Sequence specificity at targeting double-stranded DNA with a γ-PNA oligomer modified with guanidinium G-clamp nucleobases

摘要

γ-PNA, a new class of peptide nucleic acids, promises to overcome previous sequence limitations of double-stranded DNA (dsDNA) targeting with PNA. To check the potential of γ-PNA, we have synthesized a biotinylated, pentadecameric γ-PNA of mixed sequence carrying three guanidinium G-clamp nucleobases. We have found that strand invasion reactions of the γ-PNA oligomer to its fully complementary target within dsDNA occurs with significantly higher binding rates than to targets containing single mismatches. Association of the PNA oligomer to mismatched targets does not go to completion but instead reaches a stationary level at or below 60%, even at conditions of very low ionic strength. Initial binding rates to both matched and mismatched targets experience a steep decrease with increasing salt concentration. We demonstrate that a linear DNA target fragment with the correct target sequence can be purified from DNA mixtures containing mismatched target or unrelated genomic DNA by affinity capture with streptavidin-coated magnetic beads. Similarly, supercoiled plasmid DNA is obtained with high purity from an initial sample mixture that included a linear DNA fragment with the fully complementary sequence. Based on the results obtained in this study we believe that γ-PNA has a great potential for specific targeting of chosen duplex DNA sites in a sequence-unrestricted fashion.