The three-dimensional context of a double helix determines the fluorescence of the internucleoside-tethered pair of fluorophores.

We report a general phenomenon of the formation of either a fluorescent or an entirely quenched oligodeoxynucleotide (ODN) duplex system by hybridizing pairs of complementary ODNs with identical chemical composition. The ODNs carried internucleoside tether-linked cyanines, where the cyanines were chosen to form a Förster's resonance energy transfer (FRET) donor-acceptor pair. The fluorescent and quenched ODN duplex systems differed only in that the cyanines linked to the respective ODNs were linked either closer to the 5'- or 3'-ends of the molecule. In either case, however, the dyes were separated by an identical number (7 or 8) of base pairs. Characterization by molecular modeling and energy minimization using a conformational search algorithm in a molecular operating environment (MOE) revealed that linking of the dyes closer to the 5'-ends resulted in their reciprocal orientation across the major groove which allowed a closely interacting dye pair to be formed. This overlap between the donor and acceptor dye molecules resulted in changes in absorbance spectra consistent with the formation of H-aggregates. Conversely, dyes linked closer to 3'-ends exhibited emissive FRET and formed a pair of dyes that interacted with the DNA helix only weakly. Induced CD spectra analysis suggested that interaction with the double helix was weaker than in the case of the closely interacting cyanine dye pair. Linking the dyes such that the base pair separation was 10 or 0 favored energy transfer with subsequent acceptor emission. Our results suggest that when interpreting FRET measurements from nucleic acids, the use of a "spectroscopic ruler" principle which takes into account the 3D helical context of the double helix will allow more accurate interpretation of fluorescence emission.

[1]  P. Sigler,et al.  Structure of NF-κB p50 homodimer bound to a κB site , 1998, Nature.

[2]  Benedetta Mennucci,et al.  Fretting about FRET: failure of the ideal dipole approximation. , 2009, Biophysical journal.

[3]  Andrea E. Holmes,et al.  3,3′-Diethylthiatricarbocyanine Iodide: A Highly Sensitive Chiroptical Reporter of DNA Helicity and Sequence , 2011, International journal of molecular sciences.

[4]  Fred Russell Kramer,et al.  Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes. , 2002, Nucleic acids research.

[5]  A. Bogdanov,et al.  Hairpin-like fluorescent probe for imaging of NF-κB transcription factor activity. , 2011, Bioconjugate chemistry.

[6]  D. Lilley,et al.  Orientation dependence in fluorescent energy transfer between Cy3 and Cy5 terminally attached to double-stranded nucleic acids , 2008, Proceedings of the National Academy of Sciences.

[7]  S. Burley.  X-ray crystallographic studies of eukaryotic transcription initiation factors. , 1996, Philosophical transactions of the Royal Society of London. Series B, Biological sciences.

[8]  Surong Zhang,et al.  Fluorescence resonance energy transfer in near-infrared fluorescent oligonucleotide probes for detecting protein–DNA interactions , 2008, Proceedings of the National Academy of Sciences.

[9]  Sabine Müller,et al.  Accurate distance determination of nucleic acids via Förster resonance energy transfer: implications of dye linker length and rigidity. , 2011, Journal of the American Chemical Society.

[10]  D. E. Wolf,et al.  Detection of nucleic acid hybridization by nonradiative fluorescence resonance energy transfer. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[11]  A M Gewirtz,et al.  Real time detection of DNA.RNA hybridization in living cells. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[12]  J Christopher Love,et al.  Cell-surface sensors for real-time probing of cellular environments. , 2011, Nature nanotechnology.

[13]  D. Lilley,et al.  The structure of cyanine 5 terminally attached to double-stranded DNA: implications for FRET studies. , 2008, Biochemistry.

[14]  J. Lakowicz.  Principles of fluorescence spectroscopy , 1983 .

[15]  G. Ghosh,et al.  Mechanism of κB DNA binding by Rel/NF-κB dimers* , 2000, The Journal of Biological Chemistry.

[16]  B. Armitage.  Cyanine Dye–DNA Interactions: Intercalation, Groove Binding, and Aggregation , 2005 .

[17]  S. Burley,et al.  2.1 Å resolution refined structure of a TATA box-binding protein (TBP) , 1994, Nature Structural Biology.

[18]  P. Zamecnik,et al.  A Novel Thymidine Phosphoramidite Synthon for Incorporation of Internucleoside Phosphate Linkers During Automated Oligodeoxynucleotide Synthesis , 2008, Nucleosides, nucleotides & nucleic acids.

[19]  D. Lilley,et al.  The structure of sulfoindocarbocyanine 3 terminally attached to dsDNA via a long, flexible tether. , 2012, Biophysical journal.

[20]  Surong Zhang,et al.  Sensing of transcription factor binding via cyanine dye pair fluorescence lifetime changes. , 2012, Molecular bioSystems.

[21]  L. Stryer,et al.  Energy transfer: a spectroscopic ruler. , 1967, Proceedings of the National Academy of Sciences of the United States of America.

[22]  Gang Bao,et al.  Dual FRET molecular beacons for mRNA detection in living cells. , 2004, Nucleic acids research.

[23]  Sanjay Tyagi,et al.  Molecular Beacons: Probes that Fluoresce upon Hybridization , 1996, Nature Biotechnology.

[24]  Paul Labute,et al.  LowModeMD - Implicit Low-Mode Velocity Filtering Applied to Conformational Search of Macrocycles and Protein Loops , 2010, J. Chem. Inf. Model..

[25]  Ralph Weissleder,et al.  Synthesis and properties of fluorescent NF-kappa B-recognizing hairpin oligodeoxyribonucleotide decoys. , 2004, Bioconjugate chemistry.

[26]  A. Meller,et al.  The Effect of dye-dye interactions on the spatial resolution of single-molecule FRET measurements in nucleic acids. , 2010, Biophysical journal.

[27]  D. Lilley,et al.  Orientation of cyanine fluorophores terminally attached to DNA via long, flexible tethers. , 2011, Biophysical journal.

[28]  R. Knox.  Förster's resonance excitation transfer theory: not just a formula. , 2012, Journal of biomedical optics.

[29]  Surong Zhang,et al.  Near-infrared fluorescent oligodeoxyribonucleotide reporters for sensing NF-kappaB DNA interactions in vitro. , 2008, Oligonucleotides.