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...

Translocation of single-stranded DNA through alpha-hemolysin (alpha-HL) channels is investigated in glycerol/water mixtures containing 1 M KCl. Experiments using glass nanopore membranes as the lipid bilayer support demonstrate that the translocation velocities of poly(deoxyadenylic acid), poly(deoxycytidylic acid), and poly(deoxythymidylic acid) 50-mers are decreased by a factor of approximately 20 in a 63/37 (vol %) glycerol/water mixture, relative to aqueous solutions. The ion conductance of alpha-HL and the entry rate of the polynucleotides into the protein channel also decrease with increasing viscosity. Precise control of translocation parameters by adjusting viscosity provides a potential means to improve sequencing methods based on 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 (10–15 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.

A method is described for fabricating 25-75 mum thick fused quartz membranes containing a single conical shaped nanopore (orifice radius ranging from 10 to 1000 nm). The quartz nanopore membrane (QNM) provides an excellent solid support structure for lipid bilayers in ion channel recordings due to the large electrical resistivity of fused quartz. Electrical measurements demonstrate that the leakage current through 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) bilayers suspended across a 500-1000 nm radius QNM orifice is immeasurably small, corresponding to a bilayer resistance greater than 10(12) ohms. Translocation of single-stranded DNA oligomers (poly dA 50-mer and poly dA 20-mer) through a protein ion channel (alpha-hemolysin) reconstituted in a DPhPC bilayer suspended across the QNM orifice is demonstrated.

Ion current rectification (ICR), defined as an increase in ion conduction at a given polarity and a decrease in ion conduction for the same voltage at the opposite polarity, i.e., a deviation from a linear ohmic response, occurs in conical shaped pores due to the voltage dependent solution conductivity within the aperture. The degree to which the ionic current rectifies is a function of the size and surface charge of the nanopore, with smaller and more highly charged pores exhibiting greater degrees of rectification. The ICR phenomenon has previously been exploited for biosensing applications, where the level of ICR for a nanopore functionalized with an analyte-specific binding molecule (e.g., an antibody, biotin, etc.) changes upon binding its target analyte (e.g., an antigen, streptavidin, etc.) due to a resulting change in the size and/or charge of the aperture. While this type of detection measurement is typically qualitative, for the first time, we demonstrate that the rate at which the nanopore ICR response changes is dependent on the concentration of the target analyte introduced. Utilizing a glass nanopore membrane (GNM) internally coated with a monoclonal antibody specific to the cleaved form of synaptosomal-associated protein 25 (cSNAP-25), creating the antibody-modified glass nanopore membrane (AMGNM), we demonstrate a correlation between the rate of ICR change and the concentration of introduced cSNAP-25, over a range of 500 nM–100 μM. The methodology presented here significantly expands the applications of nanopore ICR biosensing measurements and demonstrates that these measurements can be quantitative in nature.