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