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

开发以用户为中心的新型 RNA 检测和单分子分析技术

• 批准号: 10238117
• 负责人: Ken A Halvorsen
• 金额: $ 38.63万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT ALBANY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10238117
• 关键词: 3' Untranslated Regions; Binding; Biological; Biological Assay; Biological Markers; Biology; Catalytic RNA; Code; Collaborations; Complex; DNA; Detection; Development; Devices; Disease; Ensure; Equipment; Feedback; Genome; Goals; Health; Human; Messenger RNA; Methods; MicroRNAs; Microscope; Molecular; Molecular Conformation; Monitor; Proteins; RNA; RNA Sequences; RNA analysis; Reaction; Reagent; Research; Research Personnel; Role; Sampling; Structure; Techniques; Technology; Therapeutic; Time; Training; Transcript; Untranslated RNA; cost effective; experimental study; gel electrophoresis; improved; instrument; interest; nanoscale; nanoswitch; new technology; novel; single molecule; technology development; therapeutic target; tool; transcription termination; undergraduate student; user-friendly; viral RNA

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.

我们对RNA及其在生物学中复杂作用的理解不断发展。我们知道， 基因组被转录成RNA，但只有一小部分转录物编码蛋白质。的 其余的非编码RNA的功能在很大程度上仍然是未知的，但越来越多的证据表明， 重要的监管作用。因此，人们越来越关注利用RNA来改善肿瘤的生长。 人类健康，使用RNA作为潜在的疾病生物标志物、治疗剂或治疗靶点。我的实验室 专注于开发新的，以用户为中心的技术，用于检测和分析RNA，以加速 加快RNA研究的发现步伐，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、无仪器的RNA检测。 这种试剂由我们称为DNA纳米开关的纳米器件组成。DNA纳米开关 通过DNA杂交组装成分子开关，其在结合时改变构象， 感兴趣的分子这些构象可以很容易地确定使用一个最常见的实验室 工具-凝胶电泳。结果是，用户将能够从生物样品中检测RNA序列。 使用一步反应对样品进行处理。在接下来的五年里，我们将优化这些用于microRNA的工具， mRNA、长链非编码RNA和病毒RNA。 其次，我们正在开发高通量RNA单分子分析方法。这将是 通过持续开发微力显微镜（CFM）和相关分析来实现 用于RNA分析。CFM集成了离心机和显微镜，可以同时进行数千次 作为拴系珠的单分子实验被真实的实时监控。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工作台上进行 离心机，使这样的实验用户友好，即使在本科水平。在未来五 在接下来的几年里，我们将为三个不同的项目开发CFM功能和单分子检测：1）机制 转录终止，2）治疗性核酶优化，和3）结构/功能探测 a mRNA中的3' UTR。 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，而无需昂贵的 设备或特殊培训。对于每一个技术开发项目，我们都参与了 区域RNA研究人员合作提供材料，生物背景和反馈， 确保我们实现我们的目标，并产生尽可能广泛的影响。