Publication

New methods for the investigation of RNA refolding by NMR spectroscopy

Philipp Wenter
2006
EPFL thesis
Abstract

A detailed characterization of the time scales and mechanisms by which RNA molecules change their folding structure upon binding of metabolites or during catalysis is important for the understanding of the different biological functions of RNA. The folding from an unfolded, denaturated state has been extensively studied, establishing hierarchical pathways, in which fast formation of secondary structural elements precedes the folding of tertiary elements. In contrast, only a few examples of time-resolved RNA refolding in the native state have been reported so far. Specifically, NMR spectroscopy has not yet been employed for the investigation of RNA refolding despite its intrinsic sensibility to dynamic structural changes. In the project presented here, new tools for the identification of RNA secondary structures and the quantification of RNA refolding processes by NMR spectroscopy have been developed and employed. Preparation of 15N-labeled phosphoramidites and their sequence-selective introduction into RNA sequences allowed a straightforward identification of base pairs and imino protons by HNN-COSY and 15N HSQC experiments, respectively. Synthesis and introduction of NPE-protected uridines and guanosines into bistable RNA sequences provided a powerful method for the photoinduced preparation of selected RNA folds far from equilibrium. When introduced into bistable RNA sequences they allow to disrupt selected base pairs, thereby destabilizing the associated secondary structures. As a consequence, one out of usually two coexisting fold could be prepared selectively in its caged form. Upon photolysis, the native sequence was released under physiological conditions with completely retained, preselected secondary structure arrangement. Refolding into the thermodynamic equilibrium was subsequently followed by real-time imino proton NMR spectroscopy, providing quantitative, time-resolved and structural information for the refolding processes of three bistable RNA sequences and a Mg2+-induced secondary and tertiary structure refolding. Depending on their topology, these RNA sequences refold either via a dissociative mechanism in which the disruption of existing base pairs precedes the formation of new ones or via an associative mechanism in which new base pairs are formed from initially unpaired sequence regions simultaneously to the detachement of existing base pairs. In the transition state approximately half of the base pairs are disrupted. The investigation of a bistable RNA sequence designed to undergo a topologically favored refolding processes was carried out by exchange sensitive NMR experiments. The introduction of sequence specific 15N-labeles into RNA sequences allowed the implementation of exchange sensitive 1D and 2D 1H/15N-heteronuclear NMR methods which were designed to map very slow exchange. We found that a 34mer RNA sequence exhibits two folds which exchange on the observable time scale (τobs = T1{15N}

About this result
This page is automatically generated and may contain information that is not correct, complete, up-to-date, or relevant to your search query. The same applies to every other page on this website. Please make sure to verify the information with EPFL's official sources.

Graph Chatbot

Chat with Graph Search

Ask any question about EPFL courses, lectures, exercises, research, news, etc. or try the example questions below.

DISCLAIMER: The Graph Chatbot is not programmed to provide explicit or categorical answers to your questions. Rather, it transforms your questions into API requests that are distributed across the various IT services officially administered by EPFL. Its purpose is solely to collect and recommend relevant references to content that you can explore to help you answer your questions.