Fluorescent probe for visualizing guanine-quadruplex DNA by fluorescence lifetime imaging microscopy

Abstract. The importance of guanine-quadruplex (G4) is not only in protecting the ends of chromosomes for human telomeres but also in regulating gene expression for several gene promoters. However, the existence of G4 structures in living cells is still in debate. A fluorescent probe, 3,6-bis(1-methyl-2-vinylpyridinium) carbazole diiodide (o-BMVC), for differentiating G4 structures from duplexes is characterized. o-BMVC has a large contrast in fluorescence decay time, binding affinity, and fluorescent intensity between G4 structures and duplexes, which makes it a good candidate for probing G4 DNA structures. The fluorescence decay time of o-BMVC upon interaction with G4 structures of telomeric G-rich sequences is ∼2.8  ns and that of interaction with the duplex structure of a calf thymus is ∼1.2  ns. By analyzing its fluorescence decay time and histogram, we were able to detect one G4 out of 1000 duplexes in vitro. Furthermore, by using fluorescence lifetime imaging microscopy, we demonstrated an innovative methodology for visualizing the localization of G4 structures as well as mapping the localization of different G4 structures in living cells.

[1]  I Berger,et al.  In vitro generated antibodies specific for telomeric guanine-quadruplex DNA react with Stylonychia lemnae macronuclei , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[2]  S. Balasubramanian,et al.  Single-molecule conformational analysis of G-quadruplex formation in the promoter DNA duplex of the proto-oncogene c-kit. , 2007, Journal of the American Chemical Society.

[3]  Giridharan Gokulrangan,et al.  Orientational dynamics and dye-DNA interactions in a dye-labeled DNA aptamer. , 2005, Biophysical journal.

[4]  S. Neidle,et al.  Highly prevalent putative quadruplex sequence motifs in human DNA , 2005, Nucleic acids research.

[5]  Zi-Fu Wang,et al.  A Novel Method for Screening G-quadruplex Stabilizers to Human Telomeres , 2011 .

[6]  N. Maizels,et al.  Gene function correlates with potential for G4 DNA formation in the human genome , 2006, Nucleic acids research.

[7]  J. Chaires.  Analysis and interpretation of ligand-DNA binding isotherms. , 2001, Methods in enzymology.

[8]  A. Phan,et al.  Structure of the human telomere in K+ solution: a stable basket-type G-quadruplex with only two G-tetrad layers. , 2009, Journal of the American Chemical Society.

[9]  Edward S Yeung,et al.  Dynamics of single biomolecules in free solution. , 2004, Annual review of physical chemistry.

[10]  A. Phan,et al.  Stacking of G-quadruplexes: NMR structure of a G-rich oligonucleotide with potential anti-HIV and anticancer activity , 2011, Nucleic acids research.

[11]  Wen-Hwa Lee,et al.  A cis-element with mixed G-quadruplex structure of NPGPx promoter is essential for nucleolin-mediated transactivation on non-targeting siRNA stress , 2012, Nucleic acids research.

[12]  D. Davies,et al.  Helix formation by guanylic acid. , 1962, Proceedings of the National Academy of Sciences of the United States of America.

[13]  W D Wilson,et al.  Effects of compound structure on carbazole dication-DNA complexes: tests of the minor-groove complex models. , 2000, Biochemistry.

[14]  Daniela Rhodes,et al.  G-quadruplex structures: in vivo evidence and function. , 2009, Trends in cell biology.

[15]  D. Bearss,et al.  Direct evidence for a G-quadruplex in a promoter region and its targeting with a small molecule to repress c-MYC transcription , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[16]  Ľuboš Bauer,et al.  Structural features of intra- and intermolecular G-quadruplexes derived from telomeric repeats. , 2010, Biochemistry.

[17]  P. Lou,et al.  Verification of antiparallel G-quadruplex structure in human telomeres by using two-photon excitation fluorescence lifetime imaging microscopy of the 3,6-Bis(1-methyl-4-vinylpyridinium)carbazole diiodide molecule. , 2006, Analytical chemistry.

[18]  Cheng-Chung Chang,et al.  A fluorescent carbazole derivative: high sensitivity for quadruplex DNA. , 2003, Analytical chemistry.

[19]  S. Balasubramanian,et al.  Quantitative visualization of DNA G-quadruplex structures in human cells. , 2013, Nature chemistry.

[20]  Roger A. Jones,et al.  Human telomeric sequence forms a hybrid-type intramolecular G-quadruplex structure with mixed parallel/antiparallel strands in potassium solution , 2006, Nucleic acids research.

[21]  Dinshaw J. Patel,et al.  Structure of the human telomere in K+ solution: an intramolecular (3 + 1) G-quadruplex scaffold. , 2006, Journal of the American Chemical Society.

[22]  P. Lou,et al.  Detection of quadruplex DNA structures in human telomeres by a fluorescent carbazole derivative. , 2004, Analytical chemistry.

[23]  A. Phan,et al.  Propeller-type parallel-stranded G-quadruplexes in the human c-myc promoter. , 2004, Journal of the American Chemical Society.

[24]  J. Correia,et al.  Not so crystal clear: the structure of the human telomere G-quadruplex in solution differs from that present in a crystal , 2005, Nucleic acids research.

[25]  A. Phan,et al.  Giardia telomeric sequence d(TAGGG)4 forms two intramolecular G-quadruplexes in K+ solution: effect of loop length and sequence on the folding topology. , 2009, Journal of the American Chemical Society.

[26]  Cheng-Chung Chang,et al.  Investigation of spectral conversion of d(TTAGGG)4 and d(TTAGGG)13 upon potassium titration by a G-quadruplex recognizer BMVC molecule , 2007, Nucleic acids research.

[27]  Shankar Balasubramanian,et al.  Prevalence of quadruplexes in the human genome , 2005, Nucleic acids research.

[28]  W. Gilbert,et al.  Formation of parallel four-stranded complexes by guanine-rich motifs in DNA and its implications for meiosis , 1988, Nature.