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

描述(申请人提供)：尽管商业测序仪的成本和产量在过去5年中持续改善，但仍有必要进一步降低测序成本，提高产量和测序精度，并降低与样品制备相关的成本。单分子方法，如太平洋生物科学或纳米孔技术，有可能减少样品制备的瓶颈，但存在非常高的原始错误率。我们正在开发激活器测序技术，以实现低错误率的单分子测序。该方法适用于荧光、发光、pH传感和电化学等多种读出，其中许多读出可以在类似Ion Torrent的一次性芯片平台上使用。如果成功，激活器测序将在一个可扩展的平台上实现低成本、长读取长度、高精度的测序，该平台能够利用半导体行业的技术诀窍和投资，根据摩尔定律类型的特征尺寸下降来产生持续的年度性能提升。激活剂测序使用“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多个拷贝。具体地说，使用用酶激活剂标记在磷酸末端的dNTPs进行合成测序。一旦将dNTP掺入到预置模板上，就会释放一种激活剂，它可以将工程化的酶开关从“关”变为“开”构象。每一种被激活的酶都可以迅速产生大量可检测的产物，从而放大原始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才能产生可检测到的pH信号，但从单个dNTP分子释放的激活剂可以在几秒钟内启动单个酶分子产生数万个质子。报告的多个拷贝的产生使检测核苷酸掺入变得更容易，从而允许以低噪声进行单分子测序。这种单分子测序将简化样品准备，并通过消除去相限制而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，工程酶开关可以起到这样的“分子放大器”的作用。拟议的第一阶段SBIR赠款将展示激活器测序的能力，使用工程酶开关利用荧光检测进行高精度的单分子测序。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。

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.