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以让研究人员跟踪大量新发现的、不同的非蛋白编码（nc） rna，因为它们在细胞周围实现了许多生物学功能，更不用说在单分子水平上了。该项目将从根本上克服这一限制，开发一种新的探针概念，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。当开发一种新的细胞显微镜技术时，为了确定性能参数，在生物系统上进行实际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）的新基因调控范式相关，其中一条途径是微RNA （miRNAs）抑制所有多细胞真核生物（包括人类）的内源性基因。一个创始类成员是let-7，或致死-7，这是一个进化上保守的miRNA，从秀丽隐杆线虫到人类。已发现其可调控疾病相关转录效应物的表达，其中高迁移率组AT-hook 2 （HMGA2）蛋白参与转录调控，与多种癌症和饮食性肥胖相关。HMGA2 mRNA的表达受7个不寻常的let-7a-1结合位点控制。作为细胞内探针技术的原理验证，我们将与Sunney Xie（哈佛大学）和David Bartel（怀特黑德研究所/麻省理工学院）的团队合作，通过追求以下里程碑，检测let-7 miRNA， HMGA2 mRNA和RNAi蛋白组装成单个活性微rna -蛋白（miRNP）复合物：(1)我们将在完全可控的样品上设计，合成和测试培养细胞中的单分子检测。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缓慢扩散，就达到细胞内检测单个组装的miRNP复合物所需的信噪比阈值（放大水平）。(2)我们将在现实世界的Let-7a/HMGA2 mRNA探针/靶系统上测试开发的探针技术，并独特地解决有关miRNAs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”探针技术的范围和局限性。也就是说，与目标1和目标2并行，我们会问：多路复用（即并行检测多个目标）可能吗？DNA靶标中的多个串联重复序列能否被检测到？蛋白质组装，特别是在蛋白质错误折叠疾病如阿尔茨海默氏症和朊病毒疾病中发生的蛋白质组装，能否通过我们新颖的“分子圣诞树”探针概念检测到？DNA重复序列和蛋白质聚合检测的下限是什么？这些下限是否可以进一步提高？是否可以同时检测多种不同的生物聚合物？这对检测限有何影响？

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?