Luminescent polymer-PNA optical assays for the detection of nucleotide mutations associated with resistant leukaemia.

Lead Research Organisation: Durham University
Department Name: Physics

Abstract

As we explore the human genome we are finding more and more that single nucleotide mutations of the DNA gene code can cause major illnesses. Detecting a single base error amongst hundreds of thousands of bases can be daunting; however because of the unique coding of the base sequence, it is possible to identify a unique strand of DNA using a small length of complimentary DNA or analogous protein nucleic acid (PNA). The problem then becomes how to detect that the PNA has correctly bound to its target DNA strand. Here we use a new technique. A highly fluorescent water soluble conjugated polymer is used as an optical antenna. These polymers bind electrostatically to DNA and when we shine light onto them they absorb very strongly. If the PNA has also bound to the target DNA, and the PNA has a chemically attached fluorescent label, we can design the polymer and fluorescent label in such a fashion that the polymer readily transfers its excited state energy to the fluorescent label. This only happens though over very short distances, much less than the length of the DNA and so the energy transfer can only occur when the PNA is bound to the DNA/polymer pair. We than measure emission from the PNA s label to tell that the PNA has read the nucleotide sequence of the DNA. Because the polymer chain can be long and has very high absorption strength we can achieve optical amplification for the detection of approaching 1 million, which gives us a very sensitive tool indeed to read DNA sequences.

Technical Summary

Luminescent polymers are driving forward a new generation of cheap, flexible displays based on their excellent photoluminescence properties combined with high stability and simple chemical tailoring. These properties can be exploited in other fields and, through the advent of water soluble luminescent polymers, applications in the area of biological sciences are now possible. Both anionic and cationic polymers can be synthesised and, via their charged nature, can bind readily electrostatically to DNA. With the addition of appropriate peptide nucleic acid (PNA) it becomes possible to ?detect? the specific nucleotide sequence of a segment of the DNA, using cheap, simple and fast optical detection. The polymer acts as an antenna for the system, strongly absorbing light in the violet, with the exciton thus created able to migrate along the length of the polymer chain. If a complimentary, fluorophore-labelled, PNA is also bound to the DNA strand, rapid and efficient Forster Energy transfer can occur, transferring the excitation energy from the polymer to the PNA label. This emission can then be detected and used to signal that the PNA has specifically bound to its target DNA. Because of the optical properties of the polymer, ?optical? amplification of up to one million-fold is possible for the entire assay. Currently in the Physics Department at Durham we are working to understand and optimise the full photophysics of this complex system. In order to extend the work into real application areas we need to understand the hybridisation interactions between DNA and PNA, how to optimise this in the presence of the luminescent polymer and how we should improve the polymer to optimise it for the assay application. Our initial assay is tailored specifically at tackling well known problems in nucleotide mutation detection so we need to gain experience working with it in a clinical environment. A Discipline Hopping award would be an ideal opporunity to take us on this next step and to maximize the potential of the physics when applied to biological analysis in an area of real clinical need.

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