Spectroscopical Investigations To Determine Rna Ligand Interactions And Rna Dynamics

RNA-Ligand Interactions, Part A focuses on structural biology methods. Major topics covered include semisynthetic methodologies (RNA synthetic methods and derivatization of RNA); RNA structure determination (X-ray crystallography, NMR, EM); techniques for monitoring RNA conformation and dynamics (solution methods and electrophoretic and spectroscopic methods); and modeling tertiary structure: Part B, its companion Volume 318 of Methods in Enzymology, focuses on molecular biology methods. The critically acclaimed laboratory standard for more than forty years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Since 1955, each volume has been eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with more than 300 volumes (all of them still in print), the Series contains much material still relevant today--truly an essential publication for researchers in all fields of life sciences.

This volume looks at the different spectroscopic and biophysical methods used by researchers to study the structure and folding of RNA, and to follow their interactions with proteins. The chapters in this book cover topics such as single-molecule spectroscopy of multiple RNA species; surface plasmon resonance, MS or microcalorimetry for investigating molecular interactions with RNA; FTIR, SAXS, SANS and SRCD spectroscopies to analyze RNA structure; use of fluorescent nucleotides to map RNA-binding sites on proteins surfaces or CryoEM; and much more. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and comprehensive, RNA Spectroscopy: Methods and Protocols is a valuable resource for anyone interested in learning more about this developing field.

RNA-Ligand Interactions, Part B focuses on molecular biology methods. Major topics covered include: solution probe methods, tethered-probe methodologies, in vitro affinity selection methodologies, genetic methodologies for detecting RNA-protein interactions, protein engineering methodologies useful for RNA-protein interaction studies, and cell biology methods.RNA-Ligand Interactions, Part A, its companion, VOLUME 317 focuses on structural biology methods.The critically acclaimed laboratory standard for more than forty years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Since 1955, each volume has been eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with more than 300 volumes (all of them still in print), the series contains much material still relevant today--truly an essential publication for researchers in all fields of life sciences.

This volume looks at the different spectroscopic and biophysical methods used by researchers to study the structure and folding of RNA, and to follow their interactions with proteins. The chapters in this book cover topics such as single-molecule spectroscopy of multiple RNA species; surface plasmon resonance, MS or microcalorimetry for investigating molecular interactions with RNA; FTIR, SAXS, SANS and SRCD spectroscopies to analyze RNA structure; use of fluorescent nucleotides to map RNA-binding sites on proteins surfaces or CryoEM; and much more. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and comprehensive, RNA Spectroscopy: Methods and Protocols is a valuable resource for anyone interested in learning more about this developing field.

RNA-protein interactions play a fundamental role in gene expression and protein synthesis. Recent research into the role of RNA in cells has elucidated many more vital interactions with proteins. This book provides an up-to-date and comprehensive guide to a wide range of laboratory procedures to investigate the interactions between RNA and proteins. - ;RNA-protein interactions play a vital role in gene transcription and protein expression. Interactions such as the synthesis of mRNA by RNA polymerases, to the essential modification of RNA by the proteins of the spliceosome complex, and the highly catalytic action of the ribosome in protein synthesis, are established as being fundamental to the function of RNA. Recent research into, for example, the role of RNA as a catalyst, has elucidated many more interactions with proteins that are vital to cell function. RNA - Protein Interactions: A Practical Approach provides a clear and comprehensive guide to the experimental procedures used in studying RNA - protein interactions. The approaches covered range from those initially used to detect a novel RNA-protein interaction, various biochemical and genetic approaches to purifying and cloning RNA binding proteins, through to methods for an in depth analysis of the structural basis of the interaction. The volume includes a number of procedures that have not previously been covered in this type of manual. These include the production of site-specifically modified RNAs by enzymatic and chemical methods and in vivo screening for novel RNA - protein interactions in yeast and E. coli . This is the first volume to gather in one place this wide array of approaches for studying RNA - protein interactions. As is customary for the Practical Approach series, the writing is characterized by a clear explanatory style with many detailed protocols. This informative book will be a valuable aid to laboratory workers in biochemistry and molecular biology - graduate students, postdoctoral and senior scientists - whose research encompasses this field. -

RNA is a structurally flexible and dynamic molecule, and rapidly interconverts between multiple conformations. Despite the significant advances in traditional biophysical techniques like NMR spectroscopy and X-ray crystallography it is difficult to resolve the structures of dynamically inter-converting states as these methods measure the average property of an ensemble of molecules. It is necessary to resolve and get detailed descriptions of the multiple conformational structures, their quantitative distribution and their related dynamics in order to understand the ruggedness of the energy landscape. The present study attempts to link the conformational dynamics of RNA to its structure and biological function. Ultrafast time-resolved fluorescence spectroscopy is used to probe the distribution of multiple conformations and their dynamics that occur on a very fast timescales. This tool uses the resolution of time domain behavior of 2-aminopurine fluorescence decay on the ultrafast timescale to capture the characteristics of different structures. The RNAs are labeled with the fluorescent base analog 2-aminopurine to probe the base stacking patterns in RNA. Base stacking interactions is a unique feature that defines nucleic acid structures compared to proteins and are the major stabilizing force in RNA. Chapter 1 discusses the RNA folding mechanism and the relationships between sequence, structure, dynamics and function and explains the advantage of using ultrafast time resolved spectroscopy in defining structure-function relationships in RNA. Chapter 2 discusses the conformational heterogeneity and dynamics in purine-sensing riboswitches. We observed multiple conformations where the ligand is significantly stacked with the bases of the binding pocket and shows a dynamic balance between base stacking interactions and hydrogen bonding to achieve specificity and high affinity by the aptamer domain. Chapter 3 explores the conformational heterogeneity of GAAA tetraloop receptor and the impacts of the tertiary interactions between the receptor and the GAAA tetraloop, where we probe the isolated and GAAA-tetraloop bound receptor. The presence of any bound conformation in the free receptor state leads us to propose the conformational capture mechanism in tetraloop-receptor recognition. Chapter 4 summarizes findings of employing the ultrafast spectroscopy approach in studying riboswitches and the GAAA tetraloop-receptor. Overall our research is an effort to understand the role of conformational dynamics in RNA folding, tertiary interactions, recognition and functions.