Factors Impacting Invader-Mediated Recognition of Double-Stranded DNA.

The development of chemically modified oligonucleotides enabling robust, sequence-unrestricted recognition of complementary chromosomal DNA regions has been an aspirational goal for scientists for many decades. While several groove-binding or strand-invading probes have been developed towards this end, most enable recognition of DNA only under limited conditions (e.g.

Serine-γPNA, Invader probes, and chimeras thereof: three probe chemistries that enable sequence-unrestricted recognition of double-stranded DNA.

Three probe chemistries are evaluated with respect to thermal denaturation temperatures, UV-Vis and fluorescence characteristics, recognition of complementary and mismatched DNA hairpin targets, and recognition of chromosomal DNA targets in the context of non-denaturing fluorescence hybridization (nd-FISH) experiments: (i) serine-γPNAs (SγPNAs), , single-stranded peptide nucleic acid (PNA) probes that are modified at the γ-position with ()-hydroxymethyl moieties, (ii) Invader probes, , DNA duplexes modified with +1 interstrand zippers of 2'--(pyren-1-yl)methyl-RNA monomers, a molecular arrangement that results in a violation of the neighbor exclusion principle, and (iii) double-stranded chimeric SγPNAs:Invader probes, , duplexes between complementary SγPNA and Invader strands, which are destabilized due to the poor compatibility between intercalators and PNA:DNA duplexes. Invader probes resulted in efficient, highly specific, albeit comparatively slow recognition of the model DNA hairpin targets. Recognition was equally efficient and faster with the single-stranded SγPNA probes but far less specific, whilst the double-stranded chimeric SγPNAs:Invader probes displayed recognition characteristics that were intermediate of the parent probes.