Medusa(TM) Sequencer

Medusa(TM) 测序仪

• 批准号: 7592920
• 负责人: Thomas Schneider
• 金额: $ 25.49万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7592920
• 关键词: Base Sequence; Binding; Cells; Characteristics; Clinic; Code; Count; DNA; DNA Primers; DNA-Directed RNA Polymerase; Devices; Diffuse; Energy Transfer; Goals; Hereditary Disease; Individual; Legal patent; Life; Medical; Messenger RNA; Microscope; Mutation; Nucleic acid sequencing; Nucleotides; Object Attachment; Organism; Patients; Polymerase; RNA; Rate; Reading; Reagent; Series; Signal Transduction; Structure; Technology; Testing; Upper arm; Walkers; analog; base; cost; fluorophore; improved; single molecule; tripolyphosphate

The project is described at http://www.ccrnp.ncifcrf.gov/toms/patent/medusa/ The MedusaTM Sequencer is a single-molecule sequencing device that consists of a DNA (or RNA) polymerase attached to a set of four flexible arms. The tip of each arm carries a nonhydrolyzable nucleotide triphosphate analog and a spectrally distinct Forster Resonance Energy Transfer (FRET) acceptor fluorophore. A donor fluorophore attached to the polymerase excites an acceptor fluorophore by FRET if the fluorophores are close to each other. A MedusaTM Sequencer binds to a DNA primer hybridized to the DNA or RNA to be sequenced. The four arms with nucleotide tips "test" the polymerase pocket by diffusing in and out. The arm tip that has the nucleotide complementary to the unknown base of the template will dwell longer in the pocket than the other three that are not complementary. However, the polymerase will not incorporate the nucleotide on the tip of the arm into the nascent strand because the nucleotide triphosphate α-β bond is nonhydrolyzable. FRET between the donor on the polymerase and the acceptor at the arm tip produces a characteristic spectrum that identifies the bound base. Free hydrolyzable dNTPs (or NTPs) allow the MedusaTM Sequencer to step forward. The series of FRET signals reveals the unknown nucleotide sequence. A MedusaTM Sequencer could also be injected into a cell to read mRNA sequences inside a living organism. Coded versions of the MedusaTM Sequencer can signal when the device has been damaged. In the past year we constructed the core of the Medusa(TM) structure and are now ready to determine whether single nucleotides can be distinguished using a single molecule microscope.

该项目的描述见 http://www.ccrnp.nifcrf.gov/toms/patent/medusa/ MedusaTM 测序仪是一种单分子测序装置，由连接到一组四个柔性 DNA（或 RNA）聚合酶组成。武器。每个臂的尖端都带有不可水解的三磷酸核苷酸类似物和光谱上不同的福斯特共振能量转移（FRET）受体荧光团。如果荧光团彼此靠近，则附着在聚合酶上的供体荧光团会通过 FRET 激发受体荧光团。 MedusaTM 测序仪与 DNA 引物结合，该引物与待测序的 DNA 或 RNA 杂交。带有核苷酸尖端的四个臂通过扩散进出来“测试”聚合酶袋。具有与模板的未知碱基互补的核苷酸的臂尖将比其他三个不互补的臂尖在口袋中停留更长时间。然而，聚合酶不会将臂末端的核苷酸整合到新生链中，因为核苷酸三磷酸α-β键是不可水解的。聚合酶上的供体和臂尖上的受体之间的 FRET 会产生识别结合碱基的特征光谱。游离的可水解 dNTP（或 NTP）使 MedusaTM 测序仪能够向前迈进。这一系列 FRET 信号揭示了未知的核苷酸序列。 MedusaTM 测序仪也可以注入细胞中以读取活体内部的 mRNA 序列。 MedusaTM 测序仪的编码版本可以在设备损坏时发出信号。去年，我们构建了 Medusa(TM) 结构的核心，现在准备确定是否可以使用单分子显微镜区分单核苷酸。