title: Recognition of double-stranded DNA using energetically activated duplexes with interstrand zippers of 1-, 2- or 4-pyrenyl-functionalized O2'-alkylated RNA monomers.
abstract: Despite advances with triplex-forming oligonucleotides, peptide nucleic acids, polyamides and--more recently--engineered proteins, there remains an urgent need for synthetic ligands that enable specific recognition of double-stranded (ds) DNA to accelerate studies aiming at detecting, regulating and modifying genes. Invaders, i.e., energetically activated DNA duplexes with interstrand zipper arrangements of intercalator-functionalized nucleotides, are emerging as an attractive approach toward this goal. Here, we characterize and compare Invaders based on 1-, 2- and 4-pyrenyl-functionalized O2'-alkylated uridine monomers X-Z by means of thermal denaturation experiments, optical spectroscopy, force-field simulations and recognition experiments using DNA hairpins as model targets. We demonstrate that Invaders with +1 interstrand zippers of X or Y monomers efficiently recognize mixed-sequence DNA hairpins with single nucleotide fidelity. Intercalator-mediated unwinding and activation of the double-stranded probe, coupled with extraordinary stabilization of probe-target duplexes (ΔT(m)/modification up to +14.0 °C), provides the driving force for dsDNA recognition. In contrast, Z-modified Invaders show much lower dsDNA recognition efficiency. Thus, even very conservative changes in the chemical makeup of the intercalator-functionalized nucleotides used to activate Invader duplexes, affects dsDNA-recognition efficiency of the probes, which highlights the importance of systematic structure-property studies. The insight from this study will guide future design of Invaders for applications in molecular biology and nucleic acid diagnostics.
authors.affiliation: Department of Chemistry, University of Idaho, Moscow, ID 83844, USA. hrdlicka@uidaho.edu.
authors.forename: Saswata
authors.initials: S
authors.lastname: Karmakar
authors.forename: Andreas S
authors.initials: AS
authors.lastname: Madsen
authors.forename: Dale C
authors.initials: DC
authors.lastname: Guenther
authors.forename: Bradley C
authors.initials: BC
authors.lastname: Gibbons
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Hrdlicka
mesh_terms: D000478:Alkylation; D004247:DNA / Q000737:chemistry*; D055029:Inverted Repeat Sequences; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009943:Organophosphorus Compounds / Q000737:chemistry; D011721:Pyrenes / Q000737:chemistry*; D012313:RNA / Q000737:chemistry*; D020871:RNA Stability; D013816:Thermodynamics
chemical_list: D009943:Organophosphorus Compounds; D011721:Pyrenes; C434331:phosphoramidite; D012313:RNA; D004247:DNA; C030984:pyrene