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

描述（由申请人提供）：最有效的抗癌方法是在早期阶段，早期发现和风险评估对患者的生存至关重要。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呈爆炸式增长。然而，这些生物标记物的检测需要新的诊断技术的发展，这些技术的开发成本很高，而且需要几年的时间。需要一种能够检测当前和即将发现的癌症生物标志物的通用诊断工具。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，或称适体酶，能够依赖配体的指数扩增。该酶可在生理范围内交替检测茶碱和黄素单核苷酸。与RT-PCR等其他诊断方法不同，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，目前的分析需要几个小时才能完成，使用有害的放射性物质，并且一次只能检测一个配体。提出了一种实时、无害、多路复用的荧光酶。Fvrster（荧光）共振能量转移（FRET）方法将用于检测指数扩增荧光标记的适体酶。FRET将耦合到适配体酶的活性，从而实时检测配体浓度。作为原理验证，将开发两种针对癌症生物标志物前列腺特异性抗原（PSA）和p21活化激酶（PAK1）的适配酶。现有的PSA和PAK1适配体基序将被整合到适配体酶中，每个适配体将与一个独特的FRET对耦合，为每个配体产生不同的FRET信号。不同的FRET对将有最小的光谱重叠，使多路检测配体。不同浓度的PSA和PAK1将在同一样品中同时实时检测。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，多重功能允许对每个患者进行单独评估并监测患者对治疗的反应。针对新靶标的适配体可以很容易地合成，适配体酶内的适配体区域是可互换的。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，并且可以避免进一步严格的检测开发。

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.