Trekking with the Ribognome: Single Molecule Microscopy of Intracellular miRNPs

描述（由申请人提供）：目前，没有合适的显微镜工具可供研究人员跟踪细胞周围大量新发现的、多样化的非蛋白质编码（nc）RNA，因为它们实现了许多生物学功能，更不用说在单分子水平上了。该项目将通过开发一种新的探针概念来从根本上克服这一限制，该探针概念被优化用于检测活细胞内的单个小ncRNA分子。预计我们的“分子圣诞树”探针技术将随后转移到其他生物聚合物。在开发新的细胞显微镜技术时，为了确定性能参数，对生物系统进行真实世界的现场测试是必不可少的。在最近发现的ncRNA中，有一些与RNA干扰（RNAi）的新基因调控模式相关，其中一种途径是微小RNA（miRNA）抑制所有多细胞真核生物（包括人类）中的内源性基因。let-7或lethal-7是一种进化上保守的C. elegans to humans人类.已发现其调节疾病相关转录效应物的表达，其中高迁移率族AT-钩2（HMGA 2）蛋白参与转录调节并与各种癌症以及饮食诱导的肥胖症相关。HMGA 2 mRNA的表达由一个不寻常的7个let-7a-1结合位点控制。作为我们的细胞内探针技术的原理证明，我们将通过与Sunney Xie（哈佛U.）和大卫巴特尔（怀特黑德研究所/麻省理工学院）：（1）我们将设计，合成和测试培养细胞中的单分子检测在一个完全可控的样品。一旦将足够数量的标记的let-7a探针加载到单个靶标上并在复合物中一起缓慢扩散，就达到了用于单个组装的miRNP复合物的细胞内检测的必要信噪比阈值（扩增水平）。(2)我们将在真实世界的Let-7a/HMGA 2 mRNA探针/靶系统上测试开发的探针技术，并独特地解决有关miRNA细胞生物学的许多悬而未决的问题。(3)我们将定义我们的“分子圣诞树”探测技术的范围和局限性。也就是说，与目标1和2并行，我们会问：多路复用（即，并行检测多个目标）可能吗？可以检测到DNA靶点中的多个串联重复序列吗？蛋白质组装，特别是发生在蛋白质错误折叠疾病，如阿尔茨海默氏症和朊病毒疾病，可以检测到我们的新的“分子圣诞树”探针的概念？DNA重复序列和蛋白质聚合检测的下限是什么，这些限制是否可以进一步推动？多个不同的生物聚合物可以同时检测吗？这对检测限有什么影响？

DESCRIPTION (provided by applicant): Currently, there are no suitable microscopy tools available that would allow researchers to follow the vast number of newly discovered, diverse non-protein coding (nc)RNAs around the cell as they fulfill their numerous biological functions, let alone at the single molecule level. The proposed project will fundamentally overcome this limitation by developing a novel probe concept optimized for detecting single small ncRNA molecules inside living cells. It is expected that our "molecular Christmas tree" probe technology will subsequently be transferable to other biopolymers. When developing a new cell microscopy technique, real-world field testing on a biological system for the purpose of determining performance parameters is essential. Among recently discovered ncRNAs are those associated with the new gene regulatory paradigm of RNA interference (RNAi), where in one pathway micro-RNAs (miRNAs) act to repress endogenous genes in all multicellular eukaryotes, including humans. A founding class member is let-7, or lethal-7, which is an evolutionarily conserved miRNA from C. elegans to humans. It has been found to regulate expression of disease-related transcriptional effectors, among them the High Mobility Group AT-hook 2 (HMGA2) protein involved in transcriptional regulation and associated with various cancers as well as diet-induced obesity. Expression of HMGA2 mRNA is controlled by an unusual seven let-7a-1 binding sites. As a proof-of-principle for our intracellular probe technology we will detect the assembly of let-7 miRNA, HMGA2 mRNA and RNAi proteins into single active micro-RNA-protein (miRNP) complexes, by pursuing the following milestones in collaboration with the groups of Sunney Xie (Harvard U.) and David Bartel (Whitehead Institute/MIT): (1) We will design, synthesize and test single molecule detection in cultured cells on a fully controllable sample. The necessary signal-to-noise threshold (amplification level) for intracellular detection of single assembled miRNP complexes is reached once a sufficient number of labeled let-7a probes are loaded onto a single target and slowly diffuse together in a complex. (2) We will test the developed probe technology on the real-world Let-7a/HMGA2 mRNA probe/target system and uniquely address numerous outstanding questions concerning the cell biology of miRNAs. (3) We will define the scope and limitations of our "molecular Christmas tree" probe technology. That is, in parallel to Aims 1 and 2 we will ask: Is multiplexing (i.e., the detection of multiple targets in parallel) possible? Can multiple tandem repeat sequences in a DNA target be detected? Can protein assembly, particularly that occurring during protein misfolding diseases such as Alzheimer's and prion diseases, be detected by our novel "molecular Christmas tree" probe concept? What are the lower limits for DNA repeat sequence and protein polymerization detection and can these limits be further pushed? Can multiple different biopolymers be detected in parallel and how does this affect detection limits?