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 长链寡核苷酸合成继续受到其收益递减的限制，目前最大 长度约为 250 个碱基。作为一般规则，每 100 个分子中就有一个无法耦合，这意味着平均 据说合成运行的耦合效率 (CE) 为 99%。公式 CEn，其中 n 是 在合成过程中添加的碱基表明，生成的链越长，产生的失败链就越多。 例如，合成具有 99% CE 的 40 个碱基链将生成 68% 的全长产物 (FLP)，如下所示 与合成 200 个碱基链相反，后者在相同的 CE 下会产生 13% 的 FLP。虽然还有其他 影响CE的因素（即合成参数和试剂质量），主要问题是不充分 试剂对固体基质（即聚苯乙烯珠或 可控孔玻璃）。最常见的情况是珠子包装在夹在中间的柱内 两个多孔过滤​​器之间；在这里，珠子的堆叠导致接触合成试剂的表面积减少， DNA分子因此变得不反应或仅部分反应。此外，用过的试剂和不需要的 副产物被困在载体内并进入连续的循环，进一步污染 合成运行。为了规避这些限制，我们提出了一种新方法，可以让我们控制动作 通过等离激元表面上的介电电泳来分离单个珠子。在这里，反应被完全调整到 用最小体积的试剂液滴封装每个珠子，以实现高精度合成。因为每颗珠子 在溶液中被隔离，副产物不会被捕获，并且每种副产物与所有合成都有最大程度的接触 试剂；正是珠子与试剂的这种紧密的 1:1 比例将显着提高碱添加效率 允许生产超过 1000 个碱基的超长 DNA 链。直到最近，远场光学（即光学 由于衍射限制聚焦光斑尺寸，镊子）无法在纳米尺度上应用；所以， 研究人员开始研究光波直接集中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 气蚀；然后，液滴沿着等离子体表面传输，以封装单个珠子 利用交流电场产生的介电泳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拉出的方法可用于大范围的液滴 和珠子尺寸以及适当的电极设计。我们相信最大化寡核苷酸纯度的关键 合成过程中的产量取决于确定每种试剂所需的最小体积/浓度 涂覆单个珠子的表面。通过我们提出的等离子表面合成平台，我们有 能够处理每个单独的珠子，以获得准确、优化的珠子与试剂滴的比例 规定的浓度。这些发展对于充分发挥合成生物学的潜力是必要的，通过 使整个社区都能参与大型项目，这将推动基因组生物学的发现和 药品。