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? ? ? ?
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| Rinaldi, Arlie J; Suddala, Krishna C; Walter, Nils G (2015) Native purification and labeling of RNA for single molecule fluorescence studies. Methods Mol Biol 1240:63-95 |
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| Pitchiaya, Sethuramasundaram; Androsavich, John R; Walter, Nils G (2012) Intracellular single molecule microscopy reveals two kinetically distinct pathways for microRNA assembly. EMBO Rep 13:709-15 |
| Androsavich, John R; Chau, B Nelson; Bhat, Balkrishen et al. (2012) Disease-linked microRNA-21 exhibits drastically reduced mRNA binding and silencing activity in healthy mouse liver. RNA 18:1510-26 |
| Johnson-Buck, Alexander E; McDowell, Sarah E; Walter, Nils G (2011) Metal ions: supporting actors in the playbook of small ribozymes. Met Ions Life Sci 9:175-96 |
| Hoerter, John A H; Krishnan, Vishalakshi; Lionberger, Troy A et al. (2011) siRNA-like double-stranded RNAs are specifically protected against degradation in human cell extract. PLoS One 6:e20359 |
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