Hybridization Chain Reaction: In Situ Amplification for Biological Imaging

描述（由申请人提供）：基因组数据的最近爆炸提供了一个前所未有的机会，研究发育和疾病过程的分子基础。虽然微阵列技术的基因表达谱在一个给定的组织已经变得司空见惯，用于确定确切的空间和时间范围的基因表达的方法仍然需要改进。用于基因表达研究的原位杂交技术通常具有有限的灵敏度和显著的背景信号，即使在不存在靶基因的位置。此外，现有技术没有充分利用同时成像许多基因的潜力。 拟议研究的目标是适应新开发的纳米传感器技术的基因表达的多重原位扩增。这种扩增工具，称为杂交链反应（HCR），通过仅在探针分子特异性结合其靶标时激活来减少背景。该事件触发了来自荧光标记的DMA发夹的拴系“聚合物”的自组装。并行多路复用可以简单地通过使用独立的HCR放大器为每个独特的目标物种。 研究计划包括原位HCR放大器的设计、验证和应用。具体目标是： 1：设计触发、多路复用、非线性HCR放大器，并完善HCR放大器设计的计算工具。 第二章：使用纳米光刻技术原位验证放大器的空间定位、灵敏度、特异性和多路复用，以在表面上精确地图案化靶分子。 第三章：将HCR应用于鸟类胚胎中模式基因的原位杂交，检测先前研究预测的遗传标记的共表达。 我们的目标是开发原位HCR放大器，使生物标本中的许多目标的灵敏和同时检测。如果成功，这种纳米技术将成为现代基因组和蛋白质组学工具的重要辅助工具，适用于从生物实验到组织活检的各种环境。

DESCRIPTION (provided by applicant): The recent explosion of genomic data has offered an unprecedented opportunity to study the molecular basis for developmental and disease processes. Although microarray techniques for profiling the genes expressed in a given tissue have become commonplace, methods for determining the exact spatial and temporal extent of gene expression are still in need of improvement. In situ hybridization techniques for gene expression studies often have limited sensitivity and significant background signal even in locations where no target genes are present. Furthermore, existing technologies do not fully exploit the potential for imaging many genes simultaneously. The goal of the proposed research is to adapt a newly developed nanosensor technology for multiplexed in situ amplification of gene expression. This amplification tool, termed hybridization chain reaction (HCR), reduces background by activating only when a probe molecule binds specifically to its target. This event triggers the self-assembly of a tethered 'polymer' from fluorescently-labeled DMA hairpins. Parallel multiplexing can be achieved simply by using independent HCR amplifiers for each unique target species. The research plan involves the design, validation and application of in situ HCR amplifiers. Specific aims are: 1: Design triggered, multiplexed, nonlinear HCR amplifiers, and refine the computational tools for the design of HCR amplifiers. 2: Validate the spatial localization, sensitivity, specificity and multiplexing of the amplifiers in situ using nanolithography techniques to precisely pattern target molecules on surfaces. 3: Apply HCR to in situ hybridization of patterning genes in avian embryos, testing for the co- expression of genetic markers predicted by previous studies. The objective is to develop in situ HCR amplifiers that will enable the sensitive and simultaneous detection of numerous targets in biological specimens. If successful, this nanotechnology will serve as an important adjunct to modern genomic and proteomic tools in settings ranging from biological experiments to tissue biopsies.