Enhancing the Interactions of Ion-Tagged Oligonucleotides and Magnetic Ionic Liquid Supports for the Sequence-Specific Extraction of Hepatitis B Virus DNA,Analytica Chimica Acta

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Enhancing the Interactions of Ion-Tagged Oligonucleotides and Magnetic Ionic Liquid Supports for the Sequence-Specific Extraction of Hepatitis B Virus DNA
Analytica Chimica Acta ( IF 5.7 ) Pub Date : 2024-12-18 , DOI: 10.1016/j.aca.2024.343563
Seong-Soo Lee, Derek R. Eitzmann, Jared L. Anderson

Background

Infections from the hepatitis B virus (HBV) are a major risk factor for hepatocellular carcinoma, one of the most common types of liver cancer. Circulating cell-free DNA (ccfDNA) in human plasma can be used as a non-invasive biomarker for diagnosing HBV-related liver diseases. The isolation of target ccfDNA sequences is often challenging due to the co-extraction of highly abundant non-target DNA from samples. Ion-tagged oligonucleotide (ITO) probes coupled with magnetic ionic liquids (MIL) support have emerged as a promising methodology for sequence-specific DNA extractions to address challenges associated with solid supports such as streptavidin-coated magnetic bead.

Results

ITOs, disubstituted ion-tagged oligonucleotide (DTO), and ion-tagged disubstituted oligonucleotide (ITDO) probes featuring various substituents including linear alkyl, branched alkyl, and perfluoroalkyl moieties were examined to enhance hydrophobic and fluorophilic interactions between the probes and a trihexyl(tetradecyl)phosphonium manganese(II) hexafluoroacetylacetonate ([P₆₆₆₁₄⁺][Mn(hfacac)₃⁻]) MIL support. The ITO-MIL approach was optimized by adjusting the annealing temperature (45 °C), molar ratio of target DNA to ITO probe (1:100), and ionic strength (200 mM NaCl) to maximize the extraction of target DNA. The 3-allyl-1-(23-bis(pentylthio)propyl)imidazolium bromide ([AI(C₅S)₂⁺][Br^-])-ITO probe featuring a branched alkyl substituent yielded a higher loading efficiency (48.05 ± 6.72%) due to increased hydrophobic ITO-MIL interactions, compared to that of 24.57 ± 4.40% for the 3-allyl-1-(3-(pentylthio)propyl)imidazolium bromide ([AIC₅S⁺][Br^-])-ITO probe with a linear alkyl substituent. The high affinity of ITO-MILinteractions minimized the loss of probe during successive wash steps, yielding an approximate 10-fold greater amount of extracted target DNA using the [AI(C₅S)₂⁺][Br^-])-ITO probe compared to [AIC₅S⁺][Br^-])-ITO probe.

Significance

This study provides novel synthetic approaches for tailoring probe substituents using thiol-ene and thiol-yne “click” chemistry. Hydrophobic and fluorophilic ITO-MIL interactions were systematically investigated. Optimal conditions for the ITO-MIL method significantly improved the amount of extracted DNA. The ITO-MIL method using the [AI(C₅S)₂⁺][Br^-]-HBV-ITO probe demonstrated selective extraction of target HBV DNA in both DI water and diluted human plasma containing a high abundance of background DNA.

更新日期：2024-12-19

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