A Homogeneous Fluorescence Assay for Rapid and Sensitive Quantification of the Global Level of Abasic Sites in Genomic DNA

Intact DNA sequences can be efficiently amplified and detected by using polymerase chain reaction (PCR) technology, even only a single copy is present. In contrast, it is still a challenge to identify and quantify the multifarious lesions in genomic DNA (gDNA) that cannot be amplified via PCR. Apurinic/apyrimidinic sites or abasic sites (AP sites) are one of the most common DNA lesions with the DNA base lost while the phosphodiester backbone remaining intact. It is estimated that the spontaneous hydrolysis of nucleobases may generate 10~50 (×103) AP sites/cell per day [1]. As the key intermediate of base excision repair (BER) pathway, AP sites level further elevated when exposed to exogenous damage agents, such as reactive alkylating agents or oxygen species [2], [3]. If unrepaired, these noninformative lesions would inhibit DNA duplication and transcription, resulting in cytotoxicity and mutagenicity [4]. Accumulation of AP sites was reported to be associated with aging and various diseases, such as Parkinsonism, Alzheimer’s disease, and cancer.

Strategies with covalently labeling have been developed for the detection of AP sites, like the aldehyde reactive probe (ARP)-based ELISA-like assay [5], [6], [7], [8] and LC-MS-based approaches [9], [10]. Although a significant advance in AP site detection, both methods relied on tedious separation steps and long assay time (>24 h). Furthermore, the LC-MS/MS assay needed a large sample input (>10 μg), while the ARP labeling methods suffered from the cross-reactivity with the naturally occurring formylated-bases, such as 5-formylcytosine (5-fC) and 5-formyluracil (5-fU) and high background caused by the intracellular biotin [6], [11], [12], [13]. A new molecule O-2-propynylhydroxylamine hydrochloride (AA3) has been synthesized which showed lower background and higher sensitivity for AP site detection. However, the tagged DNA need to be further purified by precipitation and column filtration which increased the risk of target loss [14].

To address these issues, we attempted to develop a homogeneous signal amplification system for rapid detection of these DNA lesions with high chemical selectivity and lower gDNA sample input. For this purpose, an oligodeoxynucleotide (ODN) strand with known sequence was employed as a surrogate for the AP sites, which can be easily amplified and generate multiple fluorescence signals. Most importantly, we observed that with the assistance of T4 pyrimidine dimer glycosylase (T4 PDG), a hydroxylamine-modified oligonucleotide strand can be stably and specifically ligated to the 3'-α, β-unsaturated aldehyde resulted from the AP sites with high discrimination effects over the 5-fC and 5-fU bases.

To integrate the following steps into a homogeneous reaction system so that no additional separation or purification procedures were needed, we utilized a novel property of lambda exonuclease (λ exo) which could smartly excise a specific DNA strand in a mixture solution via the variation of its 5′-terminal structures in a DNA duplex [15], [16], [17]. It not only offered a powerful tool to clear the unwanted excessive DNA labeling strands as desired, but also allowed further triggering a molecular conversion machine by the labeled DNA strands to amplified fluorescence signals in the same reaction buffer. The whole process was compact and highly efficient, which greatly simplified the quantitative detection procedures of the AP sites in gDNA. The method enabled quantification of AP sites in gDNA from both normal cells and cells exposed to external damaging agents, showing the variation of AP sites level along with damaging and repair processes.

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