Direct and Single‐Molecule Visualization of the Solution‐State Structures of G‐Hairpin and G‐Triplex Intermediates

Rajendran, Arivazhagan; Endo, Masayuki; Hidaka, Kumi; Sugiyama, Hiroshi

doi:10.1002/ange.201308903

Cited by 46 publications

(48 citation statements)

References 39 publications

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“…Previously, we identified four different conformations that the human telomere sequence adopts in an aqueous KCl solution: hybrid-1 ( Fig. 2A), hybrid-2, and their corresponding triplexes with the double-chain reversal loop unfolded (33), in agreement with other singlemolecule studies and computational work (34,35). Current-time (i-t) traces of the events that correlated with the hybrid-folded DNA showed intermediate current levels (I M = 37% or 44%; Fig.…”

Section: Resultssupporting

confidence: 86%

Single-molecule investigation of G-quadruplex folds of the human telomere sequence in a protein nanocavity

Fleming

Middleton

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

102

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Human telomeric DNA consists of tandem repeats of the sequence 5′-TTAGGG-3′ that can fold into various G-quadruplexes, including the hybrid, basket, and propeller folds. In this report, we demonstrate use of the α-hemolysin ion channel to analyze these subtle topological changes at a nanometer scale by providing structuredependent electrical signatures through DNA-protein interactions. Whereas the dimensions of hybrid and basket folds allowed them to enter the protein vestibule, the propeller fold exceeds the size of the latch region, producing only brief collisions. After attaching a 25-mer poly-2′-deoxyadenosine extension to these structures, unraveling kinetics also were evaluated. Both the locations where the unfolding processes occur and the molecular shapes of the G-quadruplexes play important roles in determining their unfolding profiles. These results provide insights into the application of α-hemolysin as a molecular sieve to differentiate nanostructures as well as the potential technical hurdles DNA secondary structures may present to nanopore technology.α-hemolysin nanopore | single-molecule detection N ucleic acids can fold into a myriad of secondary structures that depend on the primary sequence as well as the physical conditions in which the structures are prepared and characterized. One prime example of a multistructural sequence is human telomeric DNA comprising the repeat sequence 5′-TTAGGG-3′. This guanine-rich single-stranded sequence is known to fold into highly ordered nanostructures in the form of G-quadruplexes that feature the coordination of two alkali cations to three layers of G-tetrads formed by Hoogsteen hydrogen-bonded assemblies of four guanine bases ( Fig. 1A) (1, 2). G-quadruplexes provide a fascinating case in which the cation and physical context play critical roles in defining the overall structural topology as well as the stability of the fold. Guanine-rich sequences also are known to present challenges to PCR amplification and sequencingby-synthesis methods that require processive analysis of singlestranded DNA (ssDNA) because of the high thermodynamic stability of these folded structures.The following studies highlight the cation and contextdependent conditions in which the topology of the natural human telomere sequence 5′-TAGGG(TTAGGG) 3 TT-3′ is affected. In NaCl solution, NMR studies revealed a structure referred to as the basket fold (Fig. 1A) (4). Key features of this structure include an antiparallel strand arrangement with alternating syn and anti orientations of the guanosine glycosidic bonds and three tetrads linked by two edgewise loops and one diagonal loop. In contrast, NMR-based studies in KCl solution show that the same sequence folds predominantly to the hybrid-1 and hybrid-2 structures (Fig. 1A) (5-8). Characteristics of this structure are an antiparallel strand orientation with a 3+1 core of syn and anti G nucleotides yielding three tetrads. The loop topology of the hybrid fold consists of a double-chain reversal loop and two edgewise loops, in which hybr...

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Section: Resultssupporting

confidence: 86%

Single-molecule investigation of G-quadruplex folds of the human telomere sequence in a protein nanocavity

Fleming

Middleton

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

102

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“…This structure was first characterized as an intermediate form of the G-quadruplex but with a stable conformation. Its existence in solution has been directly visualized (50), and its three-dimensional structure in solution has been determined. The correct formation of G3 used in this study was confirmed by CD and EMSA (data not shown).…”

Section: Resultsmentioning

confidence: 99%

G-quadruplexes Significantly Stimulate Pif1 Helicase-catalyzed Duplex DNA Unwinding

Duan

Liu

Yang

et al. 2015

Journal of Biological Chemistry

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Background: G-quadruplexes (G4s) play a variety of roles in DNA transactions. Results: Pif1-catalyzed duplex DNA unwinding was greatly stimulated by G4s in several aspects, including the unwinding rate and amplitude and the chemical-mechanical coupling efficiency. Conclusion: G4s significantly activate helicase Pif1-catalyzed duplex DNA unwinding through a mechanism of G4-induced dimerization. Significance: The G4-activating effect may be implicated in the rescue of stalled replication forks, activating of replication origins, and lagging strand maturation.

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“…The nature of these states is however still debated. The folding pathway may include a collapse of the unfolded species into hairpin-like structures with G:G base pairs (44–47), which then fold into an antiparallel G-quadruplex (37,48), then interconvert progressively into triplexes intermediates (49–51) before folding properly into a G-quadruplex (37). This model is sequential, and therefore does not consider side-reactions in the mechanism, i.e.…”

Section: Introductionmentioning

confidence: 99%

Folding and misfolding pathways of G-quadruplex DNA

Marchand¹,

Gabelica²

2016

Nucleic Acids Res

160

245

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G-quadruplexes adopt various folding topologies, but information on their folding pathways remains scarce. Here, we used electrospray mass spectrometry to detect and quantify the specifically bound potassium ions, and circular dichroism to characterize the stacking topology of each ensemble. For human telomeric (hTel) sequences containing the d((GGGTTA)3GGG) core, K+ binding affinity and cooperativity strongly depends on the chosen construct. The shortest sequences bind only one K+ at low KCl concentration, and this 2-quartet G-quadruplex is antiparallel. Flanking bases increase the K+ binding cooperativity. To decipher the folding pathways, we investigated the kinetics of K+ binding to telomeric (hybrid) and c-myc (parallel) G-quadruplexes. G-quadruplexes fold via branched pathways with multiple parallel reactions. Up to six states (one ensemble without K+, two ensembles with 1-K+ and three ensembles with 2-K+) are separated based on their formation rates and ion mobility spectrometry. All G-quadruplexes first form long-lived misfolded structures (off-pathway compared to the most stable structures) containing one K+ and two quartets in an antiparallel stacking arrangement. The results highlight the particular ruggedness of G-quadruplex nucleic acid folding landscapes. Misfolded structures can play important roles for designing artificial G-quadruplex based structures, and for conformational selection by ligands or proteins in a biological context.

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Direct and Single‐Molecule Visualization of the Solution‐State Structures of G‐Hairpin and G‐Triplex Intermediates

Cited by 46 publications

References 39 publications

Single-molecule investigation of G-quadruplex folds of the human telomere sequence in a protein nanocavity

Single-molecule investigation of G-quadruplex folds of the human telomere sequence in a protein nanocavity

G-quadruplexes Significantly Stimulate Pif1 Helicase-catalyzed Duplex DNA Unwinding

Folding and misfolding pathways of G-quadruplex DNA

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