Abstract
RNA exists in cells as dynamic, three dimensional entities, and determination of their
structure can be an essential step in understanding their function. With the introduction of
next generation sequencing, it has become possible to study the structure of thousands of
RNAs in a single experiment. A highly successful method to probe RNA structure is
Selective 2’-Hydroxyl Acylation analyzed by Primer Extension (SHAPE), however, this
method is limited by high background rates arising from non-probed molecules and pretermination
in the reverse transcription. In this thesis I describe the development of highthroughput
SHAPE-based approaches to investigate RNA structure based on novel
SHAPE reagents that permit selection of full-length cDNAs. The SHAPE Selection
(SHAPES) method is applied to the foot-and-mouth disease virus (FMDV) plus strand RNA
genome, and the data is used to construct a genome-wide structural map of the virus. I
have used the data to discover stable structures de novo, including previously
characterized structural elements, such as the internal ribosome entry site (IRES), and I
show that three of the novel structures have been conserved through evolution, indicating
that they are functional. The SHAPES method is further applied to the hepatitis C virus
(HCV), where the data is used to refine known and predicted structures. Over the past
years, the interest of studying RNA structure in their native environment has been
increased, and to allow studying RNA structure inside living cells using the SHAPE
Selection approach, I introduce a biotinylated probing reagent. This chemical can cross
cell membranes and reacts with RNA inside the cells, allowing the structural conformations
to be studied in the context of physiological relevant conditions in living cells. The methods
and results presented in this thesis represent important steps forward in studying RNA
structures with high-throughput technologies, and the selection approach could be key to
obtaining high quality sequencing-based probing data in experiments with a high
background.
structure can be an essential step in understanding their function. With the introduction of
next generation sequencing, it has become possible to study the structure of thousands of
RNAs in a single experiment. A highly successful method to probe RNA structure is
Selective 2’-Hydroxyl Acylation analyzed by Primer Extension (SHAPE), however, this
method is limited by high background rates arising from non-probed molecules and pretermination
in the reverse transcription. In this thesis I describe the development of highthroughput
SHAPE-based approaches to investigate RNA structure based on novel
SHAPE reagents that permit selection of full-length cDNAs. The SHAPE Selection
(SHAPES) method is applied to the foot-and-mouth disease virus (FMDV) plus strand RNA
genome, and the data is used to construct a genome-wide structural map of the virus. I
have used the data to discover stable structures de novo, including previously
characterized structural elements, such as the internal ribosome entry site (IRES), and I
show that three of the novel structures have been conserved through evolution, indicating
that they are functional. The SHAPES method is further applied to the hepatitis C virus
(HCV), where the data is used to refine known and predicted structures. Over the past
years, the interest of studying RNA structure in their native environment has been
increased, and to allow studying RNA structure inside living cells using the SHAPE
Selection approach, I introduce a biotinylated probing reagent. This chemical can cross
cell membranes and reacts with RNA inside the cells, allowing the structural conformations
to be studied in the context of physiological relevant conditions in living cells. The methods
and results presented in this thesis represent important steps forward in studying RNA
structures with high-throughput technologies, and the selection approach could be key to
obtaining high quality sequencing-based probing data in experiments with a high
background.
| Originalsprog | Engelsk |
|---|
| Forlag | Department of Biology, Faculty of Science, University of Copenhagen |
|---|---|
| Antal sider | 133 |
| Status | Udgivet - 2015 |
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