Title:

Investigations on Nucleic Acids Structures by AFM

Description:  We would like to establish new methods to characterize secondary structure of nucleic acids-protein-complexes and sense-antisense RNA complexes by Scanning Force Microscopy (or other SXM techniques).
Author:Bonin, Ewert, Oberstraß, Oesterschulze, Nellen, Kassing
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ISBN: 3100870301   ISBN: 3100870301   ISBN: 3100870301   ISBN: 3100870301 
 
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First Results towards
Investigations on Nucleic Acids Structures by AFM

M. Bonin1, K. Ewert2, J. Oberstraß1, E. Oesterschulze2, W. Nellen1, R. Kassing2

1 Department of Genetics, 2 Institute of Technical Physics
University of Kassel, Heinrich-Plett-Straße 40, 34125 Kassel


Our Aim

We would like to establish new methods to characterize secondary structure of nucleic acids-protein-complexes and sense-antisense RNA complexes by Scanning Force Microscopy (or other SXM techniques). The Technical Physics group involved in cantilever development and the Genetics group interested in antisense mechanisms and signaltransduction started working together in the promising field of analysing structural features with the AFM and complementing these results with biochemical and molecular biology data. A further goal is to probe inter- and intramolecular hybridization of RNA molecules by measuring attractive forces.

We expect this to provide a deeper insight into structural features of biomolecules, their alterations and maybe even the kinetics of structeral changes or interactions.

First Tests

As a first step we investigated well known objects like circular and linearized DNA, then we went a step further trying to visualize protein-nucleic acid interactions. In order to find out whether single and double stranded RNA could be distinguished, we decorated double stranded RNA with antibodies.





   Circular and Linearized DNA   

Contact Mode Imaging
of Circular DNA

Tapping Mode Imaging
of Linearized DNA

Gelelectrophoresis
Contact Mode Imaging of Circular DNATapping Mode Imaging of Linearized DNAGelelectrophoresis
Quiagen® purified plasmid DNA
(pAS2, 8.5 kb = 2.8 µm on mica)
Plasmid pGEM3Z digested by NdeI
(2743 bp or 910 nm)
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   Visualization of protein-nucleic acid interactions   

DNA with T7 RNA Polymerase
 
T7 RNA Polymerase bound to linearized pGEM3Z plamid DNA (3D)T7 RNA Polymerase bound to linearized pGEM3Z plamid DNA (2D)Gelelectrophoretic shift of a 280 bp fragment of pGEM3Z
T7 RNA polymerase bound to pGEM3Z plasmid DNA linearized with NdeI. The transcription was initiated by addition of rGTP. The measured position of the molecule is in good agreement with the expected binding site. Note the bending of DNA at the polymerase binding site, presumably indicating the start of transcription.
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   Double stranded and single stranded RNA   

DNA template and ssRNA

700 nts ssRNA and 260 nts dsRNA

260 nts dsRNA
3.4 kb DNA template (*) and
700 nts single stranded RNA
Zoom on 700 nts single stranded RNA and
260 nts double stranded RNA (above).
260 nts double stranded RNA

Complementary RNAs (sense and antisense) of 260 nts were transcribed in vitro and hybridized. A 700 nts single standed RNA was added for size comparison. So we had two different DNA templates, ssRNA and dsRNA in our solution, all of different lenghts. SsRNA and dsRNA can so far not be distinguished by their width.
 
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   Sense/Antisense Structures and Mobility of Different Conformations   

Predicted/Calculated Secondary Structures

Gelelectrophoresis

RNA Secondary Structure (sense)

RNA Secondary Structure (antisense)

Gelelectrophoresis of the RNA species
Secondary structures of sense (left) and antisense (right) RNA of a PSV-A gene fragment (Dictyostelium discoideum) were predicted by M-fold (M. Zuker et al., www-homepage: http://www.ibc.wustl.edu/~zuker/seqanal).

A future aim is to distinguish these structures by AFM.

Native Gelelectrophoresis of sense, antisense ssRNA and the dsRNA hybrid of these two (the same was used for the AFM figures above). The different mobilities of sense and antisense are due to differences in secondary structure (see figures on the left).
 
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   Double stranded RNA with J2 Antibody   

Double stranded RNA with J2 Antibody
 
dsRNA and J2 Antibody overviewdsRNA and JS2 Antibody detaildsRNA Gelretardation with J2 Antibody
Double stranded-RNA-specific monoclonal antibody J2 binds to 260 bp ds RNA (RNA concentration: 2µg/ml, antibody con.:10-7 molar). We found one or two mAb molecules bound to the RNA.
 
Gelretardation of dsRNA and J2 antibody. Most likely, the complex is too large to be separated in the native gel. Degradation of the RNA can be excluded since a control ssRNA, which is not bound to the antibody, remains intact.
 
 
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Acknowledgements

We thank A. Schaper and T. Jovin, MPI for Biophysical Chemistry, Göttingen for valuable discussions and help to start with DNA preparations for AFM; the Companies Park Scientific (last two images) and DI (rest of the AFM images) for the opportunity of testing their AFMs; N. Lukacs, Institute for Plant Biology, Research Center of the Hungarian Academy of Science, for providing the anti-dsRNA antibodies.


  
Entschlüsselt: Mein Genom, mein Leben
Siehe auch:
Die Doppelhelix: Ein persönlicher Bericht über …
Meine Gene - mein Leben: Auf dem Weg zur personalisierten Medizin
Wittgensteins "Tractatus": Ein Kommentar
Sonstige Artikel:
The Subjection of Women (Dover Thrift Editions)
 
   
 
     

This web site is a part of the project StudyPaper.com.
We are grateful to Bonin, Ewert, Oberstraß, Oesterschulze, Nellen, Kassing for contributing this article.

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