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

廉价和快速的DNA测序技术的发展仍然是一个重大的挑战，广泛的科学兴趣。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的经验，以及Halcyon分子在操纵DNA和进行TEM的能力。两种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功能化的支架，可以掺入额外的锇、铂（ZPt = 78）或铀（ZU = 92）原子。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。

The development of inexpensive and rapid DNA sequencing technology remains a major challenge of broad scientific interest. Preliminary work 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.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 complementary pyrimidine label, osmium tetraoxide bipyridine will be the selective binding agent for 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.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.