DNA with tandem repeats of guanines folds into G-quadruplexes made of a stack of G-quartets. In vitro, G-quadruplex formation inhibits telomere extension, and POT1 binding to the singlestranded telomeric DNA enhances telomerase activity by disrupting the G-quadruplex structure, highlighting the potential importance of the G-quadruplex structure in regulating telomere length in vivo. We have used single-molecule spectroscopy to probe the dynamics of human telomeric DNA. Three conformations were observed in potassium solution, one unfolded and two folded, and each conformation could be further divided into two species, long-lived and short-lived, based on lifetimes of minutes vs. seconds. Vesicle encapsulation studies suggest that the total of six states detected here is intrinsic to the DNA. Folding was severely hindered by replacing a single guanine, showing only the shortlived species. The long-lived folded states are dominant in physiologically relevant conditions and probably correspond to the parallel and antiparallel G-quadruplexes seen in high-resolution structural studies. Although rare under these conditions, the shortlived species determine the overall dynamics because they bridge the different long-lived species. We propose that these previously unobserved transient states represent the early and late intermediates toward the formation of stable G-quadruplexes. The major compaction occurs between the early and late intermediates, and it is possible that local rearrangements are sufficient in locking the late intermediates into the stably folded forms. The extremely diverse conformations of the human telomeric DNA may have mechanistic implications for the proteins and drugs that recognize G-rich sequences.FRET ͉ G-quadruplex ͉ single molecule ͉ telomere ͉ vesicle encapsulation E ukaryotic chromosomes have a novel nucleoprotein structure at both ends called a telomere. Telomeres are indispensable for protection against genome degradation and have implications in cellular aging and cancer (1-3). In many organisms, a telomeric DNA is composed of tandem repeats of short DNA sequences. In vitro, this guanine-rich region forms G-quadruplex (4), which blocks the binding of telomerase (5). The potential importance of Gquadruplex structure was further implied by the recent demonstration in vitro that the human POT1, which binds to the singlestranded form of human telomeric DNA (6), facilitates the telomerase activity by disrupting G-quadruplex (7). Human disease helicases, BLM and WRN, also were shown to disrupt Gquadruplexes in vitro (8,9). Because extension of the telomere is critical for generation and growth of cancer cells, the human telomeric G-quadruplex is considered a target for anticancer agents. Structure-based design of chemotherapeutic drugs would require an understanding of a range of conformations that can be adopted by the G-rich sequences, and indeed it was shown to be possible to design molecules that are specific to different types of G-quadruplex structures (10). Although there is no demonstr...

Guanine-rich DNA sequences can form G-quadruplexes. These four-stranded structures are known to form in several genomic regions and to influence certain biological activities. Sometimes, the instability of G-quadruplexes causes the abnormal biological processes. Mutation is a culprit for the destabilization of G-quadruplexes, but the details of mutated G-quadruplexes are poorly understood. In this article, we investigated the conformational dynamics of single-base mutated human telomeric G-quadruplexes in the presence of K+ with single-molecule FRET spectroscopy. We observed that the replacement of single guanine by thymine in a G-track induces various folded structures, i.e. structural polymorphism. Moreover, direct observation of their dynamics revealed that a single-base mutation causes fast unfolding of folded states under physiological conditions. Furthermore, we found that the degree of destabilization varies according to mutation positions. When the central guanine of a G-track is replaced, the G-quadruplexes unfold quickly at any K+ concentrations and temperature. Meanwhile, outer-quartet mutated G-quadruplexes have heterogeneous dynamics at intermediate K+ concentrations and longstanding folded states at high K+ concentrations. Several factors such as base-stacking interaction and K+ coordination are responsible for the different dynamics according to the mutation position.