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.

摘要 长链寡核苷酸合成继续受到其回报递减的限制，目前为最大 长度约为250个碱基。一般来说，每100个分子中就有一个分子无法偶联，这意味着平均 合成运行据说具有99%的耦合效率(CE)。公式CEN，其中n是 在合成过程中加入碱基，表明链产生的时间越长，产生的失效链就越多。 例如，合成具有99%CE的40碱基链将产生68%的全长产物(FLP) 与合成200个碱基链相反，后者将产生13%的FLP，具有相同的CE。虽然还有其他人 可能影响CE的因素(即合成参数和试剂质量)，主要问题是不充分 试剂对固体衬底表面上的每个分子的可及性(即聚苯乙烯珠子或 可控孔玻璃)。最常见的情况是将珠子包装在夹在中间的柱子中 在两个多孔过滤器之间；在这里，堆积珠子导致接触合成试剂的表面积减少， 使DNA分子变得未反应或只有部分反应。此外，用完的试剂和不需要的 副产物被困在支架内并进入连续的循环，进一步污染 合成运行。为了绕过这些限制，我们提出了一种新的方法，允许我们控制动作 通过等离子体表面的介电泳法对单个珠子进行分离。在这里，反应被调整到完全 用最小体积的试剂液滴包裹每个珠子，以实现高精度的合成。因为每一颗珠子 被隔离在溶液中，副产物不会被捕获，并且每一种都与所有合成有最大程度的接触 试剂；正是这种紧密的珠子与试剂的1：1比例将显著提高碱基添加效率 允许生产超长DNA链和1000个碱基。直到最近，远场光学(即光学 镊子)由于衍射有限的聚焦光斑尺寸而不能在纳米尺度上应用；因此， 研究人员开始研究光波直接集中的等离子体纳米结构的效应 放在珠子上。在我们的平台中，通过脉冲激光形成了精确的体积和浓度的试剂液滴 空化；然后液滴沿着等离子体表面传输，通过以下方式包裹单个珠子 利用交流电场产生的介电泳力克服表面张力障碍。因此，这一点 将珠子封装成液滴并将其拔出的方法可以用于大范围的液滴 和珠子大小与适当的电极设计。我们相信使寡核苷酸纯度最大化的关键 而合成过程中的产率取决于确定所需的每个试剂的最小体积/浓度 在单个珠子的表面涂上一层。通过我们提出的在等离子体表面合成的平台，我们已经 能够处理每个单独的珠子，以获得准确、优化的珠粒与试剂液滴的比率 限定的浓度。这些发展对于实现合成生物学的全部潜力是必要的，通过 让整个社区都能接触到大型项目，这些项目将推动基因组生物学和 医药。