Expanded DNA, In Vitro Selection, Aptamers, and Cancer

描述(由申请人提供)：科学家、临床医生和内科医生长期以来都想要一种技术，这种技术可以常规地输送分子，这些分子可以结合对他们的研究重要的靶点、诊断生物标志物和对患者疾病发展至关重要的分子。目前，大分子按需结合分子(BMOD)是通过抗体技术最快获得的。如今，抗体正在治疗剂中占据一席之地，而以前抗体主要是利用药物化学的“艰苦过程”产生的小分子。25年前，科学家们提出，DNA和RNA(XNA)的可复制性和进化性可能为大分子BMOD提供一种替代途径。这里，可以从XNA分子的文库中选择XNA“适配子”，以通过体外选择(SELEX)与靶结合。适配子可能在抗体不起作用的条件下发挥作用，特别是在蛋白质展开的环境中。它们最终可能取代抗体或成为治疗剂，就像今天的许多抗体一样。尽管SELEX取得了成功，但我们现在了解到，它使用的四核苷酸XNA的官能团太少，序列多样性太小，来自天然XNA的干扰太多，无法以最广泛的形式满足这一愿景。因此，我们建议在这里使用人工扩展的遗传信息系统(Aegis)来扩展SELEX，这是一种DNA，它将标准DNA中的四个核苷酸与八个独立配对的核苷酸相加。这项拟议的工作将立即向SELEX添加两个Aegis核苷酸(Z和P，形成独立于标准C：G和T：A对的Z：P对)，使我们能够立即使用Aegis-SELEX来创建与肺癌、乳腺癌和肝癌细胞结合的GACTZP适配子。立即取得进展是可能的，因为两个合作的申请实验室(FfAME的Steven Benner和UF的Wehong Tan)已经将细胞SELEX、聚合酶技术和宙斯盾测序方面的突破结合在一起，生产出第一个宙斯盾适配子。仅在短短几周内从12轮SELEX中获得了这30个名摩尔适配子，为这项工作提供了“中心假设”：因为宙斯盾XNA文库具有更丰富的多样性，它们比标准XNA文库更富含高亲和力适配子。通过允许XNA适配子获得高达60%的抗体序列多样性，Aegis-SELEX进一步提供了最终实现SELEX技术目标的机会。宙斯盾-SELEX还应该有助于在新的方向上扩展蛋白质-核酸相互作用和分子识别的科学。为了实现这一愿景，必须做三件事，初步工作证明都是可行的：(1)我们必须提高复制宙斯盾DNA的聚合酶的保真度。(2)改进宙斯盾DNA测序技术。(3)我们必须在Z和P上增加更多已被证明的宙斯盾核苷酸；以及(4)我们必须将宙斯盾-SELEX与标准SELEX进行比较。遵循“以二换一”的策略，我们将通过创建针对循环中的肝、乳腺和肺癌细胞的有用的适体来进行这一基准测试。

DESCRIPTION (provided by applicant): Scientists, clinician, and physicians alike have long wanted a technology that can routinely deliver molecules that bind to targets important to their research, to diagnostic biomarkers, and to molecules essential to the progression of patient diseases. Currently, macromolecular "binding molecules on demand" (BMODs) are most rapidly available by way of antibody technology. Today, antibodies are gaining niches among therapeutic agents, previously dominated by small molecules generated using the "hard slog" of medicinal chemistry. A quarter century ago, scientists suggested that the replicability and evolvability of DNA and RNA (xNA) might offer an alternative route to macromolecular BMODs. Here, xNA "aptamers" might be selected from libraries of xNA molecule to bind to a target via in vitro selection (SELEX). Aptamers might work under conditions where antibodies do not, especially in environments where proteins unfold. They might eventually displace antibodies or become therapeutic agents, as are many antibodies today. Despite the successes of SELEX, we now understand that the four-nucleotide xNA that it uses has too few functional groups, too little sequence diversity, and too much interference from natural xNA, to meet this vision in its broadest form. Therefore, we propose here to expand SELEX using an artificially expanded genetic information system (AEGIS), a kind of DNA that adds up to eight independently pairing nucleotides to the four found in standard DNA. The proposed work will immediately add two AEGIS nucleotides to SELEX (Z and P, forming a Z:P pair independent of standard C:G and T:A pairs), allowing us to immediately use AEGIS-SELEX to create GACTZP aptamers that bind to lung, breast, and liver cancer cells. Immediate progress is possible because the two collaborating applicant laboratories (Steven Benner at the FfAME and Weihong Tan at UF) have already combined breakthroughs in cell-SELEX, polymerase technology, and AEGIS sequencing, to produce the first AEGIS aptamer. Obtained in just weeks from only 12 rounds of SELEX, this 30 namomolar aptamer offers up the "central hypothesis" for this work: Because AEGIS xNA libraries have richer diversity, they are richer reservoirs of high affinity aptamers than standard xNA libraries. By allowing xNA aptamers to gain up to 60% of the sequence diversity of antibodies, AEGIS-SELEX further offers the opportunity to finally meet the technological goals of SELEX. AEGIS-SELEX should also help expand the science of protein-nucleic acid interactions and molecular recognition in new directions. To achieve this vision, three things must be done, all shown to be feasible by preliminary work: (1) We must improve the fidelity of polymerases that copy AEGIS DNA. (2) We must improve sequencing technology for AEGIS DNA. (3) We must add more AEGIS nucleotides to the Z and P that have already been proven; and (4) we must compare AEGIS-SELEX to standard SELEX. Following a "two for the price of one" strategy, we will do this benchmarking by creating useful aptamers that target circulating liver, breast, and lung cancer cells.