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

描述（由申请人提供）：对抗癌症的最有效方法是在早期阶段，使早期检测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，新发现的指示癌症的生物标志物激增。然而，这些生物标志物的检测将需要新的诊断开发，这可能是昂贵的，并需要几年的时间来开发。需要一种通用的诊断工具，可以检测当前和即将发现的癌症生物标志物。提出了一种新的通用诊断方法，可以实时检测多种癌症生物标志物。最近，设计了一种基于核酸的核酶或适体酶，能够进行配体依赖性指数扩增。适体酶已显示可互换地检测生理范围内的茶碱和黄素单胞苷肽（FMN）。与其他诊断方法（如RT-PCR）不同，该系统是等温和自我维持的，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才能完成，使用危险的放射性物质，并且一次只能检测一种配体。 提出了一种新的实时、无害、多重的荧光法适体酶。Fvrster（荧光）共振能量转移（FRET）方法将用于通过荧光标记适体酶来检测指数扩增。FRET将与适体酶的活性偶联，使得能够实时检测配体浓度。作为原理证明，将开发两种对癌症生物标志物前列腺特异性抗原（PSA）和p21活化激酶（PAK 1）具有特异性的适体酶。PSA和PAK 1的现有适体基序将并入适体酶中，并且各自将偶联至独特的FRET对，从而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，使得能够进行配体的多重检测。将在同一样本中同时实时测定不同浓度的PSA和PAK 1。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，多路复用的能力允许对每个患者进行个体评估并监测患者对治疗的反应。可以容易地制备对新靶标特异性的适体，并且适体酶内的适体区域是可互换的。本文提出的适体酶系统具有结合任何癌症生物标志物的潜力，并避免了进一步严格的测定开发。 公共卫生相关性：癌症生物标志物的检测可以通过促进个体化诊断和早期治疗来降低患者的死亡率。很可能会发现新的生物标志物，因此可以检测所有生物标志物（无论是蛋白质、小分子还是核酸）的通用诊断工具将避免及时和昂贵的测定开发。(1-4)本申请中提出的自我维持系统具有实时通用和多重检测当前和新发现的癌症生物标志物的潜力。

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. PUBLIC HEALTH RELEVANCE: Detection of cancer biomarkers can reduce mortality rates in patients by facilitating individualized diagnosis and early treatment. It is likely that new biomarkers will be discovered, thus a general diagnostic tool that can detect all biomarkers (whether protein, small molecule, or nucleic acid) would circumvent timely and costly assay development.(1-4) The self-sustained system proposed in this application has the potential for universal and multiplexed detection of current and newly discovered cancer biomarkers in real-time.