Real-time detection of cancer biomarkers using a self-sustained aptazyme

使用自我维持的适体酶实时检测癌症生物标志物

• 批准号: 8537131
• 负责人: Charles Olea
• 金额: $ 5.39万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-24 至 2014-09-23
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8537131
• 关键词: Binding; Biological; Biological Assay; Biological Markers; Cancer Detection; Catalytic RNA; Coupled; Detection; Development; Devices; Diagnosis; Diagnostic; Diagnostic Neoplasm Staging; Early Diagnosis; Early treatment; Explosion; Flavin Mononucleotide; Fluorescence; Fluorescence Resonance Energy Transfer; Goals; Growth; Hour; Individual; Label; Ligands; Malignant Neoplasms; Measures; Methods; Molecular; Nucleic Acids; Patient Monitoring; Patients; Physiological; Prostate-Specific Antigen; Proteins; Proteomics; Radioactive; Reverse Transcriptase Polymerase Chain Reaction; Risk Assessment; Sampling; Screening for cancer; Signal Transduction; Staging; System; Systems Development; Theophylline; Time; aptamer; assay development; base; cancer risk; cancer therapy; design; fighting; instrumentation; macromolecule; metabolomics; molecular marker; mortality; novel diagnostics; p21 activated kinase; response; small molecule; time use; tool

DESCRIPTION (provided by applicant): The most effective way to fight cancer is at early stages, making early detection and risk assessment essential for patient survival. With recent advances in proteomics and metabolomics, there has been an explosion of newly discovered biomarkers indicative of cancer. However, detection of these biomarkers will require new diagnostic development that can be costly and take several years to develop. A universal diagnostic tool that can detect current and soon-to-be discovered cancer biomarkers is needed. Development of a new general diagnostic that can detect multiple cancer biomarkers in real-time is proposed. Recently a nucleic acid-based ribozyme, or aptazyme, capable of ligand-dependent exponential amplification was designed. The aptazyme has been shown to interchangeably detect theophylline and flavin mononucleotide (FMN) within physiological ranges. Unlike other diagnostics, such as RT-PCR, the system is isothermal and self-sustained which does not require other biological macromolecules and additional instrumentation. However, the current assay takes hours to perform, uses hazardous radioactive materials, and can detect only one ligand at a time. A new real-time, non-hazardous, and multiplexed aptazyme using fluorescence methods is proposed. Fvrster (fluorescence) resonance energy transfer (FRET) methods will be used to detect exponential amplification by fluorescently labeling the aptazyme. FRET will be coupled to activity of the aptazyme enabling real-time detection of ligand concentrations. As proof-of-principle, two aptazymes specific for cancer biomarkers prostate-specific antigen (PSA) and p21-activated kinase (PAK1) will be developed. Existing aptamer motifs for PSA and PAK1 will be incorporated into the aptazyme and each will be coupled to a unique FRET pair producing a distinct FRET signal for each ligand. The distinct FRET pairs will have minimal spectral overlap enabling multiplexed detection of ligands. Varying concentrations of PSA and PAK1 will be simultaneously assayed in the same sample in real-time. This system has the potential to quantitatively detect cancer biomarkers whether protein, small molecule, or nucleic acid. In addition, the ability to multiplex allows for individual assessment for each patient and monitoring patient response to treatment. Aptamers specific for new targets can be made with ease and the aptamer region within the aptazyme is interchangeable. The aptazyme system proposed here has the potential to bind any cancer biomarker and circumvents further rigorous assay development.

描述(申请人提供)：抗击癌症最有效的方法是在早期阶段，使早期发现和风险评估对患者生存至关重要。随着蛋白质组学和代谢组学的最新进展，新发现的癌症生物标志物呈爆炸式增长。然而，检测这些生物标记物将需要新的诊断开发，这可能是昂贵的，并需要几年的开发。需要一种通用的诊断工具来检测当前和即将发现的癌症生物标记物。提出了一种新的可实时检测多种癌症生物标志物的通用诊断方法。最近，一种基于核酸的核酶或aptazyme被设计出来，它能够进行配体依赖的指数扩增。端粒酶已被证明在生理范围内可互换地检测茶碱和黄素单核苷酸(FMN)。与RT-PCR等其他诊断方法不同，该系统是恒温和自我维持的，不需要其他生物大分子和额外的仪器。然而，目前的检测需要几个小时的时间，使用危险的放射性材料，而且一次只能检测到一个配体。提出了一种新的实时、无毒、多重荧光方法检测aptazyme的方法。Fvrster(荧光)共振能量转移(FRET)方法将用于通过荧光标记端粒酶来检测指数扩增。FRET将与aptazyme的活性相连，从而实现对配体浓度的实时检测。作为原理证明，将开发两种针对癌症生物标记物前列腺特异性抗原(PSA)和p21激活激酶(PAK1)的aptazyme。现有的PSA和PAK1的适体基序将被整合到aptazyme中，每个适体基序都将连接到一个独特的FRET对，为每个配体产生不同的FRET信号。不同的FRET对将具有最小的光谱重叠，从而能够对配体进行多路复用检测。不同浓度的PSA和PAK1将在同一样本中实时同时检测。该系统具有定量检测癌症生物标志物的潜力，无论是蛋白质、小分子还是核酸。此外，多路传输的能力允许对每个患者进行单独评估，并监测患者对治疗的反应。可以很容易地制造针对新靶点的适体，并且适体酶内的适体区域是可互换的。这里提出的aptazyme系统具有结合任何癌症生物标记物的潜力，并绕过了进一步严格的检测开发。