Ferrocene-peptide adducts for DNA binding: Towards sequence-selective electrochemical DNA sensors

DNA is crucial to life on earth, as it contains the genetic information which defines the characteristics of any living thing, from plankton to humans. It is made up of sequences of smaller building blocks, called nucleobases. This sequence defines both the genetic code the DNA contains and also its physical structure, which is most commonly a double helix - a bit like a ladder, coiled into a spiral.

Scientists have successfully decoded the entire sequence of human DNA and in turn have been able to link specific genes - discrete stretches of a DNA sequence - to particular diseases. This offers the potential to selectively identify genes which cause disease and offer new therapies. However, as the human genome alone contains around 20,000 genes, it is very important to be able to selectively and reliably recognise and signal the presence of individual genes. The type of molecule or device that allows us to do this is called a Sensor.

Sensors typically comprise two parts; recognition units which are highly selective for the desired target, and a reporter group which allows a measureable and quantifiable output. Chemists have had significant success in developing sensors to analyse small molecules, for instance pollutants and drug molecules. However, these molecules typically interact with sensors on the atomic level, whereas DNA and other biomolecules interact at a level 10 times larger than this, so a new class of sensor needs to be developed.

Nature uses DNA-binding proteins to "read" the genetic information stored in the DNA sequence. Proteins are long chains of smaller building blocks called amino acids that fold into well-defined structures, which are important for biomolecular recognition. Though these proteins are large and complicated molecules, the majority bind to the major groove of double helix DNA using a relatively small helical sequence, which is cylindrical in shape.

It is our intention to capture this DNA binding strength and selectivity in a significantly simpler (easily synthesised) system, by preparing a minimalist protein sequence derived from a DNA-binding helix. This will then be coupled to a chemical reporter group to provide the user with a measurable output sensitive to DNA binding.

Due to its attractive properties and ease of modification, ferrocene is our reporter group of choice. We propose to use a core ferrocene unit and couple it to two DNA-binding helices which will be taken from the protein GCN4. This coupling will be achieved through the side chain of cysteine, a natural amino acid which we will introduce into the GCN4 sequence. This synthesis will lead to the development of novel, sequence-selective DNA biosensors, and provide an approach which is more widely applicable.

These miniature ferrocene-protein biosensors would offer advantages over current DNA sensors as they would be capable of selecting for both particular sequences and for double helix DNA. Other advantages would include being able to mimic the biological function of the DNA binding protein from which the sensor is derived, while providing a signal output, allowing us to monitor important biological processes taking place in real time.

These new sensors will drive increased understanding of how nature recognises genes within DNA, and in doing so will contribute greatly to efforts to develop new medical treatments and diagnostics.

In this research we will develop bioorganometallic electrochemical sensors for sequence selective detection of duplex DNA. Our unique approach utilises the sensor unit, here based on ferrocene, as the dimerisation domain of two biomolecules necessary for specific and strong binding to the target. The proposed functional peptides offer advantages over current electrochemical DNA sensors, the majority of which rely on hybridisation with a complementary single strand of DNA to achieve sequence selectivity. Our approach differs in that intact duplex DNA will be detected sequence selectively.

This is a proof-of-concept proposal and as such the immediate impact is scientific advancement and new knowledge, particularly in the fields of peptide design, DNA sensing and bioorganometallic chemistry. The PI, PDRA and undergraduate MSci students will be the direct beneficiaries of this research, with the PI using this grant, applied for under the First-Grant Scheme, to launch her independent research programme and academic career. However, we also anticipate non-academic beneficiaries.

With the recent sequencing of the human genome, and with links increasingly being established between specific genes and disease states, there is significant need to- and interest in-developing sequence-selective DNA sensors for analytical and diagnostic applications. The development of a new product capable of sequence-selectively detecting human, viral or bacterial DNA, would have huge potential for commercial exploitation. The very choice of an electrochemical sensor (as opposed to a fluorescent sensor) should allow for the development of low cost, user friendly, portable devices for use as diagnostic tools. In addition to the need for DNA sensors, there is also a potential therapeutic opportunity in developing small artificial transcription factor mimics capable of promoting or suppressing the expression of important genes associated with a disease. The advantages of synthetic peptides are such that we anticipate their incorporation into diagnostics and therapeutics will become routine. Clearly this work could have medical, diagnostic and economic impact in the long-term (ca. 20 years).

Any exploitable intellectual property as a result of this one year proof-of-concept proposal, will be discussed with Alta Innovations Ltd, the technology transfer company for the University of Birmingham, who aid in the commercialisation of intellectual property. Efforts will be taken to secure intellectual property for our work where appropriate and to ensure that suitable industrial partners and end-user groups are identified in consultation with Alta Innovations and the School of Chemistry's Advantage West Midlands Science City Business Engagement Manager.

In addition to the new knowledge and potential diagnostics and therapeutics described above, society will benefit from the training of a highly skilled PDRA in a very multidisciplinary project. The PDRA is expected to obtain the necessary skill set for a career in academia or industry. MSci undergraduate project students will also benefit by working on related projects, providing "added value", and will obtain important scientific training and exposure to academic research.