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

基于电子显微镜的DNA测序项目的碱基选择性重原子标记摘要/摘要廉价和快速的DNA测序技术的发展仍然是广泛的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）可以用来获得超高速、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)，因此可以用重原子标记单链DNA （ssDNA）。为了测试这种趋势的局限性，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团簇合成方面的经验，以及Halcyon Molecular在操纵DNA和执行TEM方面的能力。对于这项提案，我们专注于DNA碱基的选择性标记和开发适当的分析来评估我们的成功。将研究两种一般的合成方法，以制定不同的标记方案。首先，将探索三价钐（ZOs = 76）、四价铱（ZIr = 77）和三价金（ZAu = 79）簇系在一个基团上，选择性地与（烷基化试剂）或结合（铂二胺配合物）嘌呤碱基反应。还将探索金（ZAu = 79）和汞（ZHg = 80）原子通过直接金属-金属键与锇原子的结合。在这种情况下，标签会在TEM光谱中表现为强烈的斑点。对于互补的嘧啶标签，四氧化二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支架，可以加入额外的锇、铂（ZPt = 78）或铀（ZU = 92）原子。金属原子的线性排列将允许一个指向相应碱基的位置矢量。概念验证实验将使用使用单个DNA碱基的核磁共振（NMR）光谱进行。如果成功，测试将在单个DNA链上进行，并使用TEM进行测序。这些方法的成功将使金属原子DNA的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。使用我们的中尺度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，并且在分子沉积和读取中缺乏序列特异性偏差的潜力。随后，使用该技术的全人类基因组测序的商业可用性（预计在<10分钟/基因组内达到99.9999%的一致性准确性和完整性，成本< 100美元）将为转化医学带来新的机会，并促进NIH研究人员的许多新发现。

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).