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Mathematisch-Naturwissenschaftliche Fakultät - Jahrgang 2019

 

Titel Site-Directed Spin Labeling of Large Riboswitches Using Click Chemistry
Autor Mark Kerzhner
Publikationsform Dissertation
Abstract Due to the conformational variability of riboswitches, their structural analysis is of a great importance. The most frequently used techniques to investigate dynamic processes within the riboswitches are NMR and FRET. EPR spectroscopy in combination with site-directed spin labeling has also been applied for elucidation of structural dynamics of riboswitches, but such studies are not very common. One of the underlying reasons for this is an absence of efficient and inexpensive spin-labeling approaches. The existing spin-labeling methods are quite tedious and time consuming. In addition, the majority of the known riboswitches exceed the size of synthetically accessible RNA. The currently known spinlabeling approaches for long RNAs have low yields and are therefore less attractive. Thus, alternative, more efficient methods for site-directed spin labeling of long riboswitches are required.
Using copper(I)-catalyzed azide-alkyne cycloaddition (click chemistry) and the spin label dŲ, two efficient spin labeling approaches for RNA were established.
In the first approach, the click reaction was performed on CPG solid support, on which protected RNA remained after solid phase synthesis. The performance of the dŲ label was benchmarked on a self-complementary RNA duplex with Electron-Electron Double Resonance (PELDOR). In addition, this approach was compared with another established method that employs the rigid spin label Çm for RNA labeling. The influence of both labels on RNA was analyzed using CD spectroscopy, thermal denaturation measurements, cw- EPR as well as PELDOR. The results obtained by PELDOR measurements demonstrated that both labels dŲ and Çm provide a set of orientation-selective time traces, yielding information about the inter-label distances and the relative orientations of the labels.
In the second approach, the click reaction was carried out in solution using fully deprotected RNA. In combination with enzymatic ligation, this method provides a highly efficient labeling of complex and long RNAs. Using this approach, aptamer domains of 34 nt long preQ1 riboswitch from Fusobacterium nucleatum as well as of 81 nt long TPP riboswitch from E. coli were spin labeled. Furthermore, the results presented in this work demonstrated that the conformational exchange within the aptamer domain of preQ1 riboswitch can be monitored using cw-EPR and PELDOR.
In addition, an efficient assembling strategy of the entire TPP riboswitch comprising only one ligation step was elaborated and implemented in this work. This strategy can be used for site-specific spin labeling of the stems P1, P2 and P3 in future studies of TPP riboswitch.
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© Universitäts- und Landesbibliothek Bonn | Veröffentlicht: 14.02.2019