On-Chip Templated Biosynthesis of RNA Aptamer and Unnatural Protein Microarrays for SPR Imaging

用于 SPR 成像的 RNA 适体和非天然蛋白质微阵列的片上模板生物合成

• 批准号: 8964189
• 负责人: ROBERT M CORN
• 金额: $ 33.14万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8964189
• 关键词: Adenosine; Adsorption; Aftercare; Alkynes; Amber; Amino Acids; Anabolism; Antibodies; Benzophenones; Binding; Binding Sites; Biological; Biological Assay; Biological Markers; Biosensing Techniques; Blood; Cancer Detection; Catalytic RNA; Chemistry; Codon Nucleotides; Coupled; DNA; DNA Microarray Chip; DNA-Directed RNA Polymerase; Detection; Development; Disease; Elements; Fluorescence; Genetic Transcription; Goals; Grant; Green Fluorescent Proteins; Heart Arrest; Image; Immunity; In Vitro; Kinetics; Label; Light; Liquid substance; Location; Measurement; Measures; Methodology; Methods; Microfluidics; N.I.H. Research Support; Nucleic Acids; Patient Monitoring; Phase; Positioning Attribute; Protein Microchips; Proteins; Protocols documentation; RNA; RNA Sequences; RNA biosynthesis; Reaction; Research; Research Project Grants; Saliva; Sampling; Specificity; Surface; Surface Plasmon Resonance; TATA-Box Binding Protein; Technology; Time; Transfer RNA Aminoacylation; Translations; aptamer; biochip; design; fluorescence imaging; imaging modality; nanoparticle; novel; protein protein interaction; public health relevance; small molecule; tool; transcriptome sequencing

 DESCRIPTION (provided by applicant): The overall goal of this research project is to develop on-chip, multiplexed templated biosynthesis methods for the fabrication of (i) RNA aptamer microarrays and (ii) unnatural protein microarrays (uPMs) for use in surface plasmon resonance imaging (SPRI) bioaffinity sensing. SPRI has become a mainstay tool for multiplexed detection of DNA, RNA, and protein biomarkers, and recent advances in SPRI such as surface plasmon resonance phase imaging (SPR-PI) and nanoparticle-enhanced SPR-PI have increased the sensitivity of these measurements for in vitro biomarker detection at extremely low (picomolar to femtomolar) concentrations in microliter sample volumes. Most multiplexed SPRI protein biosensing measurements employ either antibody or DNA aptamer microarrays; the use of RNA aptamer microarrays has been limited due to the significant effort required for the off-chip synthesis and purification of multiple RNA sequences. In this research effort, on-chip methods for the fabrication of self-assembled RNA aptamer microarrays with multiplexed RNA polymerase surface transcription reactions in a small volume microfluidic format will be developed for use with SPR-PI to detect protein biomarkers at picomolar concentrations; RNA split-aptamer assays will be used with nanoparticle-enhanced SPR-PI to detect protein biomarkers at sub-picomolar concentrations and also to detect small molecule metabolites such as ATP. Additionally, we will develop novel on-chip methods for the templated biosynthesis of uPMs - protein microarrays where each of the immobilized proteins contains one or more unnatural amino acids (uAAs). The incorporation of uAAs into proteins can be used to enhance the stability of protein-protein interactions and to incorporate additional reactive moieties, such as alkyne-containing uAAs for surface click attachment chemistry or benzophenone-containing uAAs for the photocapture of bioaffinity targets. The uPMs will be fabricated from DNA templates in an on-chip multiplexed biosynthesis that will employ a combination of ribozyme charging of tRNA with uAAs and a modified in vitro transcription and translation (IVTT) protocol. These uPMs will be coupled to SPRI to measure the enhanced bioaffinity adsorption of target proteins. The first demonstration projects will use both SPRI and fluorescence (where appropriate) to study the fabrication of modified EGFP/CFP microarrays, the incorporation of click attachment chemistry for IVTT protein microarrays, and the photocapture of a bioaffinity target protein with a probe protein by incorporating a benzophenone-modified UAA near the specific protein- protein interaction region. These two simple yet powerful on-chip synthesis methods for creating of RNA aptamer and unnatural protein microarrays will enhance the specificity and selectivity of our SPRI bioaffinity sensing measurements.

 描述（由申请人提供）：该研究项目的总体目标是开发片上多重模板生物合成方法，用于制造（i）RNA适体微阵列和（ii）用于表面等离子体的非天然蛋白质微阵列（uPM）共振成像 (SPRI) 生物亲和感测已成为 DNA、RNA 和蛋白质生物标志物多重检测的主要工具，并且在该领域取得了最新进展。表面等离振子共振相位成像 (SPR-PI) 和纳米颗粒增强型 SPR-PI 等 SPRI 提高了微升样品体积中极低（皮摩尔至飞摩尔）浓度的体外生物标志物检测的灵敏度。生物传感测量采用抗体或 DNA 适体微阵列；由于芯片外合成和纯化需要大量工作，RNA 适体微阵列的使用受到限制。在这项研究工作中，将开发在小体积微流体格式中使用多重 RNA 聚合酶表面转录反应制造自组装 RNA 适体微阵列的芯片方法，以与 SPR-PI 一起使用来检测蛋白质生物标志物。皮摩尔浓度；RNA 分裂适体检测将与纳米粒子增强的 SPR-PI 一起使用，以检测亚皮摩尔浓度的蛋白质生物标志物，并检测小分子此外，我们将开发用于 uPM 模板生物合成的新型芯片方法，其中每种固定化蛋白质都含有一种或多种非天然氨基酸 (uAA)，可将 uAA 掺入蛋白质中。增强蛋白质-蛋白质相互作用的稳定性并掺入额外的反应部分，例如 作为用于表面点击附着化学的含炔的 uAA 或用于生物亲和目标光捕获的含二苯甲酮的 uAA，uPM 将在片上多重生物合成中由 DNA 模板制成，该生物合成将采用 tRNA 与 uAA 的核酶充电相结合。修改后的体外转录和翻译 (IVTT) 方案将这些 uPM 与 SPRI 结合以测量增强的生物亲和力。第一个示范项目将使用 SPRI 和荧光（如果适用）来研究改良的 EGFP/CFP 微阵列的制造、IVTT 蛋白质微阵列的点击附着化学的结合以及生物亲和力目标蛋白质的光捕获。通过在特定的蛋白质-蛋白质相互作用区域附近掺入二苯甲酮修饰的 UAA 来形成探针蛋白质，这两种简单而强大的芯片上合成方法可用于创建 RNA 适体和非天然蛋白质。微阵列将增强我们的 SPRI 生物亲和力传感测量的特异性和选择性。