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Preparation of Aminoacylated t-RNAs and Tools for Investigating RNA-Folding

English title Preparation of Aminoacylated t-RNAs and Tools for Investigating RNA-Folding
Applicant Pitsch Stefan
Number 105460
Funding scheme Project funding (Div. I-III)
Research institution Institut des sciences et ingénierie chimiques EPFL - SB - ISIC
Institution of higher education EPF Lausanne - EPFL
Main discipline Organic Chemistry
Start/End 01.10.2004 - 31.03.2006
Approved amount 162'149.00
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Keywords (7)

RNA; AMINOACYLATED T-RNAS; RNA-FOLDING; RNA-MODIFICATIONS; SUPPRESSOR T-RNAS; RNA-STRUCTURE;

Lay Summary (English)

Lead
Lay summary
Aminoacylated t-RNAs:
We have developed new methods for the chemicalsynthesis of aminoacylated t-RNAs and started to prepare the buildingblocks required for the introduction of naturally occurring modifiednucleosides. In the context of the proposed research, we are planning tosynthesize more of these modified building blocks and to optimize themethods for their introduction into t-RNAs. These compounds are veryimportant tools for studying the precise biological role of theseubiquitous modifications in the context of structure, dynamics, foldingpathways, editing, interaction with aminoacyl t-RNA synthetases,translation factors and m-RNA interactions. In the context of the latteraspect (codon anticodon interaction), we are planning to prepare a newmodel system which should allow to investigate the influence of modifiedanticodon-loop nucleotides on the strength and fidelity of thecodon/anticodon interaction in a detailed fashion. Additionally, it isplanned to investigate the influence of anticodon modifications on theefficiency of incorporation of unnatural amino acids into proteins by thesuppressor t-RNA approach.

RNA-Folding:
The known pathways ofRNA folding are characterized by a great heterogenity both on thestructural level (missfolded, kinetically trapped and coexistingconformers), and on the time scale of the different folding events(microseconds to hours). In the context of this project, we have developedsynthetic tools which allow the time resolved analysis of RNA folding byNMR- and fluorescence spectroscopy. For the characterization of fastprocesses (t < 0.5s), we will carry out exchange-sensitive NMR-experiments(e.g. CPMG-type and inversion-recovery) with bistable, selectively15N-labeled RNA-sequences. For studying slow processes (t > 10s) we willcreate non-equilibrium states by introducing caged nucleosides intobistable RNA-sequences; upon photolysis, folding could be followed byrepeated 1D-NMR-experiments. A combination of selective 15N-labeling andselective blocking of structures could even open the possiblity to studythe folding of biologically relevant RNAs. In order to study foldingevents occurring between 0.5s < t _____ we are developing tools forfluorescence spectroscopy. A variety of different new conjugation sitesallows the introduction of fluorophores/quenchers at almost any positionwithin RNA-sequences. By photolysis of caged nucleosides, folding ratescould be determined by steady-state FRET; by (single-molecule) FCSexperiments, folding rates could be determined from fluctuations aroundthe equilibrium.
Direct link to Lay Summary Last update: 21.02.2013

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Associated projects

Number Title Start Funding scheme
68090 Preparation of Aminoacylated t-RNAs and Tools for Investigating RNA-Folding 01.10.2002 Project funding (Div. I-III)

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