phosphoramidite and pyrene

Charge transfer across double stranded DNA was observed for the first time about 20 years ago, and ever since it has been the subject of a large number of studies. RNA has been hardly investigated in this regard, which not least is due to the lack of suitably functionalized ribonucleotide building blocks to serve as electron sources upon incorporation into oligoribonucleotides. We have synthesized two uridine derivatives carrying pyrene or dimethylaminopyrene linked to C5 of the nucleobase. The key to successful synthesis was the adaptation of Suzuki-Miyaura conditions to the coupling of the pyrene moiety with the ribonucleoside. Final decoration of the pyrenylated nucleosides with standard 5'-O- and 2'-O-protecting groups and subsequent 3'-O-phosphitylation delivered the building blocks for incorporation into RNA. Spectroscopic analysis of the two pyrenylated uridines and of the accordingly modified oligonucleotides showed that in particular the dimethyaminopyrene functionalized nucleoside is a promising candidate as an electron source for RNA charge transport studies.

Topics: Base Sequence; Chemistry Techniques, Synthetic; Electron Transport; Electrons; Methylation; Organophosphorus Compounds; Pyrenes; RNA; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Uridine

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.

Topics: Alkylation; DNA; Inverted Repeat Sequences; Models, Molecular; Nucleic Acid Conformation; Organophosphorus Compounds; Pyrenes; RNA; RNA Stability; Thermodynamics