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

Abstract

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...

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

“…S1A). Previous efforts suggested that the Δ%I∕I o of DNA native bases fall into the range of 0-1.2% Δ%I∕I o relative to C (21,28), and thus unfortunately, the AP site yielded a current blockage almost identical to G under these circumstances. This suggests that an AP site would produce a false readout as G in sequencing efforts with wild-type α-HL, and given the multiple sources and abundance of AP sites in cells, this would be problematic.…”

“…In previous work, we showed that adducts to 8-oxo-7,8-dihydro-2′-deoxyguanosine (OG) produced different current levels than native bases while being immobilized inside of the α-HL ion channel. However, all of these OG adducts failed to generate resolvable current signatures during free translocations (21). In this work, we demonstrate that the selective attachment of an 18-crown-6 (18c6) moiety to an AP site produces very unique current amplitude signatures that allow the identification of individual sites along a single homopolymeric or heteropolymeric DNA strand as a result of the specific interactions between the crown ether and electrolyte cations.…”

“…Due to the limited size of the constriction of α-HL (∼1.4 nm) (16), the translocation of ssDNA through the channel significantly reduces the electrolyte ion flow, resulting in a deep current blockage, potentially revealing the identity of the nucleotide sequence (17)(18)(19). Recently, this method has also been explored to examine epigenetic markers (20), DNA oxidative damage products (21,22), secondary structures (23), enzymatic activity (24)(25)(26), and chemical reactions (27).…”

Nanoporentechniken zur Sequenzierung und Detektion von Nukleinsäuren

“…Weil das angehängte Protein zu groß für den aHL-Kanal ist, verursacht es eine längere Aufenthaltszeit des ssDNA-Strangs im aHL (Abbildung 3 c). Diese Strategie ermçglichte die Unterscheidung und Identifizierung einzelner substituierter Basen, [21,22,34] einer epigenetischen DNA-Modifikation, [35] einer oxidativen Einzelbasenläsion [36] und abasischer Segmente. [37] Nanoporen Eine doppelsträngige DNA (dsDNA), die mit ihrer Füh-rungssequenz in der aHL-Nanopore immobilisiert ist, wird unter einem anliegenden Potential im Vorhof der aHL aufgespalten.…”

Sequence selective tagging of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) using PNAs

“…21,22 Nanopore sequencing is another elegant method for detecting lesions as specific positions within DNA, and significant advances have been made in recent years. [23][24][25] Even more recently, deep sequencing has been combined with restriction enzymes to ascertain DNA lesion location. 26 Our research group has developed methods for quantifying 8-oxodGuo in DNA by taking advantage of the ability to trap ('tag') an oxidized form of it with nitrogen nucleophiles first reported by the Burrows group (Scheme 1).…”