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

ABSTRACT: 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 ?-hemolysin ion channel to analyze these subtle topological changes at a nanometer scale by providing structure-dependent 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 ?-hemolysin as a molecular sieve to differentiate nanostructures as well as the potential technical hurdles DNA secondary structures may present to nanopore technology.

SUBMITTER: An N

PROVIDER: S-EPMC4209999 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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

An Na N Fleming Aaron M AM Middleton Eric G EG Burrows Cynthia J CJ

Proceedings of the National Academy of Sciences of the United States of America 20140915 40

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 structure-dependent electrical signatures through DNA-protein interactions. Whereas the dimensions of hybrid and basket folds allowed them to enter the protein vestibule, the propeller f ...[more]

PMID: 25225404

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Project description:Telomere shortening occurs during oxidative and inflammatory stress with guanine (G) as the major site of damage. In this work, a comprehensive profile of the sites of oxidation and structures of products observed from G-quadruplex and duplex structures of the human telomere sequence was studied in the G-quadruplex folds (hybrid (K(+)), basket (Na(+)), and propeller (K(+) + 50% CH3CN)) resulting from the sequence 5'-(TAGGGT)4T-3' and in an appropriate duplex containing one telomere repeat. Oxidations with four oxidant systems consisting of riboflavin photosensitization, carbonate radical generation, singlet oxygen, and the copper Fenton-like reaction were analyzed under conditions of low product conversion to determine relative reactivity. The one-electron oxidants damaged the 5'-G in G-quadruplexes leading to spiroiminodihydantoin (Sp) and 2,2,4-triamino-2H-oxazol-5-one (Z) as major products as well as 8-oxo-7,8-dihydroguanine (OG) and 5-guanidinohydantoin (Gh) in low relative yields, while oxidation in the duplex context produced damage at the 5'- and middle-Gs of GGG sequences and resulted in Gh being the major product. Addition of the reductant N-acetylcysteine (NAC) to the reaction did not alter the riboflavin-mediated damage sites but decreased Z by 2-fold and increased OG by 5-fold, while not altering the hydantoin ratio. However, NAC completely quenched the CO3(•-) reactions. Singlet oxygen oxidations of the G-quadruplex showed reactivity at all Gs on the exterior faces of G-quartets and furnished the product Sp, while no oxidation was observed in the duplex context under these conditions, and addition of NAC had no effect. Because a long telomere sequence would have higher-order structures of G-quadruplexes, studies were also conducted with 5'-(TAGGGT)8-T-3', and it provided oxidation profiles similar to those of the single G-quadruplex. Lastly, Cu(II)/H2O2-mediated oxidations were found to be indiscriminate in the damage patterns, and 5-carboxamido-5-formamido-2-iminohydantoin (2Ih) was found to be a major duplex product, while nearly equal yields of 2Ih and Sp were observed in G-quadruplex contexts. These findings indicate that the nature of the secondary structure of folded DNA greatly alters both the reactivity of G toward oxidative stress as well as the product outcome and suggest that recognition of damage in telomeric sequences by repair enzymes may be profoundly different from that of B-form duplex DNA.

Dataset Information

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

Publications

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

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