Enzyme Switch: many reporter molecules from a single-molecule-sequencing product

酶开关：来自单分子测序产品的许多报告分子

• 批准号: 8901270
• 负责人: Javier Farinas
• 金额: $ 25.5万
• 依托单位: CAERUS MOLECULAR DIAGNOSTICS, INC.
• 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 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8901270
• 关键词: Achievement; Amplifiers; Base Sequence; Binding; Biological; Cells; Chemistry; Coupled; DNA; DNA Sequence; Detection; Electrochemistry; Engineering; Enzyme Activators; Enzymes; Error Sources; Evaluation; Fluorescence; Future; Generations; Goals; Government; Grant; Health; Human; Hydrolysis; Image; Industry; Investments; Ions; Label; Lactamase; Laws; Length; Maltose; Measures; Medical Research; Methods; Molecular; Molecular Conformation; Noise; Nucleic acid sequencing; Nucleotides; Performance; Persons; Phase; Preparation; Protons; Reaction; Reaction Time; Reading; Reagent; Reporter; Research Personnel; Sampling; Scheme; Semiconductors; Signal Transduction; Small Business Innovation Research Grant; Source; System; Technology; Technology Transfer; Testing; Translational Research; Work; base; cost; enzyme activity; fluorescence microscope; improved; inorganic phosphate; instrumentation; luminescence; maltose-binding protein; mammalian genome; nanopore; sensor; single molecule

DESCRIPTION (provided by applicant): Even as the cost and throughput of commercial sequencers has continued to improve over the last 5 years, there is still a need to further reduce sequencing costs, to increase throughput and sequencing accuracy and to reduce the costs associated with sample preparation. Single molecule methods such as the Pacific Biosciences or nanopore technologies have the potential to reduce sample preparation bottlenecks but suffer from very high raw error rates. We are developing the Activator Sequencing technology for single molecule sequencing with low error rates. The method is applicable to a variety of read outs such as fluorescence, luminescence, pH sensing and electrochemistry, many of which can be used in a disposable CMOS chip platform similar to that of Ion Torrent. If successful, Activator Sequencing would enable low-cost, long read length, high accuracy sequencing on a scalable platform capable of leveraging semiconductor industry know-how and investments to yield continued yearly increases in performance based on Moore's Law type decreases in feature size. Activator Sequencing uses a "molecular amplifier" to convert the products of a single-molecule sequencing reaction into many copies of a readily detectable reporter molecule. Specifically, sequencing-by-synthesis is performed using dNTPs labeled at the terminal phosphate with an enzyme activator. Upon incorporation of a dNTP onto a primed template, an activator is released which can turn an engineered enzyme switch from an "off" to an "on conformation. Each activated enzyme can rapidly generate a multitude of detectable products thereby amplifying the detectable signal from the original dNTP incorporation. For example, while the Ion Torrent system needs many template copies to generate a detectable pH signal, an activator released from a single dNTP molecule can turn on a single enzyme molecule to generate tens of thousands of protons in a few seconds. The generation of multiple copies of a reporter makes it easier to detect nucleotide incorporation thereby allowing single molecule sequencing with low noise. Such single molecule sequencing would simplify sample preparation and enable very long read lengths by eliminating dephasing limitations. If combined with low-cost, highly parallel CMOS sensors, instrumentation costs would be greatly reduced compared to fluorescence instrumentation. Our preliminary results demonstrate that an engineered enzyme switch can function as such a "molecular amplifier." The proposed Phase I SBIR grant will demonstrate the ability of Activator Sequencing to use an engineered enzyme switch to perform single molecule sequencing with high accuracy using fluorescence detection. Future work would focus on transferring the technology to a scalable, integrated CMOS sensor.

描述（由申请人提供）：即使商业测序仪的成本和通量在过去5年中持续改善，仍然需要进一步降低测序成本，增加通量和测序准确性，并降低与样品制备相关的成本。单分子方法如Pacific Biosciences或纳米孔技术具有减少样品制备瓶颈的潜力，但遭受非常高的原始错误率。我们正在开发用于单分子测序的低错误率激活剂测序技术。该方法适用于各种读出，如荧光，发光，pH传感和电化学，其中许多可以用于一次性CMOS芯片平台类似的离子激流。如果成功的话，Activator Sequencing将在一个可扩展的平台上实现低成本、长读长、高准确度的测序，该平台能够利用半导体行业的专业知识和投资，根据摩尔定律类型的特征尺寸减小，产生持续的性能年度增长。 激活子测序使用“分子放大器”将单分子测序反应的产物转化为易于检测的报告分子的许多拷贝。具体地，使用在末端磷酸处用酶活化剂标记的dNTP进行合成测序。在将dNTP掺入到引发的模板上时，释放激活剂，其可以将工程化的酶开关从“关闭”构象转变为“开启”构象。每种活化的酶可以快速产生大量可检测的产物，从而放大来自原始dNTP掺入的可检测信号。例如，虽然Ion Torrent系统需要许多模板拷贝来产生可检测的pH信号，但从单个dNTP分子释放的激活剂可以打开单个酶分子，从而在几秒钟内产生数万个质子。报告基因的多个拷贝的产生使得更容易检测核苷酸掺入，从而允许具有低噪声的单分子测序。这种单分子测序将简化样品制备，并通过消除移相限制实现非常长的读取长度。如果与低成本、高度并行的CMOS传感器相结合，与荧光仪器相比，仪器成本将大大降低。我们的初步结果表明，工程酶开关可以作为这样的“分子放大器”。“拟议的第一阶段SBIR赠款将证明激活剂测序的能力，使用工程酶开关进行单分子测序与高精度使用荧光检测。未来的工作将集中在将该技术转移到可扩展的集成CMOS传感器上。