Abstract
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.
Highlights
Base modified 20 -deoxyribonucleoside-50 -triphosphates in which a specific reporter group, ligand or catalytic moiety is tethered via a linker to the nucleobase allow the preparation of functionalised nucleic acids that have a wide variety of applications in biotechnology and chemical biology [1]
The deoxyribonucleoside-50 -triphosphates (dNTPs) 5 is a substrate for Taq DNA polymerase [21], whilst nucleotides of general structure 6, which have ODNs of differing length tethered to the base are incorporated into DNA by the engineered polymerase KlenTaq [12]
We have described the chemical synthesis and characterisation of four C5-modified dUTP and two C5-modified dCTP analogues that are functionalised with polyanionic side chains linked to the nucleosides with PEG linkers of differing lengths
Summary
Base modified 20 -deoxyribonucleoside-50 -triphosphates (dNTPs) in which a specific reporter group, ligand or catalytic moiety is tethered via a linker to the nucleobase allow the preparation of functionalised nucleic acids that have a wide variety of applications in biotechnology and chemical biology [1]. DNTPs modified at the C5-position of pyrimidines or C7-position of 7-deazapurine bases are good substrates for most DNA polymerases since the modification does not interfere with Watson Crick base pairing. In addition these modifications are in the major groove whilst most protein-DNA interactions during nucleotide incorporation arise in the minor groove [2]. The Marx group have described [11] the successful incorporation of a dendrimer modified dUTP into an oligodeoxynucleotide (ODN) using KlenTaq DNA polymerase and a C5-ODN-modified dUTP using Therminator DNA polymerase [12]
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