Expanded DNA, In Vitro Selection, Aptamers, and Cancer

扩展 DNA、体外选择、适体和癌症

• 批准号: 8748649
• 负责人: STEVEN A BENNER
• 金额: $ 39.31万
• 依托单位: FOUNDATION FOR APPLIED MOLECULAR EVOLUTN
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8748649
• 关键词: Affinity; Antibodies; Antibody Diversity; Appearance; Benchmarking; Binding; Biological Markers; Biotechnology; Blood; Breast Cancer Cell; Cancer Diagnostics; Cells; Chemicals; Chemistry; Collaborations; DNA; DNA-Protein Interaction; Diagnostic; Disease; Disease Progression; Ensure; Environment; Funding; Future; Genetic; Goals; In Vitro; Information Systems; Institutes; Knowledge; Laboratories; Libraries; Malignant Neoplasms; Malignant neoplasm of liver; Malignant neoplasm of lung; Maps; Medicine; Metric; Molecular Evolution; National Institute of General Medical Sciences; Nucleic Acids; Nucleotides; Outcome; Patients; Performance; Pharmaceutical Chemistry; Physicians; Polymerase; Price; Probability; Procedures; Proteins; RNA; Research; Route; Science; Scientist; Series; Signal Transduction; Survivors; Technology; Testing; Therapeutic Agents; United States National Institutes of Health; Vision; Work; aptamer; base; cancer cell; cancer therapy; functional group; improved; innovation; malignant breast neoplasm; meetings; molecular recognition; practical application; programs; public health relevance; research study; small molecule; success; synthetic biology; tool

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.

描述（由申请人提供）：科学家、临床医生和内科医师等长期以来一直希望有一种技术能够常规地递送分子，这些分子与对其研究重要的靶标、诊断生物标志物以及对患者疾病进展至关重要的分子结合。目前，大分子“按需结合分子”（BMOD）可以通过抗体技术最快速地获得。如今，抗体在治疗药物中占据一席之地，而此前，抗体是通过药物化学的“艰难过程”产生的小分子占据主导地位。 四分之一个世纪前，科学家提出 DNA 和 RNA (xNA) 的可复制性和可进化性可能为大分子 BMOD 提供替代途径。在这里，xNA“适体”可以从xNA分子文库中选择出来，通过体外选择（SELEX）与靶标结合。适体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。它们最终可能会取代抗体或成为治疗剂，就像当今的许多抗体一样。 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 的功能基团太少，序列多样性太少，天然 xNA 的干扰太多，无法以最广泛的形式实现这一愿景。因此，我们在这里建议使用人工扩展的遗传信息系统（AEGIS）来扩展SELEX，这是一种DNA，在标准DNA中发现的四个独立配对核苷酸的基础上增加了八个独立配对的核苷酸。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 适体。 立即取得进展是可能的，因为两个合作申请实验室（FfAME 的 Steven Benner 和佛罗里达大学的 Weihong Tan）已经结合了细胞SELEX、聚合酶技术和 AEGIS 测序方面的突破，生产出了第一个 AEGIS 适体。这种 30 纳摩尔适体仅在几周内通过 12 轮 SELEX 获得，为这项工作提供了“中心假设”：由于 AEGIS xNA 文库具有更丰富的多样性，因此它们是比标准 xNA 文库更丰富的高亲和力适体库。通过让 xNA 适体获得高达 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。 AEGIS-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，所有这些都被初步工作证明是可行的：（1）我们必须提高复制 AEGIS DNA 的聚合酶的保真度。 （2）改进AEGIS DNA测序技术。 (3)我们必须在已经被证明的Z和P上添加更多的AEGIS核苷酸； (4) 我们必须将 AEGIS-SELEX 与标准 SELEX 进行比较。遵循“一价二价”的策略，我们将通过创建针对循环肝癌、乳腺癌和肺癌细胞的有用适体来进行基准测试。