Synthesis of new building blocks for use in supramolecular DNA architectures

New nucleoside building blocks for the synthesis of functional DNA are presented. A porphyrin-bis nucleoside dU-porphyrin-dU was synthesised from a di-acetylene-substituted porphyrin using Sonogashira coupling with 5-iodo deoxy uridine. The same strategy was used to obtain a new terpy-functionalised nucleoside dUterpy. This building block can be metallated with ruthenium(II) either to make a mono-nucleoside ruthenium complex (dUterpy)RuII(terpy), or to connect two building blocks to create a bis-nucleoside (dUterpy)2RuII. The terpy nucleoside building block dUterpy was incorporated into short strands of DNA to give TXT, TXXT and TXXXT as sequences (X = dUterpy). The functionalised DNA has the potential to create supramolecular assemblies through metal complexatio

Discrimination against the cytosine analog tC by Escherichia coli DNA polymerase IV DinB.

The cytosine analog 1,3-diaza-2-oxophenothiazine (tC) is a fluorescent nucleotide that forms Watson-Crick base pairs with dG. The Klenow fragment of DNA polymerase I (an A-family polymerase) can efficiently bypass tC on the template strand and incorporate deoxyribose-triphosphate-tC into the growing primer terminus. Y-family DNA polymerases are known for their ability to accommodate bulky lesions and modified bases and to replicate beyond such nonstandard DNA structures in a process known as translesion synthesis. We probed the ability of the Escherichia coli Y-family DNA polymerase DinB (Pol IV) to copy DNA containing tC and to incorporate tC into a growing DNA strand. DinB selectively adds dGTP across from tC in template DNA but cannot extend beyond the newly formed G:tC base pair. However, we find that DinB incorporates the tC deoxyribonucleotide triphosphate opposite template G and extends from tC. Therefore, DinB displays asymmetry in terms of its ability to discriminate against the modification of the DNA template compared to the incoming nucleotide. In addition, our finding that DinB (a lesion-bypass DNA polymerase) specifically discriminates against tC in the template strand may suggest that DinB discriminates against template modifications in the major groove of DNA

CG base pair recognition within DNA triple helices by modified N-methylpyrrolo-dC nucleosides.

3-Aminophenyl-modified analogues of the bicyclic nucleoside N-methyl-3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one were synthesised and incorporated directly into triplex-forming oligonucleotides in order to utilise their extended hydrogen bonding motif for recognition of the CG base pair. All analogues demonstrated strong binding affinity and very good selectivity for CG from pH 6.2 to 7.0; a marked improvement on previous modifications

DNA as supramolecular scaffold for porphyrin arrays on the nanometer scale

Tetraphenyl porphyrin substituted deoxyuridine was used as a building block to create discrete multiporphyrin arrays via site specific incorporation into DNA. The successful covalent attachment of up to 11 tetraphenyl porphyrins in a row onto DNA shows that there is virtually no limitation in the amount of substituents, and the porphyrin arrays thus obtained reach the nanometer scale (~10 nm). The porphyrin substituents are located in the major groove of the dsDNA and destabilize the duplex by Tm 5-7 °C per porphyrin modification. Force-field structure minimization shows that the porphyrins are either in-line with the groove in isolated modifications or aligned parallel to the nucleobases in adjacent modifications. The CD signals of the porphyrins are dominated by a negative peak arising from the intrinsic properties of the building block. In the single strands, the porphyrins induce stabilization of a secondary helical structure which is confined to the porphyrin modified part. This arrangement can be reproduced by force-field minimization and reveals an elongated helical arrangement compared to the double helix of the porphyrin-DNA. This secondary structure is disrupted above ~55 °C (Tp) which is shown by various melting experiments. Both absorption and emission spectroscopy disclose electronic interactions between the porphyrin units upon stacking along the outer rim of the DNA leading to a broadening of the absorbance and a quenching of the emission. The single-stranded and double-stranded form show different spectroscopic properties due to the different arrangement of the porphyrins. Above Tp the electronic properties (absorption and emission) of the porphyrins change compared to room temperature measurements due to the disruption of the porphyrin stacking at high temperature. The covalent attachment of porphyrins to DNA is therefore a suitable way of creating helical stacks of porphyrins on the nanometer scale