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 Molecular的初步工作表明，透射电子显微镜（TEM）可用于获得超快、超低成本的DNA序列。由于有效的电子散射到检测器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簇合成方面的经验，以及Halcyon Molecular在操纵DNA和进行TEM方面的能力。对于这个提议，我们专注于DNA碱基的选择性标记和开发适当的检测方法来评估我们的成功。两个一般的合成方法将进行研究，以开发不同的标记协议。首先，triosmium（ZO = 76），tetrairidium（ZIr = 77）和trigold（ZAu = 79）集群栓系到一个基团，选择性反应（烷基化试剂）或绑定（铂二胺络合物）嘌呤碱将进行探索。金（ZAu = 79）和汞（ZHg = 80）原子通过直接的金属-金属键结合到锇原子也将被探索。在这种情况下，标记将在TEM光谱中显示为强斑点。对于互补的嘧啶标记，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用于官能化的支架，可以掺入另外的锇、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朝向相应的碱基绘制。概念验证实验将使用核磁共振（NMR）光谱法使用单个DNA碱基进行。如果成功，将对单链DNA进行检测，并使用TEM进行测序。这些方法的成功将使用金属原子对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（> 150 kb）读数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，使用该技术的全人类基因组测序的商业可用性（预期在<10分钟/基因组内具有>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。 公共卫生相关性：该计划旨在开发用于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。

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).