Towards electrochemically controlled nucleic acid-amplification strategies

One of the critical functions of DNA is its ability to undergo conformational change, more precisely the association (hybridisation) and disassociation (denaturation) of the double helix. While not only indispensable inside the cell, many invaluable molecular biology technologies, across many different disciplines, that exploit and detect DNA rely on this reversible function. These include for example the polymerase chain reaction (PCR, DNA biosensor and next generation sequencing for diagnostics for healthcare, biomedical research, forensics, environmental monitoring, and food and agricultural industry.

Many of these DNA-based technologies rely on the large quantities of the genetic material. This can be achieved via various biochemical reactions, such PCR. Technologies exploiting production of large quantities of DNA is a rapidly growing area in life sciences in which the dominating technology is PCR. However, to copy and produce large quantities of DNA, PCR requires considerable technical instrumentation. This is because the biochemical reaction, based on the reversible association and dissociation of DNA, is driven by precise regulation of three distinct elevated temperatures. This prerequisite significantly compromises the use of PCR outside well equipped laboratories. As there is an increasing demand in making DNA-based testing portable and available outside a centralised laboratory setting, the development of these technologies is driven towards miniaturisation and integration of standard laboratory procedures into lab-on-a-chip systems. However, integration of the standard temperature-regulated PCR reaction has proven to be challenging due to the requirement and complexity of the precise temperature regulation to drive the reaction. This has thus far precluded the realisation of truly decentralised miniaturised DNA-based analytical systems.

We have recently demonstrated that we can reversibly control the association and dissociation of DNA, at a constant temperature, by means of electrochemistry. The fundamental control is based on an electrochemically switchable small DNA binding chemical compound named daunomycin. As no extreme conditions were implicated, and the precise temperature-regulation of reversible association and dissociation of DNA was circumvented, this finding has great potential to simplify future developments of miniaturised portable DNA-based analytical systems.

This project proposes a radically new way of controlling the association and dissociation of DNA as a new tool to control all types of biological reactions that rely on reversible DNA hybridisation events. A wide range of biological applications can make use of this new tool and, thus, addresses any of the strategic priorities of BBSRC relying on detection and testing based on DNA. To pump prime these developments and to show the proof-of-principle, we aim to apply this new tool to develop electrochemically controlled PCR (ePCR).

An initial study in our research group showed that the conditions utilised for the electrochemical control of association and dissociation of DNA was compatible with the standard PCR reaction conditions. Furthermore, it proved that the PCR reaction was not inhibited by the presence of the electroactive DNA binding compound daunomycin.

This early-stage investigation of a novel concept, we believe, is vital for the development and commercial success of a low-energy consuming portable DNA-based analytical platforms. As the current method is based electrochemical control, it offers a simpler, integration-friendly and cost-effective alternative to current technologies.

Nucleic hybridisation is core to many biological processes and protocols used in molecular biology such as nucleic acid amplification, e.g. by PCR. This project aims to radically simplify nucleic acid amplification by driving the reaction via means of electrochemistry. To fulfil this aim, specialised expertise in biosensors, physical chemistry, biophysics and microsystems engineering is brought together.

We have recently demonstrated reversible, isothermal control of DNA denaturation and renaturation by means of electrochemistry using the electroactive DNA intercalator daunomycin. The operational principle has been successfully demonstrated, with UV-vis and circular dichroism spectroelectrochemistry, using a model system of complementary synthetic DNA strands.

We propose a radically new way of controlling reversible hybridisation of nucleic acids as a novel tool to control potentially all types of biological reactions involving nucleic acid base pairing. A wide range of biological applications can make use of this new tool and, thus, addresses any of the strategic priorities of BBSRC relying on nucleic acid-based testing. To pump prime these developments and to show the proof-of-principle, we aim to apply this new tool to develop electrochemically controlled PCR (ePCR). Preliminary investigations confirmed that daunomycin does not inhibit PCR.

The proof-of-principle will be demonstrated on screen-printed electrodes via electrolysis. Standard PCR carried out in thermal cyclers will be employed as benchmark for comparison and assessment of ePCR. Gel electrophoresis will be utilised as the main method for amplification analysis. Furthermore, for optimisation, in-depth biophysical characterisation of the intercalator-DNA complex will be carried out using isothermal calorimetry and NMR. A suitable real-time electrochemical detection method will be indentified.

The proposed ePCR project is targeting the field of nucleic acid amplification by polymerase chain reaction (and related technologies): one of the most fundamental and central areas to modern life sciences.

The ePCR principle is taking a radically different view on an established method many life science researchers use in their daily life. As for PCR, ePCR and connected areas will enable researchers to enhance their understanding and knowledge of the method itself, the systems they are investigating and enable researchers to develop new diagnostic tools all supporting a continued development towards a sustainable bioeconomy. We furthermore expect ePCR to be simpler and less resource demanding making it likely that the method will be adopted in resource poor environments. Because of its widespread applicability, ePCR is well suited to be used in teaching and training in order to enhance knowledge and expertise of current and future researchers. As our interdisciplinary proposal demonstrates, ePCR is a method involving researchers from biological, biochemical, physical, chemical and engineering disciplines.

The economic impact of ePCR relates to its use as a tool for basic research and/or as part of a diagnostic application. The strongest commercial potential for ePCR is likely to be in the field of DNA amplification technologies. Gene Amplification Technology or Nucleic Acid Amplification Testing (NAAT) is a rapidly growing area in life sciences and the market for PCR is projected in the multibillion US$ range. A simplified NAAT test using ePCR, with less instrumental requirements, would be of significant competition to established PCR tests. With an increasing demand of making molecular diagnostics portable and available outside a clinical setting, e.g for Global Health, we would expect that ePCR would initially address the Point of Care Testing market segment. ePCR based NAAT systems would enter a market with major global players such as Abbott, Becton Dickinson, bioMerieux, Bio-Rad, Cepheid, Life Technologies, Qiagen, Roche Diagnostics, Rubicon Genomics, Takara Bio, etc.. These companies are also major potential partners for deals ranging from licensing to acquisition. We have already established close contact with several companies, including Axis Shield/Alere, SELEX Galileo, LifeScan, Mölnlycke Health Care Scotland and Life Technologies and will include these in our activities to maximise the impact of the proposed study. In addition we are part of the £10m Sensor and Imaging Systems Innovation Centre (CENSIS) which is one of three newly launched Scottish Innovations Centres aiming to bridge the gap between academic research and economic impact.

In addition to raising awareness for alternative PCR methods in scientific and business community, we will use our established routes for public engagement and dissemination of the ePCR project outcomes in the wider community. This will be as a part of the occasional series of events open to the general public, and publicised to local schools, held by the Division of Pathway Medicine, UoE. Furthermore, we will make use of the Beltane Public Engagement Network at the University of Edinburgh for public outreach.