况下，标记将在透射电子显微镜光谱中显示为强烈的斑点。对于赠送的嘧啶标记，四氧化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，可以加入额外的Os、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拉向相应的碱基。概念验证实验将使用使用单个DNA碱基的核磁共振(核磁共振)光谱进行。如果成功，将在单链DNA上进行测试，并使用电子显微镜进行测序。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、超低成本的DNA测序技术。用我们的中试规模的仪器组装整个人类基因组可以证明，在分子沉积和读出方面，电子显微镜测序具有高一致性准确性、极长(&gt；150kb)读数和缺乏序列特异性偏差的潜力。随后，使用这项技术的全人类基因组测序的商业可用性(预计10分钟/基因组的一致准确率和完整性为99.9999%，成本为100美元)将为转化医学带来新的机会，并促进国家卫生研究院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单链DNA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的透射电子显微镜的超低成本DNA测序技术。

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).