Ligands that stabilize the formation of telomeric DNA G-quadruplexes have potential as cancer treatments, because the G-quadruplex structure cannot be extended by telomerase, an enzyme over-expressed in many cancer cells. Understanding the kinetic, thermodynamic and mechanical properties of small-molecule binding to these structures is therefore important, but classical ensemble assays are unable to measure these simultaneously. Here, we have used a laser tweezers method to investigate such interactions. With a force jump approach, we observe that pyridostatin promotes the folding of telomeric G-quadruplexes. The increased mechanical stability of pyridostatin-bound G-quadruplex permits the determination of a dissociation constant Kd of 490 ± 80 nM. The free-energy change of binding obtained from a Hess-like process provides an identical Kd for pyridostatin and a Kd of 42 ± 3 μM for a weaker ligand RR110. We anticipate that this single-molecule platform can provide detailed insights into the mechanical, kinetic and thermodynamic properties of liganded bio-macromolecules, which have biological relevance.
We have investigated new folding pathways of human telomeric type-1 and type-2 G-quadruplex conformations via intermediate hairpin and triplex structures. The stabilization energies calculated by ab initio methods evidenced the formation of a hairpin structure with Hoogsteen GG base pairs. Further calculations revealed that the G-triplet is more stable than the hairpin conformation and equally stable when compared to the G-tetrad. This indicated the possibility of a triplex intermediate. The overall folding is facilitated by K(+) association in each step, as it decreases the electrostatic repulsion. The K(+) binding site was identified by molecular dynamics simulations. We then focused on the syn/anti arrangement and found that the anti conformation of deoxyguanosine is more stable than the syn conformation, which indicated that folding would increase the number of anti conformations. The K(+) binding to a hairpin near the second lateral TTA loop was found to be preferable, considering entropic effects. Stacking of G-tetrads with the same conformation (anti/anti or syn/syn) is more stable than mixed stacking (anti/syn and vice versa). These results suggest the formation of type-1 and type-2 G-quadruplex structures with the possibility of hairpin and triplex intermediates.
We herein report the real-time observation of G-quadruplex formation by monitoring the G-quadruplex-induced global change of two duplexes incorporated in a DNA nanoscaffold. The introduced G-rich strands formed an interstrand (3 + 1) G-quadruplex structure in the presence of K(+), and the formed four-stranded structure was disrupted by removal of K(+). These conformational changes were visualized in a nanoscaffold in real-time with fast-scanning atomic force microscopy.
Intramolecular folding in three tandem guanine repeats of human telomeric DNA has been investigated using optical-tweezers, MD simulation and circular dichroism. A mechanically and thermodynamically stable species in this sequence shows a structure consistent with a triplex conformation. A similar species has also been observed to coexist with a G-quadruplex in a DNA sequence with four tandem guanine repeats.
In addition to the Watson-Crick double helix, secondary DNA structures are thought to play important roles in a variety of biological processes. One important example is the G-quadruplex structure that is formed at the chromosome ends, which inhibits telomerase activity by blocking its access to telomeres. G-quadruplex structures represent a new class of molecular targets for DNA-interactive compounds that may be useful to target telomeres. Here, we reported the first example of enantioselective recognition of quadruplex DNA by a chiral cyclic helicene. We propose a new ligand-binding cleft between two telomeric human G-quadruplexes linked by a TTA linker. We found that the cyclic helicene M1 exhibited potent inhibitory activity against telomerase.
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.
Human telomeres have guanine-rich sequences at the end of chromosomes. 1 They play an important role in genome stability and cell growth by protecting chromosome ends. 2 The human telomere DNA, ∼5-8 kbp long, consists of tandem repeats of the sequence TTAGGG, with a single-stranded 3′ overhang of 100-200 nt. 3 The 3′ overhang strand is proposed to encroach into the double-stranded region of the telomeric tract. This structure, called a T-loop, is thought to participate in the protection of the chromosome end in the case of mammals. 4 In normal cells, each cell division results in a 50-200 bp loss of the telomere, which induces senescence and finally apoptosis. 5 In contrast, cancer cells maintain the telomere length to achieve immortality. Reverse transcriptase, called telomerase, is activated in 80-85% of cancer cells and extends the telomeric sequence. 6 It is known that human telomere sequences can fold in a variety of ways to form G-quadruplexes in vitro, and small molecules stabilizing the G-quadruplex, such as telomestatin, effectively inhibit telomerase activity. 7 Therefore, human telomeric Gquadruplex is a potential target for tumor chemotherapy. Recently, three groups including our group determined the topology of the human telomeric G-quadruplex in K + solution, having two lateral loops and one external loop. 8 This structure contains the (3 + 1) hybrid G-quadruplex topology, in which three strands are oriented in one direction and one is in the opposite direction.Although T-loop structure and G-quadruplex structure are proposed to form in the same telomere region, only we and Patel speculate on the possible relationship between the two structures. 8a,9 Therefore, critical evidence is demanded. Patel and colleagues suggested that the interstrand G-quadruplex formation in a sodium ion solution may participate in T-loop formation. Here we have examined the interstrand G-quadruplex structure in the more physiological potassium ion solution. Then we examined a long lariat structure having the same G-quadruplex formation as a model of the T-loop structure.We first examined the formation of an interstrand G-quadruplex with d(GGGTTAGGGTTAGGGT) (ODN1) and d(TAGGGT) (ODN2). Figure 1 shows the CD spectra of ODN1 and ODN2 in 100 mM K + ion solution with mole fraction variation. The mixture of ODN1 and ODN2 showed a strong positive Cotton effect at 290 nm with negative signals near 255 and 235 nm, which are characteristic of the hybrid G-quadruplex structure. 8a In clear contrast, ODN2 alone showed the CD spectra of an essentially unstructured single strand. Figure 1b shows the Job plot of the CD cotton effect monitored at 290 nm. A clear inflection point around 50% indicates a 1:1 stoichiometry for the formation of interstrand G-quadruplex by ODN1 and ODN2. FRET experiments using FAM-attached ODN1 and TAMRA-attached ODN2 further confirmed the formation of the interstrand G-quadruplex formation ( Figure 1S).In the G-quadruplex structure, G residues adopt unique arrangements of syn/anti conformations around indivi...
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