Cyclopentane-peptide nucleic acids for qualitative, quantitative, and repetitive detection of nucleic acids

Abstract

We report the development of chemically modified peptide nucleic acids (PNAs) as probes for qualitative and quantitative detection of DNA. The remarkable stability of PNAs toward enzymatic degradation makes this class of molecules ideal to develop as part of a diagnostic device that can be used outside of a laboratory setting. Using an enzyme-linked reporter assay, we demonstrate that excellent levels of detection and accuracy for anthrax DNA can be achieved using PNA probes with suitable chemical components designed into the probe. In addition, we report on DNA-templated cross-linking of PNA probes as a way to preserve genetic information for repetitive and subsequent analysis. This report is the first detailed examination of the qualitative and quantitative properties of chemically modified PNA for nucleic acid detection and provides a platform for studying and optimizing PNA probes prior to incorporation into new technological platforms.

Figure 1

A. General structure of aegPNA showing two nucleobases (B = A, T, G, or C). This is a partial structure that may be extended in either direction to include a specific number of nucleobases. B. Structure of cyclopentane-modified PNA.

Figure 2

Quantitative vs. qualitative detection regimes for PNA probes. In regime A, only the presence of DNA, but not the amount can be detected. In regime B, the presence and amount of DNA can be detected.

Figure 3

Schemes for the two detection methods using PNA probes. A. Sandwich: PNA probes RP1 and SP1 recognize a 27-base sequence of anthrax DNA and form a three-component, non-covalent complex on a plastic surface (step i). Probe RP1 has six biotins on the C-terminal. If the target DNA is present and the complex forms, a color can be developed by introducing a polyhorseradish peroxidase+streptavidin conjugate followed by a solution of tetramethylbenzidine and peroxide (step ii). Enzymatic oxidation initially produces a blue color at 652 nm (step iii), quenching the enzyme with sulfuric acid produces a yellow color at 450 nm (step iv). B. Crosslinked sandwich: PNA probes RP3 and SP3 have reactive maleimide and thiol groups on their N- and C-terminal ends, respectively. A disulfide with 2-pyridylthiol is used as a protecting group for the pendant thiol on SP3. This group is reduced with dithiothreitol immediately following hybridization of the PNA probes with DNA to reveal a pendant thiol that reacts with maleamide on the adjacent PNA (step v).

Figure 4

Detection of DNA using PNA probes SP1 and RP1. A. Absorbance data (652 nm) for detection of TS1 DNA over a range of concentrations and linear regression analyses of maximum velocities (VMax) for each concentration. B. Signal response plot (VMax vs. [DNA]) for each DNA in Table 2 and curve fits for each plot. C. Signal response plot (Absorbance at 450 nm vs. [TS1 DNA]) obtained after quenching of enzymatic oxidation with H₂SO₄.