A nanophotonic approach to building DNA using enzymatic synthesis

使用酶合成构建 DNA 的纳米光子方法

• 批准号: 10035169
• 负责人: Ronald Wayne Davis
• 金额: $ 53.03万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-23 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10035169
• 关键词: Address; Area; Behavior; Biology; Chemistry; Communities; Coupling; DNA; Development; Dideoxy Chain Termination DNA Sequencing; Electrodes; Encapsulated; Engravings; Exposure to; Failure; Genome; Glass; Individual; Label; Lasers; Length; Light; Liquid Chromatography; Mass Chromatography; Measurement; Measures; Medicine; Methods; Nanostructures; Nucleotides; Oils; Oligonucleotides; Optics; Pattern; Phase; Physiologic pulse; Polystyrenes; Positioning Attribute; Process; Production; Reaction; Reagent; Refuse Disposal; Research Personnel; Running; Sampling; Series; Site; Solid; Spottings; Surface; Surface Tension; System; Technology; Testing; Time; Transportation; Withdrawal; Workload; base; cost; design; electric field; gene product; gene synthesis; laser tweezer; nano; nanophotonic; nanoscale; novel; optical traps; plasmonics; qubit; synthetic biology; tripolyphosphate; wasting

Abstract Long strand oligonucleotide synthesis continues to be limited by its diminishing returns, with a current maximum length of ~ 250 bases. As a general rule, one of every 100 molecules will fail to couple, meaning that the average synthesis run is said to have a coupling efficiency (CE) of 99%. The formula, CEn, where n is the number of bases added during synthesis, states the longer the strand generated, the more failure strands will be produced. For example, synthesis of a 40 base strand with a 99% CE will generate 68% full-length product (FLP) as opposed to synthesis of a 200 base strand, which will yield 13% FLP with the same CE. While there are other factors that may influence CE (i.e. synthesis parameters and quality of reagents), the main problem is inadequate accessibility of reagent to each of the molecules on the surface of the solid substrate (i.e. polystyrene beads or controlled-pore-glass). The most common case is when beads are packaged inside a column sandwiched between two porous filters; here, stacking of beads causes reduced surface area exposure to synthesis reagents, whereby DNA molecules become unreacted or only partially reacted. Moreover, spent reagents and unwanted byproducts become trapped within the support and carry over into consecutive cycles, further contaminating the synthesis run. To circumvent these limitations, we propose a novel method that allows us to control the actions of an individual bead through dielectrophoresis on a plasmonic surface. Here, reactions are tuned to completely encapsulate each bead with minimal volume reagent droplets for high-precision synthesis. Because each bead is isolated in solution, byproducts cannot become trapped, and each has maximum contact with all synthesis reagents; it is this intimate 1:1 ratio of bead to reagent that will significantly increase the base addition efficiency allowing the production of ultra-long strands of DNA > 1000 bases. Until very recently, far-field optics (i.e. optical tweezers) could not be applied at the nano-scale due to diffraction-limited focused spot size; therefore, researchers began studying effects of plasmonic nanostructures where light waves are concentrated directly onto the bead. In our platform, reagent droplets of precise volume and concentration are formed by pulsed laser cavitation; droplets are then transported along the plasmonic surface to encapsulate individual beads by overcoming surface tension barrier using dielectrophoretic forces generated by an AC electrical field. Thus, this approach of encapsulating a bead into a droplet and pulling it out can be employed for a large range of droplet and bead sizes with the appropriate electrode design. We believe the key to maximizing oligonucleotide purity and yield during synthesis lies in determining the minimal volume/concentration of each reagent necessary to coat the surface of an individual bead. With our proposed platform of synthesis on a plasmonic surface, we have the capability to address each individual bead for an accurate, optimized ratio of bead to reagent droplet of defined concentration. These developments are necessary to realize the full potential of synthetic biology, by making large-scale projects accessible to the entire community that will fuel discoveries in genome biology and medicine.

抽象的 长链寡核苷酸的合成继续受到其回报的减少的限制，电流最大 长度约250个基部。通常，每100个分子中的一个都不会夫妇，这意味着平均 据说合成运行的耦合效率为99％。公式，cen，其中n是 在合成过程中添加的碱基，指出产生的链的时间越长，产生故障链就会越多。 例如，具有99％CE的40个基本链的合成将产生68％的全长产品（FLP）作为 与合成200个碱基的合成相反，该链将产生13％的FLP，同一CE。虽然还有其他 可能影响CE的因素（即合成参数和试剂质量），主要问题是不足 试剂对固体底物表面上每个分子的可访问性（即聚苯乙烯珠或 受控的孔玻璃）。最常见的情况是何时将珠子包装在夹杂的列内 在两个多孔过滤​​器之间；在这里，堆叠珠会导致表面积降低暴露于合成试剂， DNA分子未反应或仅部分反应。此外，花费的试剂和不必要的 副产品被困在支撑中，并将其延伸到连续的周期中，进一步污染了 合成运行。为了规避这些限制，我们提出了一种新颖的方法，使我们能够控制动作 单个珠通过等离子表面上的介电的珠子。在这里，反应被调整为完全 用最小的体积试剂液滴封装每个珠子，以进行高精度合成。因为每个珠子 在溶液中分离出来，副产品不能被困，并且每个副产品与所有合成都具有最大的接触 试剂；正是这种亲密的1：1珠子与试剂的比率将显着提高基本添加效率 允许产生超长的DNA> 1000碱基的链。直到最近，远场光学器件（即光学 由于衍射有限的聚焦点尺寸，无法在纳米尺度上应用镊子）；所以， 研究人员开始研究直接浓缩光波的等离子体纳米结构的影响 到珠子上。在我们的平台中，通过脉冲激光形成精确体积和浓度的试剂液滴 空化；然后将液滴沿等离子体表面运输，以将单个珠子封装 使用AC电场产生的电介摄取力克服表面张力屏障。因此，这个 将珠子封装到液滴中并将其拔出的方法可以用于大量液滴 并具有适当的电极设计的珠子尺寸。我们相信最大化寡核苷酸纯度的关键 合成过程中的产量在于确定每种试剂的最小体积/浓度所必需的 涂上单个珠子的表面。我们提出的在等离子表面上的合成平台，我们有 解决每个单独的珠的能力，以获得精确，优化的珠子与试剂液滴的比率 定义的浓度。这些发展对于实现合成生物学的全部潜力是必要的 使整个社区可以访问大规模项目，以助长基因组生物学的发现和 药品。