Functionalisation of Designer DNA for Nanomaterial Applications via “click” chemistry

Abstract

An enzymatic approach to extend DNA to give designer products of a predefined length, sequence, and functionality has been investigated. This method facilitates the extension of repetitive DNA units through repeated cycles of heating and cooling. The key elements of the reaction mixture encompass the predefined oligoseed, a thermostable DNA polymerase, and the deoxynucleotide triphosphates (dNTPs). By conducting 20 heat-cool cycles of this enzymatic method, long DNA strands of a pre-determined sequence that exceed 20,000 base pairs were obtained. The incorporation of an unnatural nucleotide, 5-C8-alkyne-dCTP, into the extending DNA during the self-priming PCR synthesis was reported. The C5 -alkyne modification is located in the major groove and was found to have a negligible impact on the DNA polymerase activity. Therefore, by tuning the starting oligoseed, long designer DNA with functionalities at pre determined positions can be synthesised. DNA carrying the 5- C8- alkyne-dCTP provides alkynyl anchoring points for further modification and space within the major groove. To explore the potential for additional functionalisation, click chemistry using azide-fluor-545 was examined. This self-priming PCR DNA synthesis approach in conjunction with non-standard dNTPS demonstrated the potential for the advancement of DNA modification via organic synthesis at targeted base positions along a designer DNA template. In a second series of experiments, click chemistry and the self-priming PCR synthesis method was again exploited to produce a series of DNA-based hydrogel-like materials. Click chemistry was performed to crosslink alkyne-bearing DNA, that had been loaded with a range of levels of alkynly modifications, with a series of diazide polyethylene glycols (PEG), N3[PEG]nN3 where n ranged from 1-9. Varying the length of the diazide-modified PEG crosslinker allowed for control over the properties of the DNA-based hydrogel materials. Self-priming PCR was also used to synthesise extended DNA that contained multiple repeats of the sequence that encodes for an elastin-like polypeptide (ELP). The predefined starting oligoseed contained two repeats of a DNA sequence that codes for the pentapeptide repetitive sequence VPGVG typical of ELPs. Extended ELPs-based DNA samples were carefully purified and then a range of specific lengths were isolated using a double well- gele electrophoresis set-up. Samples ranging in length from 100-1500 bp, were sent to collaborators for the biological expression of elastin-like protein. Three oligoseed sequences that contained 6, 19 and 22 base-repeats were designed based on naturally occurring sequences found inside cells and are known for G-quadraplex formation. Each sequence was extended using the same self-priming PCR approach and a detailed AFM investigation of their structure upon addition of two Pt (II) complexes that are known to stabilise G-quadraplexes was performed. As the oligoseed sequences are guanine rich, the potential to form G-tetrad structures in the absence/presence of to these extended oligoseeds was investigated. Overall, the modification of DNA that was produced from carefully designed oligoseed sequences using the self-priming PCR synthetic approach, demonstrates the potential of this synthetic strategy in the production of site-specific modified DNA, DNA hydrogel materials, novel biomaterials and as a tool to study the structural effects of metal complex binding to biologically important DNA sequences.