Fluorescence of Unmodified Oligonucleotides: A Tool to Probe G-Quadruplex DNA Structure Miguel Angel Mendez, Veronika A. Szalai Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, Baltimore, MD 21250 Received 20 February 2009; revised 12 June 2009; accepted 13 June 2009 Published online 22 June 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bip.21268 This article was originally published online as an accepted preprint. The ‘‘Published Online’’date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com INTRODUCTION M olecules of DNA rich in guanine present signifi- cantly higher fluorescence than homologous chains without consecutive guanines (G). 1 To demonstrate the usefulness of this electronic property, we have studied several sequences ca- pable of forming a particular superstructure known as a gua- nine-quadruplex (G-quadruplex, Scheme 1). G-quadru- plexes, four stranded structures mediated by hydrogen- bonded Gs (Scheme 1), have been implicated in gene control and other biological processes. 2,3 They are used as drugs for treating cancer 4 and as sources for new materials. 5–7 Use of fluorescently modified DNA, either labeled with extrinsic flu- orophores 8 or intrinsic fluorescence enhanced by metallic particles, 9 is a common means to detect and monitor the structure, hybridization, and binding properties of DNA. These technologies have enabled the creation of broadly used tools such as molecular beacons, DNA chips, and aptamer- based fluorescence sensors. 9–14 Fluorescence resonance elec- tron transfer (FRET) in molecular beacons has been exploited in the detection of folded and unfolded states of ol- igonucleotides. 15–17 These changes have been coupled with sensing capabilities to detect a variety of analytes. FRET also has been used to detect movements of nanomachines. 5 In some instances, however, addition of a fluorescent tag—often Fluorescence of Unmodified Oligonucleotides: A Tool to Probe G-Quadruplex DNA Structure Additional Supporting Information may be found in the online version of this article. Correspondence to: V. A. Szalai; e-mail: vszalai@umbc.edu ABSTRACT: Fluorescence of unmodified oligonucleotides has not been exploited for guanine-quadruplex (G-quadruplex) characterization. We observe that G-rich sequences fluoresce more strongly than duplex or single-stranded DNA but much more weakly than fluorophores like fluorescein. This increase in the intrinsic fluorescence is not due to an increase in absorption at the excitation wavelength but rather to a change in the quantum yield. We show that unlabeled oligonucleotides that form G-quadruplexes can be differentiated on the basis of their emission spectra from similar sequences that do not contain consecutive guanines. Intermolecular quadruplexes formed by the oligonucleotides 5 0 -T 4 G n T 4 -3 0 (n 5 4–10) display a nonlinear, but continuous, increase in emission intensity as the G content increases. The sequence 5 0 -GGGT-3 0 , which has been proposed to form a monomeric quadruplex and an interlocked quadruplex (Krishnan-Ghosh et al. J Am Chem Soc 2004, 126, 11009), was compared with the similar sequence 5 0 -TGGG-3 0 , the structure of which has not been characterized. Both the maximum emission intensity and the spectral shape differ for these oligonucleotides as a function of sample preparation, indicating that different types of quadruplexes form for both sequences. Our work is the first to demonstrate that the suprastructure of G-rich sequences can be probed using fluorescence signatures of unmodified oligonucleotides. # 2009 Wiley Periodicals, Inc. Biopolymers 91: 841–850, 2009. Keywords: guanine; quadruplex; fluorescence; exciplex V V C 2009 Wiley Periodicals, Inc. Biopolymers Volume 91 / Number 10 841