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

 描述（由申请人提供）：本研究项目的总体目标是开发用于制造（i）RNA适体微阵列和（ii）用于表面等离子体共振成像（SPRI）生物亲和传感的非天然蛋白质微阵列（uPM）的芯片上多重模板生物合成方法。SPRI已成为用于DNA、RNA和蛋白质生物标志物的多重检测的主要工具，SPRI的最新进展如表面等离子体共振相位成像（SPR-PI）和纳米颗粒增强的SPR-PI增加了这些测量在微升样品体积中极低（皮摩尔至飞摩尔）浓度下用于体外生物标志物检测的灵敏度。大多数多重SPRI蛋白生物传感测量采用抗体或DNA适体微阵列; RNA适体微阵列的使用由于芯片外合成和纯化多个RNA序列所需的大量工作而受到限制。在这项研究工作中，将开发用于在小体积微流体格式中利用多重RNA聚合酶表面转录反应制造自组装RNA适体微阵列的芯片上方法，用于与SPR-PI一起检测皮摩尔浓度的蛋白质生物标志物; RNA分裂适体测定将与纳米颗粒增强的SPR-PI一起使用，以检测亚细胞水平的蛋白质生物标志物。皮摩尔浓度，并且还检测小分子代谢物如ATP。此外，我们将开发新的芯片上的方法为模板的uPMs的生物合成-蛋白质微阵列，其中每个固定的蛋白质含有一个或多个非天然氨基酸（uAA）。将uAA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，并掺入另外的反应性部分，例如 作为用于表面点击连接化学的含炔uAA或用于生物亲和性靶标的光捕获的含二苯甲酮uAA。uPM将在芯片上多重生物合成中由DNA模板制造，所述芯片上多重生物合成将采用用uAA装载tRNA的核酶和改良的体外转录和翻译（IVTT）方案的组合。这些uPM将与SPRI偶联以测量靶蛋白的增强的生物亲和吸附。第一个示范项目将使用SPRI和荧光（在适当的情况下）来研究修饰的EGFP/CFP微阵列的制造，IVTT蛋白微阵列的点击连接化学的结合，以及通过在特定的蛋白质-蛋白质相互作用区域附近结合二苯甲酮修饰的UAA来用探针蛋白光捕获生物亲和性靶蛋白。这两种简单而强大的芯片上合成方法用于创建RNA适体和非天然蛋白质微阵列将增强我们的SPRI生物亲和传感测量的特异性和选择性。

 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.