A nanophotonic approach to building DNA using enzymatic synthesis
使用酶合成构建 DNA 的纳米光子方法
基本信息
- 批准号:10035169
- 负责人:
- 金额:$ 53.03万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:AddressAreaBehaviorBiologyChemistryCommunitiesCouplingDNADevelopmentDideoxy Chain Termination DNA SequencingElectrodesEncapsulatedEngravingsExposure toFailureGenomeGlassIndividualLabelLasersLengthLightLiquid ChromatographyMass ChromatographyMeasurementMeasuresMedicineMethodsNanostructuresNucleotidesOilsOligonucleotidesOpticsPatternPhasePhysiologic pulsePolystyrenesPositioning AttributeProcessProductionReactionReagentRefuse DisposalResearch PersonnelRunningSamplingSeriesSiteSolidSpottingsSurfaceSurface TensionSystemTechnologyTestingTimeTransportationWithdrawalWorkloadbasecostdesignelectric fieldgene productgene synthesislaser tweezernanonanophotonicnanoscalenoveloptical trapsplasmonicsqubitsynthetic biologytripolyphosphatewasting
项目摘要
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。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
数据更新时间:{{ journalArticles.updateTime }}
{{
item.title }}
{{ item.translation_title }}
- DOI:
{{ item.doi }} - 发表时间:
{{ item.publish_year }} - 期刊:
- 影响因子:{{ item.factor }}
- 作者:
{{ item.authors }} - 通讯作者:
{{ item.author }}
数据更新时间:{{ journalArticles.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ monograph.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ sciAawards.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ conferencePapers.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ patent.updateTime }}
Ronald Wayne Davis其他文献
Ronald Wayne Davis的其他文献
{{
item.title }}
{{ item.translation_title }}
- DOI:
{{ item.doi }} - 发表时间:
{{ item.publish_year }} - 期刊:
- 影响因子:{{ item.factor }}
- 作者:
{{ item.authors }} - 通讯作者:
{{ item.author }}
{{ truncateString('Ronald Wayne Davis', 18)}}的其他基金
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:
10705040 - 财政年份:2020
- 资助金额:
$ 53.03万 - 项目类别:
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:
10268193 - 财政年份:2020
- 资助金额:
$ 53.03万 - 项目类别:
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:
10460609 - 财政年份:2020
- 资助金额:
$ 53.03万 - 项目类别:
Molecular and single-cell immunology of myalgic encephalomyelitis/chronic fatigue syndrome
肌痛性脑脊髓炎/慢性疲劳综合征的分子和单细胞免疫学
- 批准号:
10416027 - 财政年份:2018
- 资助金额:
$ 53.03万 - 项目类别:
Molecular and single-cell immunology of myalgic encephalomyelitis/chronic fatigue syndrome
肌痛性脑脊髓炎/慢性疲劳综合征的分子和单细胞免疫学
- 批准号:
10159206 - 财政年份:2018
- 资助金额:
$ 53.03万 - 项目类别:
Ultra high-throughput DNA synthesis via nano-optical conveyer belts
通过纳米光学传送带进行超高通量 DNA 合成
- 批准号:
9379771 - 财政年份:2017
- 资助金额:
$ 53.03万 - 项目类别:
Genomic and synthetic biology tools for expressing natural product gene clusters
用于表达天然产物基因簇的基因组和合成生物学工具
- 批准号:
8702454 - 财政年份:2014
- 资助金额:
$ 53.03万 - 项目类别:
Genomic and synthetic biology tools for expressing natural product gene clusters
用于表达天然产物基因簇的基因组和合成生物学工具
- 批准号:
9340321 - 财政年份:2014
- 资助金额:
$ 53.03万 - 项目类别:
Genomic and synthetic biology tools for expressing natural product gene clusters
用于表达天然产物基因簇的基因组和合成生物学工具
- 批准号:
9316665 - 财政年份:2014
- 资助金额:
$ 53.03万 - 项目类别:
MISINCORPORATION OF AMINO ACID ANALOGS IN SELECTED HUMAN AND MURINE PROTEINS
选定的人类和鼠类蛋白质中氨基酸类似物的错误掺入
- 批准号:
8365482 - 财政年份:2011
- 资助金额:
$ 53.03万 - 项目类别:
相似国自然基金
层出镰刀菌氮代谢调控因子AreA 介导伏马菌素 FB1 生物合成的作用机理
- 批准号:2021JJ40433
- 批准年份:2021
- 资助金额:0.0 万元
- 项目类别:省市级项目
寄主诱导梢腐病菌AreA和CYP51基因沉默增强甘蔗抗病性机制解析
- 批准号:32001603
- 批准年份:2020
- 资助金额:24.0 万元
- 项目类别:青年科学基金项目
AREA国际经济模型的移植.改进和应用
- 批准号:18870435
- 批准年份:1988
- 资助金额:2.0 万元
- 项目类别:面上项目
相似海外基金
Role of Central Neurotensin Signaling in the Ventral Tegmental Area for Ingestive Behavior and Body Weight
中枢神经降压素信号在腹侧被盖区对摄入行为和体重的作用
- 批准号:
10665597 - 财政年份:2022
- 资助金额:
$ 53.03万 - 项目类别:
Role of Central Neurotensin Signaling in the Ventral Tegmental Area for Ingestive Behavior and Body Weight
中枢神经降压素信号在腹侧被盖区对摄入行为和体重的作用
- 批准号:
10536558 - 财政年份:2022
- 资助金额:
$ 53.03万 - 项目类别:
Elucidation of the functional role of neural stem cells in the area postrema in the regulation of feeding behavior
阐明后区神经干细胞在调节摄食行为中的功能作用
- 批准号:
21K15177 - 财政年份:2021
- 资助金额:
$ 53.03万 - 项目类别:
Grant-in-Aid for Early-Career Scientists
Evaluation Analysis of Networked Compact City Considering the Wandering Behavior in the Urban Function and Residential guidance Area.
考虑城市功能与居住引导区游走行为的网络化紧凑城市评价分析。
- 批准号:
21K04296 - 财政年份:2021
- 资助金额:
$ 53.03万 - 项目类别:
Grant-in-Aid for Scientific Research (C)
What is the origin of friction force depending on the sliding velocity? Approach from atomic-scale behavior in real area of contact
取决于滑动速度的摩擦力的来源是什么?
- 批准号:
20K04115 - 财政年份:2020
- 资助金额:
$ 53.03万 - 项目类别:
Grant-in-Aid for Scientific Research (C)
Defining the differential roles of Glutamatergic and GABAergic projections from the Lateral Preoptic Area to the Lateral Habenula in Reward, Aversion, and Drug-Seeking Behavior.
定义从外侧视前区到外侧缰核的谷氨酸能和 GABA 能投射在奖励、厌恶和药物寻求行为中的不同作用。
- 批准号:
10242872 - 财政年份:2019
- 资助金额:
$ 53.03万 - 项目类别:
Elucidating roles of ventral tegmental area dopaminergic neurons in motivation of appetitive goal-directed behavior
阐明腹侧被盖区多巴胺能神经元在食欲目标导向行为的激励中的作用
- 批准号:
19K03381 - 财政年份:2019
- 资助金额:
$ 53.03万 - 项目类别:
Grant-in-Aid for Scientific Research (C)
Defining the differential roles of Glutamatergic and GABAergic projections from the Lateral Preoptic Area to the Lateral Habenula in Reward, Aversion, and Drug-Seeking Behavior.
定义从外侧视前区到外侧缰核的谷氨酸能和 GABA 能投射在奖励、厌恶和药物寻求行为中的不同作用。
- 批准号:
9926602 - 财政年份:2019
- 资助金额:
$ 53.03万 - 项目类别:
Investigating the interplay between ventral tegmental area dopamine, medial orbitofrontal cortex, and ventromedial striatum in compulsive-like behavior
研究强迫样行为中腹侧被盖区多巴胺、内侧眶额皮质和腹内侧纹状体之间的相互作用
- 批准号:
9393053 - 财政年份:2018
- 资助金额:
$ 53.03万 - 项目类别:
Role of Lateral Hypothalmic Area Perineuronal Nets in the Reinstatement of Cocaine-Seeking Behavior
外侧下丘脑区神经周围网络在恢复可卡因寻求行为中的作用
- 批准号:
9598308 - 财政年份:2018
- 资助金额:
$ 53.03万 - 项目类别: