Knowledge about ribonucleic acids (RNAs) and their roles has recently been expanded by extensive studies seeking to uncover novel RNAs using biochemical techniques. These techniques visualize specific RNA in situ to understanding the regulatory mechanisms of gene expression. Lately, a variety of photo labeling techniques has been developed for imaging RNA in vivo to locate and track it with high accuracy. This study focuses on fluorescence-based methods of in vivo RNA visualization. Using fluorescent light has transformed the study of micro-molecular components due to its many advantages. Molecular beacons targeting repeat expansion of RNA can differentiate between normal repeat sizes and repeat expansions in the pathogenic range. With modification and testing, molecular beacons have the potential to serve as probes for sensing repeat structure accessibility and for use in diagnostics assays. Broccoli GFP-mimic RNAs have different instability and metal ion preferences than Baby Spinach, a truncated version of the Spinach aptamer, that are primarily associated with the sequence and structure of predicted quadruplex-flanking stem structures. Unique duplex-to-quadruplex transitions in GFP-mimic RNAs, which likely explains their sensitivity to magnesium due to its stability and fluorescence. RNA Mango is an RNA aptamer that binds thiazole Orange (TO1) dye and its derivative with nanomolar binding affinity, unlike MS2 and Spinach systems. Mango is superior in its detection of bacterial fluorescence compared with Spinach, which requires 200uM of the fluorophore. Inserting a Mango aptamer with TO1-Biotin, a derivative of TO1, genetically into the cell can be used to monitor the expression of RNA in real time by measuring the fluorescence signal that comes from the TO1-Biotin.

Methods in Enzymology: Visualizing RNA Dynamics in the Cell continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers research methods visualizing RNA dynamics in the cell, and includes sections on such topics as identification of RNA cis-regulatory sequences, IRAS, IMAGEtags, MERFISH, plant RNA labeling using MS2, and visualization of 5S dynamics in live cells using photostable corn probe. Continues the legacy of this premier serial with quality chapters authored by leaders in the field Covers research methods in visualizing RNA dynamics in the cell Contains sections on such topics as identification of RNA cis-regulatory sequences, IRAS, IMAGEtags, MERFISH, plant RNA labeling using MS2 and visualization of 5S dynamics in live cells using photostable corn probe

The study of RNA-protein interactions is crucial to understanding the mechanisms and control of gene expression and protein synthesis. The realization that RNAs are often far more biologically active than was previously appreciated has stimulated a great deal of new research in this field. Uniquely, in this book, the world's leading researchers have collaborated to produce a comprehensive and current review of RNA-protein interactions for all scientists working in this area. Timely, comprehensive, and authoritative, this new Frontiers title will be invaluable for all researchers in molecular biology, biochemistry and structural biology.

Ribonucleic acids (RNAs) serve as one of the main building blocks of organic life and with the advent of new understanding beyond an intermediary for protein coding, understanding its function becomes imperative. Previous tools study RNA expression from cell populations such as tissue or cell lines, however, an expansive area of research has developed for the individual cells. The recognition of cell heterogeneity in normal and diseased tissues further highlighted the importance of accurate RNA detection in single cells. Single molecule RNA fluorescence in situ hybridization (smRNA-FISH) has revolutionized RNA detection and quantification by visualizing single RNA transcripts. The work in this dissertation improves upon smRNA-FISH by developing quantum dot based fluorescent in situ technologies that uses hybridization of quantum dot-labeled single stranded DNA oligonucleotides to the RNA targets for visualization and quantification. In chapter 2, we demonstrate our proof-of-concept quantum dot single molecule RNA-FISH (qdot-smRNA-FISH) by comparing the sensitivity and specificity of qdot-smRNA-FISH against commercialized smRNA-FISH. In chapter 3, we describe qdot-smRNA-FISH for detection of isoforms (isoFISH), where different from smFISH that requires dozens of hybridization probes per gene, isoFISH requires only two hybridization probes per splicing variant. In chapter 4, we used the co-localization of multi-color quantum dot FISH (CoQ-FISH) to observe for the first time a fusion transcript formation of Eml4-Alk in non-small cell lung cancer biopsy samples from trans-splicing and not chromosomal rearrangement. In chapter 5, we develop cell surface qDot RNA-FISH (Surface-FISH), a method for visualization of RNAs on the cell surface. We identified RNA targets using RNA-seq on membrane coated nanoparticles from EL4 and applied Surface-FISH to observe regions of Malat1 and Neat1. In chapter 6, we developed Surface-PromFISH that uses randomized antisense probes to promiscuously label as many csRNAs as possible and made it compatible with co-staining of cell membrane proteins. We subjected the hybridization targets to RNA sequencing (Surface-FISHseq), which led to 51 putative PBMC csRNAs and we identified a potential function where antisense blocking of FNDC3B and CTSS lowers monocyte attachment.

With its complex and extensively regulated metabolism, the study of the RNA lifecycle demands tools that allow for the localization of RNAs to be observed either in an in situ setting or, preferably, under in vivo conditions. In RNA Detection and Visualization: Methods and Protocols, the best and brightest investigators provide an up-to-date and in-depth description of basic methods and protocols used for detecting and visualizing mRNAs in both fixed and live cells, from bacteria to mammals. For novices and experts alike, this mix of classic in situ hybridization and advanced live imaging techniques, cell fractionation and affinity purification procedures, and bioinformatics tools gives researchers the most complete and extensive array of research aids possible. As a volume written in the highly successful Methods in Molecular BiologyTM series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and expert tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, RNA Detection and Visualization: Methods and Protocols offers well-honed techniques in order to inspire researchers around the world to further our knowledge of the vital biological significance of RNA.

Fluorescent Analogs of Biomolecular Building Blocks focuses on the design of fluorescent probes for the four major families of macromolecular building blocks. Compiling the expertise of multiple authors, this book moves from introductory chapters to an exploration of the design, synthesis, and implementation of new fluorescent analogues of biomolecular building blocks, including examples of small-molecule fluorophores and sensors that are part of biomolecular assemblies.

Recent advances in imaging technology reveal, in real time and great detail, critical changes in living cells and organisms. This manual is a compendium of emerging techniques, organized into two parts: specific methods such as fluorescent labeling, and delivery and detection of labeled molecules in cells; and experimental approaches ranging from the detection of single molecules to the study of dynamic processes in organelles, organs, and whole animals. Although presented primarily as a laboratory manual, the book includes introductory and background material and could be used as a textbook in advanced courses. It also includes a DVD containing movies of living cells in action, created by investigators using the imaging techniques discussed in the book. The editors, David Spector and Robert Goldman, whose previous book was Cells: A Laboratory Manual,are highly respected investigators who have taught microscopy courses at Cold Spring Harbor Laboratory, the Marine Biology Laboratory at Woods Hole, and Northwestern University.