Long-Loop G‑Quadruplexes Are Misfolded Population Minorities with Fast Transition Kinetics in Human Telomeric Sequences Deepak Koirala, § Chiran Ghimire, § Christopher Bohrer, # Yuta Sannohe, ‡ Hiroshi Sugiyama,* ,‡,⊥ and Hanbin Mao* ,§ § Department of Chemistry & Biochemistry and # Department of Physics, Kent State University, Kent, Ohio 44242, United States ‡ Department of Chemistry, Graduate School of Science, and ⊥ Institute for Integrated Cell Material Sciences (iCeMS), Kyoto University, Sakyo-ku, Kyoto, Japan * S Supporting Information ABSTRACT: Single-stranded guanine (G)-rich sequences at the 3′ end of human telomeres provide ample opportunities for physiologically relevant structures, such as G-quadruplexes, to form and interconvert. Population equilibrium in this long sequence is expected to be intricate and beyond the resolution of ensemble-average techniques, such as circular dichroism, NMR, or X-ray crystallography. By combining a force-jump method at the single-molecular level and a statistical population deconvolution at the sub-nanometer resolution, we reveal a complex population network with unprecedented transition dynamics in human telomeric sequences that contain four to eight TTAGGG repeats. Our kinetic data firmly establish that G-triplexes are intermediates to G-quadruplexes while long-loop G-quadruplexes are misfolded population minorities whose formation and disassembly are faster than G-triplexes or regular G-quadruplexes. The existence of misfolded DNA supports the emerging view that structural and kinetic complexities of DNA can rival those of RNA or proteins. While G-quadruplexes are the most prevalent species in all the sequences studied, the abundance of a misfolded G-quadruplex in a particular telomeric sequence decreases with an increase in the loop length or the number of long-loops in the structure. These population patterns support the prediction that in the full-length 3′ overhang of human telomeres, G-quadruplexes with shortest TTA loops would be the most dominant species, which justifies the modeling role of regular G-quadruplexes in the investigation of telomeric structures. ■ INTRODUCTION In human cells, telomeres at the end of chromosomes consist of single-stranded 3′ overhang of ∼200 nucleotides with a consensus guanine (G)-rich repeat sequence, 5′-TTAGGG. 1-4 Four such G-rich repeats are known to form a stable DNA secondary structure, G-quadruplex (GQ). 5,6 A G-quadruplex is composed of a stack of G-quartets, each of which is held together by four guanines through Hoogsteen hydrogen bonds and further stabilized by intercalating cations such as K + or Na + . 7,8 Biological investigations suggest that these telomeric DNA secondary structures can regulate the length of telomere either by interfering with telomerase activity or by participating in events such as uncapping of telosomes. 9-11 Since telomere length is closely associated with cellular processes that lead to senescence or cancer, telomeric G-quadruplexes become an attractive target for cancer treatment. 12,13 Despite their simple repeating sequence, human telomeric G- quadruplexes exhibit a stunning structural polymorphism. At least nine conformations of telomeric G-quadruplex have been revealed in different buffers or in DNA templates that contain four G-rich repeats with varying flanking sequences. 14-21 The observation of partially folded structures either as intermediates to G-quadruplexes or as terminally folded species 22-24 added another level of structural complexity. One rationale for this structural polymorphism is that it presents a flexible regulatory mechanism for cellular processes. Particular biological functions may be regulated by prevailing structures in a population equilibrium that is dependent on cellular conditions such as pH or proteins. However, ensemble-average techniques, such as circular dichroism (CD), NMR, or X-ray crystallography, have difficulties to deconvolute individual species, especially those with insignificant population fractions or short lifetimes, formed in the same biological molecule. To resolve a structure in such a population mixture, mutations in the biological molecule are often required to selectively populate the species of interest. 20,25,26 Recently, such a practice surprisingly revealed G-quadruplex conformations that harbor (TTAGGGTTA) n in one of the loops (long-loop GQs) in human telomeric DNA fragments with more than four TTAGGG repeats. 25 This procedure, however, changes population equilibrium and distorts the transition between different species. Due to these difficulties, it is yet to clarify the population equilibrium and the Received: October 3, 2012 Published: January 17, 2013 Article pubs.acs.org/JACS © 2013 American Chemical Society 2235 dx.doi.org/10.1021/ja309668t | J. Am. Chem. Soc. 2013, 135, 2235-2241