A Novel Method of Oligonucleotide Detection Using Diels Alder Cycloaddition and SERRS
Ljiljana Fruk, Duncan Graham, W. Ewen Smith
Department of Pure and Applied Chemistry
University of Strathclyde
With the completion of the human genome project, a growing number of scientists are trying to use the information to find a way of identifying genetic diseases linked to alternations in DNA. This requires screening of different DNA sequences for specific sequence characterisation in a high throughput manner. In modern DNA analysis fluorescent labelling is the most favoured DNA detection technique. However, addition of fluorescent labels can be disadvantageous since most of these dyes have appreciable biological activity, which leads to reduced activity or even total inactivity of the probe.
Our main goal is to develop a novel method of labelling biologically important molecules for detection by surface enhanced resonance Raman scattering (SERRS). We seek to develop a method that allows real time analysis of DNA with complete selectivity at ultra low concentrations which will enable complex biological analysis. The first step of our approach was the synthesis of a number of specific azo dyes containing benzotriazole group and a maleimide moiety. Benzotriazole groups are known to complex strongly to the metal surface, which is one of the requirements for SERRS. The maleimide group in turn serves as a connection point to the molecule of interest (DNA, peptide). To achieve the coupling of the dye to DNA, a synthetic oligonucleotide was derivatised in 5’ position with furan moiety. Diels Alder reaction has been used to attach SERRS active BT maleimide dye to a furan-tagged oligonucleotide in water and reaction was followed by HPLC. The resulting modified oligonucleotide was found to be SERRS active and gave a distinctly different spectrum to that of the dye.
This is a totally new approach to biological labelling and shows excellent promise in terms of selectivity, sensitivity and simplicity. The furan tagged species can be converted to a labelled species at low concentration levels and can be followed by SERRS to show the presence of oligonucleotide sequence. The reaction is fast and selective to the tag. It is a convenient method of labelling oligonucleotides in an aqueous environment, required for biological substrates, to produce a distinctive species that does not require separation from the starting material prior to analysis.
Now we have developed a method of labelling oligonucleotides in such a way that the identity of the sequence is known. This has obvious implications in the rapidly growing DNA microarray area. Furthermore, this method can be effective for many other targets like proteins, leading to the development of effective protein arrays. Currently under investigation is the labelling of the molecular beacons, which are single stranded DNA molecules used in dynamic, real time detection of nucleic acid hybridisation both in vitro and in vivo.