Nucleic Acids Research Advance Access originally published online on August 20, 2009
Nucleic Acids Research 2009 37(18):6269-6275; doi:10.1093/nar/gkp679
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Nucleic Acids Research, 2009, Vol. 37, No. 18 6269-6275
© The Author 2009. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses?by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Structural Biology |
Direct visualization of G-quadruplexes in DNA using atomic force microscopy
1Department of Pharmacology, University of Cambridge, Cambridge CB2 1PD and 2Physics of Medicine, Cavendish Laboratory, University of Cambridge, Cambridge CB3 0HE, UK
*To whom correspondence should be addressed. Tel: +44 1223 334014; Fax: +44 1223 334100; Email: jme1000{at}cam.ac.uk
Received June 4, 2009. Revised July 14, 2009. Accepted August 1, 2009.
The formation of G-quadruplexes in G-rich regions of DNA is believed to affect DNA transcription and replication. However, it is currently unclear how this formation occurs in the presence of a complementary strand. We have used atomic force microscopy (AFM) to image stable RNA/DNA hybrid loops generated by transcription of the plasmid pPH600, which contains a 604-bp fragment of the murine immunoglobulin S
3 switch region. We show that the non-RNA-containing portion folds into G-quadruplexes, consistent with computational predictions. We also show that hybrid formation prevents further transcription from occurring, implying a regulatory role. After in vitro transcription, almost all (93%) of the plasmids had an asymmetric loop, a large asymmetric blob or a spur-like projection at the appropriate position on the DNA contour. The loops disappeared following treatment of the transcribed plasmid with RNase H, which removes mRNA hybridized with the template strand. Replacement of K+ in the transcription buffer with either Na+ or Li+ caused a reduction in the percentage of plasmids containing loops, blobs or spurs, consistent with the known effects of monovalent cations on G-quadruplex stability. The minimal sample preparation required for AFM imaging has permitted direct observation of the structural changes resulting from G-quadruplex formation.