title: Direct visualization of G-quadruplexes in DNA using atomic force microscopy.
abstract: 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 Sgamma3 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.
authors.affiliation: Department of Pharmacology, University of Cambridge, Cambridge CB2 1PD, UK.
authors.forename: Kelly J
authors.initials: KJ
authors.lastname: Neaves
authors.forename: Julian L
authors.initials: JL
authors.lastname: Huppert
authors.forename: Robert M
authors.initials: RM
authors.lastname: Henderson
authors.forename: J Michael
authors.initials: JM
authors.lastname: Edwardson
mesh_terms: D004247:DNA / Q000737:chemistry* / Q000648:ultrastructure; D054856:G-Quadruplexes; D018625:Microscopy, Atomic Force; D012313:RNA / Q000737:chemistry; D014158:Transcription, Genetic
chemical_list: D012313:RNA; D004247:DNA
references.citation: J Biol Chem. 2008 Dec 26;283(52):36743-51
references.pmid: 18986989
references.citation: Nucleic Acids Res. 2008 Nov;36(19):6260-8
references.pmid: 18832370
references.citation: Proc Natl Acad Sci U S A. 1994 May 24;91(11):4950-4
references.pmid: 8197163
references.citation: Trends Genet. 1991 Jul;7(7):230-5
references.pmid: 1887504
references.citation: Bioessays. 2007 Feb;29(2):155-65
references.pmid: 17226803
references.citation: Am J Hum Genet. 1999 Feb;64(2):346-53
references.pmid: 9973271
references.citation: Nucleic Acids Res. 2005 May 24;33(9):2908-16
references.pmid: 15914667
references.citation: EMBO J. 2007 Aug 22;26(16):3815-25
references.pmid: 17660745
references.citation: Nucleic Acids Res. 2007;35(2):406-13
references.pmid: 17169996
references.citation: Nucleic Acids Res. 2009 Apr;37(6):2053-63
references.pmid: 19223329
references.citation: J Med Chem. 2009 May 14;52(9):2863-74
references.pmid: 19385599
references.citation: Mol Biosyst. 2008 Jun;4(6):686-91
references.pmid: 18493667
references.citation: Proc Natl Acad Sci U S A. 2002 Sep 3;99(18):11593-8
references.pmid: 12195017
references.citation: Nucleic Acids Res. 2006;34(19):5402-15
references.pmid: 17012276
references.citation: Cell. 1997 Jul 25;90(2):325-33
references.pmid: 9244306
references.citation: Nucleic Acids Res. 2008 Feb;36(2):477-88
references.pmid: 18045788
references.citation: Curr Opin Chem Biol. 2009 Jun;13(3):345-53
references.pmid: 19515602
references.citation: Chem Soc Rev. 2008 Jul;37(7):1375-84
references.pmid: 18568163
references.citation: Proc Natl Acad Sci U S A. 2007 Jul 31;104(31):12755-60
references.pmid: 17646654
references.citation: Nucleic Acids Res. 2001 Nov 15;29(22):4684-90
references.pmid: 11713318
references.citation: Front Biosci. 2007 May 01;12:4336-51
references.pmid: 17485378
references.citation: J Biol Chem. 2003 Feb 14;278(7):4431-4
references.pmid: 12475983
references.citation: Nucleic Acids Res. 2007;35(22):7429-55
references.pmid: 17913750
references.citation: Biochimie. 2008 Aug;90(8):1149-71
references.pmid: 18355457
references.citation: Genes Dev. 2004 Jul 1;18(13):1618-29
references.pmid: 15231739