Na An

Crown ether–electrolyte interactions permit nanopore detection of individual DNA abasic sites in single molecules

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+ 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+, Na+ or K+) 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 single-molecule level are provided by the nanopore measurement.

Nanopore Detection of 8-Oxo-7,8-dihydro-2′-deoxyguanosine in Immobilized Single-Stranded DNA via Adduct Formation to the DNA Damage Site

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, which 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 heteropolymer 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 for detecting DNA damage, which will be an important component to any sequencing efforts.

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

Significance The bacterial protein α-hemolysin (α-HL) can form a mushroom-shaped ion channel by self-assembling across a lipid bilayer, allowing capture of a single DNA molecule inside its nanometer-scale vestibule in an electric field. Interactions between the protein nanocavity and DNA molecules generate characteristic current signals that reveal structural information. We harnessed such analytical power to investigate various G-quadruplex conformations adopted by the human telomeric sequence, namely hybrid, basket, and propeller folds that are formed under different physical conditions. Results presented here demonstrate the ability of α-HL to distinguish these G-quadruplexes based on their overall shapes and sizes and also to monitor their unraveling kinetics at different locations in the protein channel, expanding the applicability of the nanopore technology. 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 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.

Nanopore Detection of 8-Oxoguanine in the Human Telomere Repeat Sequence

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.

Sequence-specific Single-molecule Analysis of 8-Oxo-7,8- dihydroguanine Lesions in DNA based on Unzipping Kinetics of Complementary Probes in Ion Channel Recordings

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 (10-15mers) that, when base-paired with a 65mer sequence of interest, containing either G or OG at a single site, produce characteristic unzipping times that correspond well with the duplex melting temperature (T(m)). 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.

Modulation of the current signatures of DNA abasic site adducts in the α-hemolysin ion channel

Electrical current signatures of DNA adducts were investigated during immobilization of strands inside the membrane-bound α-hemolysin ion channel. The current blockages produced by these adducts were found to depend on both size and shape, providing insights into the DNA-protein interactions and the size limitation of bulky adducts to be translocated.

50 Single-molecule studies of human telomeric G-quadruplexes and the effect of oxidative damage

In the human genome, telomeric DNA has tandem repeats of the sequence 5′-TTAGGG terminating with a 3′ single-stranded overhang of 100–200 bases. These guanine-rich DNA sequences can fold into tetrastranded structures, known as G-quadruplexes. The precise fold of the G-quadruplex structure is dictated by the metal ions present which we studied through the use of the α-hemolysin ion channel. Being electrophoretically driven into the cis side of the α-hemolysin, the hybrid fold (K+) entered the vestibule mouth leading to current blockages for the duration of the time the DNA resided in the vestibule. Due to the polymorphic nature of the hybrid folds, the recorded current signatures could be correlated with the major structural topologies that exist for this fold in solution (e.g. hybrid-1, hybrid-2, and triplex; Mashimo et al., 2010). The hybrid folds were not capable of traversing to the trans side of the nanopore, while the triplex could achieve translocation. The basket fold (Na+) was also able to enter into the vestibule causing current blockages that were indicative of the orientation in which they entered into the vestibule. When the basket fold entered tail first, slow translocation events were observed. In contrast, the propeller fold (∼3.9 nm, 0.05 M K+/5 M Li+) exceeds the protein channel orifice (∼3.0 nm) producing only swift and small disturbances to the open channel current. Secondly, oxidative damage to the telomeric sequence is proposed to contribute to telomere shortening, dysfunction, and cell aging (Epel et al., 2004). Locations of the oxidative damages have different effects on the G-quadruplex folding that produced significant changes in their nanopore behavior. Placement of the guanine oxidation product, 8-oxoguanosine (OG), in a top or bottom tetrad results in destabilization of that layer, whereas the presence of OG in a middle tetrad leads to complete unfolding of the G-quadruplex. These behaviors were determined by their translocation times which correlated with the folding’s free energy supported by NIH GM093099.