DNA Hybridization by Peptide Nucleic Acids Attached to Controlled Pore Glass

Abstract

Sequence-specific hybridization between DNA in solution and a probe attached to solid support is used as a powerful tool for the detection and analysis of genetic materials. 1 DNAs are most often used as the probes fixed on the solid support.1 RNAs, peptide nucleic acids (PNAs), 2 and pyrrole/ imidazole-containing polyamides 3 are capable of sequencespecific hybridization with DNA. Pyrrole/imidazole-containing polyamides are at present useful for recognition of only short (hexamer or shorter) double-stranded DNAs. RNAs are much more difficult to handle and to synthesize compared with DNAs or PNAs. Pyrrole/imidazole-containing polyamides and RNAs are not, therefore, useful as the probes for DNA recognition.PNAs have pseudo-peptide backbones of repeating N-(2-aminoethyl)glycine units onto which the nucleobases of DNA are attached.2 PNAs form duplexes with nucleic acids, adopting Watson-Crick type base pairing and stacking patterns similar to those of DNA-DNA duplexes.2 Since the negatively charged backbone of DNA is replaced by a neutral one in PNA, PNA-DNA duplexes are free from electrostatic repulsion between the two negatively charged backbones unlike DNA-DNA duplexes. Consequently, PNA-DNA duplexes have greater stability compared with the corresponding DNA-DNA duplexes at low salt concentrations. Although formation of PNA-DNA duplexes in solution has been extensively investigated, DNA hybridization by immobilized PNA has been rarely studied.In the present study, we examined whether PNA attached to controlled pore glass (CPG) can form strong and specific complex with DNA dissolved in solution. CPG is silica glass with uniform and controlled pores. A glycerol-containing CPG derivative (CPG-glycerol) was oxidized with NaIO 4 to obtain an aldehyde-containing CPG derivative (CPG-CHO) in the present study. Then, the terminal amino group of a PNA molecule was linked to CPG-CHO by reductive amination with sodium cyanoborohydride. In Table 1, PNAs attached to CPG are summarized, together with DNAs used as substrates. HoPuPNA, HoPyPNA, and MSPNA are complementary to HoPyDNA, HoPuDNA, and MSDNA, respectively.When HoPyDNA, HoPuDNA, and MSDNA were incubated with the PNA-free CPG-glycerol under the conditions of hybridization conditions, the amount of the DNA adsorbed onto the resin was negligible. This suggests that adsorption of DNA onto the PNA-containing CPG (CPG-PNA) would occur mainly through interaction with the PNA moieties.