Development of PNA-Array Platforms for Detection of Genetic Polymorphism of Cytochrome P450 2C19

Abstract

The selective and sensitive detection of target DNA is an important issue for clinical diagnostics, including identification of single nucleotide polymorphism (SNP), as well as for fundamental studies in molecular biology. The commonly used DNA-detection techniques are mainly based on the DNA microarrays, which present probe-DNA molecules immobilized onto a solid surface in an array format for the bio-specific hybridization with the unknown target-DNA fragments. Although the DNA microarrays have been utilized as a basic platform for the clinical detection, selectivity and sensitivity are still limited toward the singlenucleotide discrimination, which precludes the accurate analysis of SNP. In addition, duplexes between the target and probe DNAs exist in a stable form only in the presence of salt because of the electrostatic repulsion between negatively charged ribose-phosphate backbones of DNA; these salt conditions also induce the secondary and tertiary structures of DNA oligomers themselves, which could disturb the hybridization between the target and probe DNAs. On the other hand, peptide nucleic acid (PNA), an artificial nucleic acid analog, has been introduced to overcome the drawbacks of natural DNA in the aspects of specificity, sensitivity, and stability, and been as a molecular tool for the identification of SNP. The neutral backbone of PNA enhances the stability of PNA-DNA duplexes due to the absence of interstrand repulsion between the negatively charged phosphate groups of DNA. In addition, the unnatural structure makes PNA not degraded by enzymatic reactions. In this work, we generated a PNA-array platform to detect the SNP of cytochrome P450 2C19 (CYP2C19) gene. The CYP2C19 gene is known to play an important role in the metabolism of therapeutic drugs, including diazepam, proguanil, lansoprazole, and omeprazole. It is noteworthy that 15-20% of the Asians are genetically deficient in CYP2C19, failing to produce enzymes for the metabolism, compared with the occurrence of 3-5% of Caucasian populations. Specifically, there exist two variant alleles leading to the mutant types. The first varient is CYP2C19*2, which has a G → A nucleotide substitution at position 681, resulting in an aberrant splicing site; the second one, CYP2C19*3, which does a G → A substitution at position 636, resulting in a premature termination codon. According to the previous report, the frequency of CYP2C19*2 and CYP2C19*3 for Koreans is 0.21 and 0.12, respectively. Onto the silicon oxide surface were aldehyde groups introduced via imine formation between amine-terminated self-assembled monolayers (SAMs) and glutaldehyde. Another imine linkage was used for immobilizing the PNA probes, and unreacted aldehyde groups were passivated with 2(2aminoethoxy)ethanol. We designed four probe-PNAs based on the clinically important SNPs of CYP2C19, and denoted them 636W and 636M for the wild and mutant types of CYP2C19*3, and 681W and 681M for CYP2C19*2, respectively (Fig. 1). The linker of the PNA probes contained repetitive ethylene glycol groups and ended with several lysine groups. The immobilization of the probe PNAs was confirmed by hybridization of target DNAs. The target DNAs (T-636W, T-636M, T-681W, and T-681M), complementary to each of the PNA probes, were tagged with Cy3 (green) for wild type and Cy5 (red) for mutant type, respectively. The hybridization was performed for 1 h at 40 C with the DNA solution containing both T-636W and T636M (100 nM), and the same conditions were used for hybridization of T-681W/M. As shown in Fig. 2a, each DNA was selectively hybridized with its complementary probe-