DNA abasic (AP) sites are one of the most frequent lesions in the genome and have a high mutagenic potential if unrepaired. After selective attachment of 2-aminomethyl-18-crown-6 (18c6), individual AP lesions are detected during electrophoretic translocation through the bacterial protein ion channel α-hemolysin (α-HL) embedded in a lipid bilayer. Interactions between 18c6 and Na þ produce characteristic pulse-like current amplitude signatures that allow the identification of individual AP sites in single molecules of homopolymeric or heteropolymeric DNA sequences. The bulky 18c6-cation complexes also dramatically slow the DNA motion to more easily recordable levels. Further, the behaviors of the AP-18c6 adduct are different with respect to the directionalities of DNA entering the protein channel, and they can be precisely manipulated by altering the cation (Li þ , Na þ or K þ ) of the electrolyte. This method permits detection of multiple AP lesions per strand, which is unprecedented in other work. Additionally, insights into the thermodynamics and kinetics of 18c6-cation interactions at a singlemolecule level are provided by the nanopore measurement.alpha-hemolysin | crown ethers | single-molecule detection | DNA damage

The ability to detect DNA damage within the context of the surrounding sequence is an important goal in medical diagnosis and therapies, but there are no satisfactory methods available to detect a damaged base while providing sequence information. One of the most common base lesions is 8-oxo-7,8-dihydroguanine that occurs during oxidation of guanine. In the work presented here, we demonstrate the detection of a single oxidative damage site using ion channel nanopore methods employing α-hemolysin. Hydantoin lesions produced from further oxidation of 8-oxo-7,8-dihydroguanine, as well as spirocyclic adducts produced from covalently attaching a primary amine to the spiroiminodihydantoin lesion, were detected by tethering the damaged DNA to streptavidin via a biotin linkage, and capturing the DNA inside an α-hemolysin ion channel. Spirocyclic adducts, in both homo-and hetero-polymer background single-stranded DNA sequences, produced current blockage levels differing by almost 10% from those of native base current blockage levels. These preliminary studies show the applicability of ion channel recordings not only for DNA sequencing, which has recently received much attention, but also to detecting DNA damage, which will be an important component to any sequencing efforts.Oxidative stress in the cell underlies multiple age-related disorders including cancer, heart and neurological diseases. 1 Reactive oxygen species (ROS) arising from metabolism, inflammation and environmental exposure to redox-active compounds lead to oxidation of many cellular components; those reactions occurring on DNA bases are of particular concern for their mutagenic potential. 2,3 Chief among these DNA base lesions is 8-oxo-7,8-dihydroguanine (OG, Figure 1), an oxidized base that exists at the level of ~1 in 10 6 bases under normal cellular conditions, 4 but at much higher levels under conditions of stress or in certain disease states. 5 Present methods for detection of OG most commonly involve (1) the comet assay, 6 which can be performed on a single cell although the lesion specificity of the assay is not high, and (2) HPLC-MS/MS methods which provide a more accurate count of specific lesions such as OG, but require complete enzymatic digestion to nucleosides before analysis. 7 Neither of these methods yield sequence information, 8 nor do they provide data on the occurrence of multiple lesions per strand, a phenomenon recognized as highly detrimental to proper DNA function. 9 In contrast, single-molecule sequencing methods such as nanopore ion channel detection 10 offer the potential to obtain both the identity and the sequence context of base damage sites burrows@chem.utah.edu and white@chem.utah on individual DNA strands as they translocate through the ion channel. Presently this method is focused on detection of the sequence of the native DNA bases (adenine (A), thymine (T), cytosine (C), and guanine (G)), in order to provide rapid genomic sequencing. 11-17 However, sequencing methods based on translocation of DNA through ion chann...

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...

Human telomeric DNA consists of tandem repeats of the sequence 5′-TTAGGG-3′, including a 3′ terminal single-stranded overhang of 100–200 nucleotides that can fold into quadruplex structures in the presence of suitable metal ions. In the presence of an applied voltage, the α-hemolysin (α-HL) protein ion channel can produce unique current patterns that are found to be characteristic for various interactions between G-quadruplexes and the protein nanocavity. In this study, the human telomere in a complete sequence context, 5′-TAGGG(TTAGGG)3TT-3′ was evaluated with respect to its multiple folding topologies. Notably, the coexistence of two interchangeable conformations of the K+-induced folds, hybrid-1 and hybrid-2, were readily resolved at a single-molecule level along with triplex folding intermediates, whose characterization has been challenging in experiments that measure the bulk solution. These results enabled us to profile the thermal denaturation process of these structures to elucidate the relative distributions of hybrid-1, hybrid-2 and folding intermediates such as triplexes. For example, at 37 °C, pH 7.9 in 50 mM aqueous KCl, the ratio of hybrid-1:hybrid-2:triplex is approximately 11:5:1 in dilute solution. The results obtained lay the foundation for utilizing the α-HL ion channel as a simple tool for monitoring how small molecules and physical context shift the equilibrium between the many G-quadruplex folds of the human telomere sequence.

b-catenin is a multifunctional protein identified to be pivotal in embryonic patterning, organogenesis and adult homeostasis. It plays a critical structural role in mediating cadherin junctions and is also an essential transcriptional co-activator in the canonical Wnt pathway. Evidence has been documented that both the canonical Wnt pathway and cadherin junctions are deregulated or impaired in a plethora of human malignancies. In the light of this, there has been a recent surge in elucidating the mechanisms underlying the etiology of cancer development from the perspective of b-catenin. Here, we focus on the emerging roles of b-catenin in the process of tumorigenesis by discussing novel functions of old players and new proteins, mechanisms identified to mediate or interact with b-catenin and the most recently unraveled clinical implications of b-catenin regulatory pathways toward tumor suppression.Since its identification as an essential and central component in the Wnt signaling cascade, and the subsequent finding of its pivotal role in cadherin-based cell-cell adhesion, b-catenin has been critically studied to elucidate the coordination of these two pathways. The significance of this coordination is substantiated in a plethora of metabolic processes, such as axis and mesoderm formation, stem cell differentiation and carcinogenesis. 1,2 Generally, the Wnt pathway is divided into four branches, namely the canonical Wnt/b-catenin pathway, and the noncanonical (or heretical) Wnt/Ca 2þ and planar cell polarity pathways. Amongst them, the canonical Wnt pathway is the best studied and is reported to be highly conserved through evolution but is frequently altered in many human malignancies such as colorectal cancers, hepatocellular carcinomas and gastric cancers. 3-6 Ca 2þ -dependent cellcell adhesion in the cadherin family of proteins is typified by an extracellular segment that consists of five distinct Ca 2þ -binding domains and a conserved cytoplasmic domain, which interacts with b-catenin and p120 catenin (herein p120); b-catenin then provides a binding site for a-catenin. Cadherin junctions, among other cell-cell adhesion complexes, are essential for cellular processes such as cell polarity and migration. 7,8 Since an indispensible morphological hallmark of malignant tumors is reduced cell-cell adhesiveness, it is predicted that the components of cadherin junctions in many human malignancies, such as breast cancer, colorectal cancer and prostate cancer, are genetically altered. 9-11 Besides cellcell adhesion, cadherin junctions function as a potent competitor of free cytosolic b-catenin. This is supported by studies of the co-crystal structures of b-catenin/cadherin and b-catenin/TCF revealing that the two b-catenin ligands shared overlapping binding regions along b-catenin. 12 The role of the cadherin junction in the subcellular distribution of b-catenin has recently been further extended as discussed below.In this review, a myriad of recently emerged mechanisms governing the signaling and adhesive activity of ...

The human telomere repeat sequence 5′-TTAGGG-3′ is a hot spot for oxidation at guanine, yielding 8-oxo-7,8-dihydroguanine (OG), a biomarker of oxidative stress. Telomere shortening resulting from oxidation will ultimately induce cellular senescence. In this study, α-hemolysin (α-HL) nanopore technology was applied to detect and quantify OG in the human telomeric DNA sequence. This repeat sequence adopts a basket G-quadruplex in the NaCl electrolyte used for analysis that enters the α-HL channel, slowly unfolds, and translocates. The basket fold containing OG disrupts the structure, leading to >10× increase in the unfolding kinetics without yielding a detectable current pattern. Therefore, detection of OG with α-HL required labeling of OG with aminomethyl-[18-crown-6] using a mild oxidant. The labeled OG yielded a pulse-like signal in the current vs time trace when the DNA strand was electrophoretically passed through α-HL in NaCl electrolyte. However, the rate of translocation was too slow using NaCl salts, leading us to further refine the method. A mixture of NH4Cl and LiCl electrolytes induced the propeller fold that unravels quickly outside the α-HL channel. This electrolyte allowed observation of the labeled OG, while providing a faster recording of the currents. Lastly, OG distributions were probed with this method in a 120-mer stretch of the human telomere sequence exposed to the cellular oxidant 1O2. Single-molecule profiles determined the OG distributions to be random in this context. Application of the method in nanomedicine can potentially address many questions surrounding oxidative stress and telomere attrition observed in various disease phenotypes including prostate cancer and diabetes.

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Translocation measurements of intact DNA strands with the ion channel α-hemolysin (α-HL) are limited to single-stranded DNA (ssDNA) experiments as the dimensions of the channel prevent double-stranded DNA (dsDNA) translocation; however, if a short oligodeoxynucleotide is used to interrogate a longer ssDNA strand, it is possible to unzip the duplex region when it is captured in the α-HL vestibule, allowing the longer strand to translocate through the α-HL channel. This unzipping process has a characteristic duration based on the stability of the duplex. Here, ion channel recordings are used to detect the presence and relative location of the oxidized damage site 8-oxo-7,8-dihydroguanine (OG) in a sequence-specific manner. OG engages in base pairing to C or A with unique stabilities relative to native base Watson-Crick pairings, and this phenomenon is used here to engineer probe sequences (10–15 mers) that when base-paired with a 65 mer sequence of interest, containing either G or OG at a single site, produce characteristic unzipping times that correspond well with the duplex melting temperature (Tm). Unzipping times also depend on the direction from which the duplex enters the vestibule if the stabilities of leading base pairs at the ends of the duplex are significantly different. It is shown here that the presence of a single DNA lesion can be distinguished from an undamaged sequence and that the relative location of the damage site can be determined based on the duration of duplex unzipping.