Three-Enzyme Cascade Bioreactor for Rapid Digestion of Genomic DNA into Single Nucleosides

Abstract

Structure-based DNA modification analysis provides accurate and important information on genomic DNA changes from epigenetic modifications to various DNA lesions. However, genomic DNA strands are often required to be efficiently digested into single nucleosides. It is an arduous task because of the involvement of multiple enzymes with different catalytic acitivities. Here we constructed a three-enzyme cascade capillary monolithic bioreactor that consists of immobilized deoxyribonuclease I (DNase I), snake venom phosphodiesterase (SVP), and alkaline phosphatase (ALPase). By the use of this cascade capillary bioreactor, genomic DNA can be efficiently digested into single nucleosides with an increasing rate of ∼20 folds. The improvement is mainly attributed to dramatically increase enzymatic capacity and activity. With a designed macro-porous structure, genomic DNA of 5−30 Kb (∼1.6−10 million Daltons) can be directly passed through the bioreactor simply by hand pushing or a low-pressure microinjection pump. By coupling with liquid chromatography-tandem mass spectrometry (LC-MS/MS), we further developed a sensitive assay for detection of an oxidative stress biomarker 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) in DNA. The proposed three-enzyme cascade bioreactor is also potentially applicable for fast identification and quantitative detection of other lesions and modifications in genomic DNA.E xposure to chemical carcinogens may result in DNA damages, a key step toward the onset of cancer. 1−4 DNA damages can occur via structural modification of the nucleosides and phosphate moieties in DNA chains. 5 It is difficult to directly search for various modifications in genomic DNA since DNA strands are long biopolymers consisting of a large number of normal nucleosides (deoxyadenosine, dA; deoxygunosine, dG; deoxycytidine, dC; and thymidine monophosphates, dT) interspersing with a small quantity of modified nucleosides (e.g., 5-methylcytosine, 5mC) or trace modifications (e.g., 5-hydroxymethylcytosine, 5-formalcytosine, and 5-carboxylcytosine). To determine these DNA modifications, genomic DNA samples were often required to be efficiently digested into mononucleotides or single nucleosides. 6−10 Liquid chromatography-mass spectrometry (LC-MS) can explore whether nucleotide is modified, and is sensitive enough to quantify these modifications on DNA chains. 9,11−15 For example, during LC-MS/MS analysis of global DNA methylation and hydroxylmethylation, genomic DNA was digested into the mixture of classical DNA nucleosides (dA, dT, dG, dC) and modified nucleosides. 16−18 Recently, we discovered a new DNA modification in drosophila, N6-methyladenine. 19 To accurately identify its structure and validate its identity, the Drosophila genomic DNA was also required to be digested into single nucleosides for high sensitivity analysis. Another example, identification and analysis of oxidative damaged DNA (e.g., 8-oxo-7,8-dihydro-2′-deoxyguanosine, 8-oxodG) completely digestion of genomic DNA to maximal release 8-oxodG w...

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