Populated Intermediates in the Thermal Unfolding of the Human Telomeric Quadruplex

Gray, Robert D.; Buscaglia, Robert; Chaires, Jonathan B.

doi:10.1021/ja307543z

Cited by 111 publications

(170 citation statements)

References 54 publications

(128 reference statements)

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“…When these overhangs were removed, only the 37% current blockage level was observed, consistent with the observation that the hybrid-1 structure predominates when the dangling ends are missing (7,36). At the end of each of the events, the current level was observed to be at an intermediate level I M immediately before returning to the open-channel current; we interpreted this to mean that hybrid-folded oligomers were too stable inside the vestibule to unravel and translocate, and the DNA exited the vestibule from the cis opening where it had entered (33).…”

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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“…Sample preparation and CD parameters were described previously. 31 Background CD and absorbance spectra were recorded using 25 mM KCl in folding buffer prepared by addition of a 1M KCl-folding buffer solution. The relatively low potassium concentration was chosen to ensure quadruplex structures are fully folded in solution, 37 while allowing the full thermal denaturation profile of the quadruplex system to be investigated.…”

Section: Methodsmentioning

confidence: 99%

“…This work includes a detailed thermodynamic characterization of ht-quadruplex unfolding, putting to use recently established unfolding mechanisms. 31 The results of the thermodynamic characterization are used to rationalize potential intermediate states and elucidate similarities between distinct quadruplex topologies. Finally, differential scanning calorimetry is used to evaluate enthalpy changes for comparison with spectral studies.…”

Section: Introductionmentioning

confidence: 99%

Thermodynamic characterization of human telomere quadruplex unfolding

2013

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The 3'-terminal extension of eukaryotic chromosomes are a unique example of functional single-stranded DNA. Human telomeres are constructed of the repeated DNA sequence 5'-d(TTAGGG). Four-repeats of human telomeric DNA have been characterized by high-resolution techniques to be capable of forming at least five distinct monomeric conformations. The predominant solution topology is influenced by solution conditions and the presence of 3'- or 5'-flanking residues. This study extends recently described sequential unfolding mechanisms for human telomeric quadruplexes to eight telomeric DNA sequences that form three unique topologies. Thermal unfolding monitored by circular dichroism is subjected to singular value decomposition to enumarate the number of significant spectral species required to model the unfolding process. Thermal denaturations are found to be best modeled by a four-state sequential mechanism with ΔGave = −8.8 kcal mol−1. The thermal unfolding was also investigated in 50% (v/v) acetonitrile. Under dehydrated conditions, quadruplex thermal denaturations are best modeled by a three-state sequential unfolding mechanism. Dehydrated quadruplexes demonstrate increased thermal stability with ΔGave = −12.7 kcal mol−1. A common spectral intermediate is shared amongst human telomere quadruplex thermal unfolding regardless of starting topology and hydration state. This unfolding intermediate was recently characterized as an ensemble of triple-helical structures for a 22-nt human telomeric quadruplex.

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“…Detailed computational and experimental (thermodynamic, kinetic) investigation of folding and ligand binding pathways of quadruplex DNA has begun very recently (17)(18)(19)(20)(21)(22). These studies leave no doubt that in solution, the Tel22 folding/unfolding process may consist of several structural steps, meaning that such solutions should be considered as equilibrium mixtures of Tel22 molecules in their folded (Q), unfolded (U), and intermediate (I) states.…”

Section: Introductionmentioning

confidence: 99%

Dominant Driving Forces in Human Telomere Quadruplex Binding-Induced Structural Alterations

Bončina

Hamon

Islam

et al. 2015

Biophysical Journal

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Recently various pathways of human telomere (ht) DNA folding into G-quadruplexes and of ligand binding to these structures have been proposed. However, the key issue as to the nature of forces driving the folding and recognition processes remains unanswered. In this study, structural changes of 22-mer ht-DNA fragment (Tel22), induced by binding of ions (K(+), Na(+)) and specific bisquinolinium ligands, were monitored by calorimetric and spectroscopic methods and by gel electrophoresis. Using the global model analysis of a wide variety of experimental data, we were able to characterize the thermodynamic forces that govern the formation of stable Tel22 G-quadruplexes, folding intermediates, and ligand-quadruplex complexes, and then predict Tel22 behavior in aqueous solutions as a function of temperature, salt concentration, and ligand concentration. On the basis of the above, we believe that our work sets the framework for better understanding the heterogeneity of ht-DNA folding and binding pathways, and its structural polymorphism.

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Populated Intermediates in the Thermal Unfolding of the Human Telomeric Quadruplex

Cited by 111 publications

References 54 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

Thermodynamic characterization of human telomere quadruplex unfolding

Dominant Driving Forces in Human Telomere Quadruplex Binding-Induced Structural Alterations

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