Electrochemical control of reversible DNA hybridisation : for future use in nucleic acid amplification

Denaturation and renaturation is indispensable for the biological function of nucleic acids in many cellular processes, such as for example transcription for the synthesis of RNA and DNA replication during cell division. However, the reversible hybridisation of complementary nucleic acids is equally crucial in nearly all molecular biology technologies, ranging from nucleic acid amplification technologies, such as the polymerase chain reaction, and DNA biosensors to next generation sequencing. For nucleic acid amplification technologies, controlled DNA denaturation and renaturation is particularly essential and achieved by cycling elevated temperatures. Although this is by far the most commonly used method, the management of rapid temperature changes requires bulky instrumentation and intense power supply. These factors so far precluded the development of true point-of-care tests for molecular diagnostics. This Thesis explored the possibility of using electrochemical means to control reversible DNA hybridisation by using electroactive intercalators. First, fluorescence-based melting curve analysis was employed to gain an in depth understanding of the reversible process of DNA hybridisation. Fundamental properties, such as stability of the double helix, were investigated by studying the effect of common denaturing agents, such as formamide and urea, pH and monovalent salt concentration. Thereafter, four different electroactive intercalators and their effect on the thermodynamic stability of duplex DNA were screened. The intercalators investigated were methylene blue, thionine, daunomycin and adriamycin. Absorbance-based melting curve analysis revealed a significant increase of the melting temperature of duplex DNA in the presence of oxidised daunomycin. This was not observed in the presence of chemically reduced daunomycin, which confirmed the hypothesis that switching of the redox-state of daunomycin altered its properties from DNA binding to non-binding. Accordingly this altered the thermodynamic stability of duplex DNA. The difference in the stability of duplex DNA, as a direct result of the redox-state of daunomycin, was exploited to drive cyclic electrochemically controlled DNA denaturation and renaturation under isothermal conditions. This proof-of-principle was demonstrated using complementary synthetic 20mer and 40mer DNA oligonucleotides. Analysis with in situ UV–vis and circular dichroism spectroelectrochemistry, as two independent techniques, indicated that up to 80 % of the duplex DNA was reversibly hybridised. Five cycles of DNA denaturation and renaturation were achieved and gel electrophoresis as well as NMR showed no degradation of DNA or daunomycin. As no extreme conditions were implicated, no covalent modification of DNA was required and isothermal conditions were kept, this finding has great potential to simplify future developments of miniaturised and portable bioanalytical systems for nucleic acid-based molecular diagnostics