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（由适用提供）：该研究项目的总体目标是开发用于制造（I）RNA APATMER微阵列的芯片，多路复用的模板生物合成方法，以及（II）不自然的蛋白质微阵列（UPMS），用于表面Plasmon Resonance Imanance Imacing（Spri）生物生物生物生物生物生物生物生物生物生物生物生物生物生物生物生物生物生物具有敏感性。 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.大多数多路复用的SPRI蛋白生物传感测量员工抗体或DNA Apatmer微阵列；由于芯片外合成和多个RNA序列的纯化所需的重大努力，RNA Apatmer微阵列的使用受到限制。在这项研究工作中，将开发出小体积的微流体格式的多路复用RNA聚合酶表面转录反应的自组装RNA APATMER微阵列的芯片方法，以与Spr-Pi一起使用，以检测PICOROL浓度下的蛋白质生物标记物； RNA裂解药物测定将与纳米颗粒增强的SPR-PI一起使用，以检测亚曲面浓度下的蛋白质生物标志物，并检测小分子代谢物（如ATP）。此外，我们将开发用于UPMS模板生物合成的新型片 - 蛋白微阵列，其中每种固定的蛋白质都包含一个或多个不自然的氨基酸（UAAS）。将UAAS掺入蛋白质中可用于增强蛋白质蛋白质相互作用的稳定性，并结合其他反应性部分，例如 作为含有炔烃的UAA，可用于表面喀哒声，或含苯甲酮的UAA，用于生物障碍靶标的光接收。 UPMS将由片上多重生物合成中的DNA模板制成，该模板将采用tRNA与UAAS的核酶充电的组合以及经过修饰的体外转录和翻译（IVTT）方案。这些UPMS将与SPRI耦合，以测量第一个演示项目的增强的生物亲和力吸附，将同时使用SPRI和荧光（在适当的情况下）研究改进的EGFP/CFP微阵列的结构，这是IVTT蛋白微阵列的结合附件，并与探测器的良好蛋白质蛋白质蛋白质构成，以使IVTT蛋白质微阵列构成蛋白质蛋白质蛋白质蛋白质蛋白质蛋白质蛋白质蛋白质蛋白质蛋白质蛋白质蛋白质蛋白质蛋白质蛋白质。在特定蛋白质相互作用区域附近的UAA。这两种简单但功能强大的片上合成方法用于创建RNA APATMER和不自然的蛋白质微阵列，将增强我们的Spri生物疗法传感测量的特异性和选择性。