Development of novel, user-centric technologies for detection and single-molecule analysis of RNA

我们对RNA及其在生物学中的复杂角色的理解是不断发展的。我们知道大多数人 基因组被转录成RNA，但只有一小部分转录产物编码蛋白质。这个 其余非编码RNA的功能在很大程度上仍不清楚，但越来越多的证据表明 重要的监管角色。因此，人们越来越有兴趣利用RNA来改善 人类健康，使用RNA作为潜在的疾病生物标记物、疗法或治疗靶点。我的实验室 专注于开发新的、以用户为中心的RNA检测和分析技术，以加速 在RNA研究中发现的速度和最终实现改善人类健康的目标。 首先，我们正在开发一种“智能试剂”，用于简单、经济有效且无需仪器的RNA检测。 这种试剂由纳米级的设备组成，我们称之为DNA纳米开关。DNA纳米开关可以自我切换 通过DNA杂交组装成分子开关，当结合到时改变构象 感兴趣的分子。使用最常见的实验室之一可以很容易地识别这些构象 工具--凝胶电泳法。其结果是，用户将能够从生物中检测出RNA序列 使用一步反应的样品。在接下来的五年里，我们将针对microRNA优化这些工具， 信使核糖核酸、非编码长链RNA和病毒核糖核酸。 第二，我们正在开发高通量的RNA单分子分析方法。这将是 通过离心力显微镜(CFM)和相关分析的不断发展而实现 用于RNA分析。CFM集成了离心机和显微镜，使数以千计的 作为拴系珠的单分子实验被实时监控。重要的是，最近 我们实验室的发展使这些实验能够在标准的台式计算机上进行 离心机，使这样的实验即使在本科生水平上也是用户友好的。在接下来的五年里 几年来，我们将为三个不同的项目开发CFM特征和单分子分析：1)机制 2)治疗性核酶的优化；3)结构/功能的探测 3‘端非编码区。 这些工具将为研究人员提供新的方法来检测和分析各种RNA，而不需要昂贵的成本 对于每个技术开发项目，我们都参与了 区域RNA研究人员合作提供材料、生物学背景和反馈 确保我们正在实现我们的目标，并尽可能产生最广泛的影响。

Our understanding of RNA and its complex roles in biology is constantly evolving. We know that most of the genome is transcribed into RNA, but only a small fraction of these transcripts code for proteins. The functions of the remaining non-coding RNAs are still largely unknown, but increasing evidence points to important regulatory roles. Thus, there is increasing interest in exploiting RNA for the improvement of human health, using RNA as potential disease biomarkers, therapeutics, or therapeutic targets. My lab is focused on developing novel, user-centric technologies for detection and analysis of RNA to accelerate the pace of discovery in RNA research and ultimate realize the goal of improving human health. First, we are developing a “smart reagent” for simple, cost effective, and instrument-free RNA detection. This reagent consists of nanoscale devices that we call DNA nanoswitches. The DNA nanoswitches self- assemble by DNA hybridization into molecular switches that change conformations upon binding to molecules of interest. These conformations can be easily identified using one of the most common lab tools – gel electrophoresis. The result is that users will be able to detect RNA sequences from biological samples using a one-step reaction. Over the next five years we will optimize these tools for microRNA, mRNA, long non-coding RNA, and viral RNA. Second, we are developing methods for high-throughput single-molecule analysis of RNA. This will be accomplished by continued development of the Centrifuge Force Microscope (CFM) and associated assays for RNA analysis. The CFM integrates a centrifuge and a microscope to enable thousands of simultaneous single-molecule experiments as tethered beads are monitored in real time. Importantly, recent developments in our lab have enabled these experiments to be performed in a standard benchtop centrifuge, making such experiments user friendly even at the undergraduate level. Over the next five years, we will develop CFM features and single molecule assays for three distinct projects: 1) mechanisms of transcription termination, 2) therapeutic ribozyme optimization, and 3) structure/function probing of a 3’ UTR in mRNA. These tools will provide new ways for researchers to detect and analyze various RNAs without expensive equipment or special training. For each of the technology development projects, we have engaged regional RNA researchers in collaborations to provide materials, biological context, and feedback to ensure that we are achieving our goals and the having the broadest possible impact.