RESUMO
Modified 2'-deoxyribonucleotide triphosphates (dNTPs) have widespread applications in both existing and emerging biomolecular technologies. For such applications it is an essential requirement that the modified dNTPs be substrates for DNA polymerases. To date very few examples of C5-modified dNTPs bearing negatively charged functionality have been described, despite the fact that such nucleotides might potentially be valuable in diagnostic applications using Si-nanowire-based detection systems. Herein we have synthesised C5-modified dUTP and dCTP nucleotides each of which are labelled with an dianionic reporter group. The reporter group is tethered to the nucleobase via a polyethylene glycol (PEG)-based linkers of varying length. The substrate properties of these modified dNTPs with a variety of DNA polymerases have been investigated to study the effects of varying the length and mode of attachment of the PEG linker to the nucleobase. In general, nucleotides containing the PEG linker tethered to the nucleobase via an amide rather than an ether linkage proved to be the best substrates, whilst nucleotides containing PEG linkers from PEG6 to PEG24 could all be incorporated by one or more DNA polymerase. The polymerases most able to incorporate these modified nucleotides included Klentaq, Vent(exo-) and therminator, with incorporation by Klenow(exo-) generally being very poor.
Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Nucleotídeos de Desoxicitosina/metabolismo , Nucleotídeos de Desoxiuracil/química , Polietilenoglicóis/químicaRESUMO
Supramolecular polymer nanowires have been prepared by using DNA-templating of 2,5-(bis-2-thienyl)-pyrrole (TPT) by oxidation with FeCl(3) in a mixed aqueous/organic solvent system. Despite the reduced capacity for strong hydrogen bonding in polyTPT compared to other systems, such as polypyrrole, the templating proceeds well. FTIR spectroscopic studies confirm that the resulting material is not a simple mixture and that the two types of polymer interact. This is indicated by shifts in bands associated with both the phosphodiester backbone and the nucleobases. XPS studies further confirm the presence of DNA and TPT, as well as dopant Cl(-) ions. Molecular dynamics simulations on a [{dA(24):dT(24)}/{TPT}(4)] model support these findings and indicate a non-coplanar conformation for oligoTPT over much of the trajectory. AFM studies show that the resulting nanowires typically lie in the 7-8â nm diameter range and exhibit a smooth, continuous, morphology. Studies on the electrical properties of the prepared nanowires by using a combination of scanned conductance microscopy, conductive AFM and variable temperature two-terminal I-V measurements show, that in contrast to similar DNA/polymer systems, the conductivity is markedly reduced compared to bulk material. The temperature dependence of the conductivity shows a simple Arrhenius behaviour consistent with the hopping models developed for redox polymers.
Assuntos
Cloretos/química , DNA/química , Compostos Férricos/química , Nanofios/química , Polímeros/química , Pirróis/química , Pirróis/síntese química , Condutividade Elétrica , Conformação Molecular , Oxirredução , Espectroscopia de Infravermelho com Transformada de Fourier , TemperaturaRESUMO
DNA templated nanowires of a pentynyl-modified poly2-(2-thienyl)-pyrrole undergo functionalisation via"click chemistry" and retain their 1D-nanostructure and conductive properties.