Book of Abstracts: Albany 2007

category image Albany 2007
Conversation 15
June 19-23 2007

Alkynylpyrene Exciplex-based Split-probe Systems for the Detection of Mutation and Single Nucleotide Polymorphisms

An exci-probe detection system comprises two probe oligonucleotides, which bind to the target nucleic acid at adjacent sites. The prototype consisted of two 8-mer oligonucleotides complementary to adjacent sites of a 16-mer oligonucleotide DNA target (5'pdGCCAAACACAGAATCG-3'), a sequence chosen to avoid formation of a secondary structure (1). The 8-mer probe oligonucleotides are labelled with exciplex partners A and B attached to their internal 5' and 3' phosphate ends, respectively. On binding to the target strand exci-partners A and B become juxtaposed to form an exciplex on irradiation at the appropriate wavelength (Figure 1).

Figure 1: The tandem split-oligonucleotide probe exciplex system, exciprobes.

If A and B are identical (i.e., A=B= pyrene) the system is described as an excimer (2, 3). An exciplex is an excited state complex of an electronically excited donor A* and a ground state acceptor B. Previous work has used 1-pyrenemethylamine and N?-methyl-N?-naphthalen-1-yl-ethanediamine as exciplex partners A and B (Figure 1), giving exciplex emission at 480 nm with a huge Stokes shift (100-150 nm) (1).

Excitation of the prototype was at 350 nm and it would be advantageous to excite out of the UV region. We now describe exciplex partners that achieve this by incorporation of an alkyne moiety into the pyrenyl partner. Phenylalkynylpyrene derivatives 1 and 2 were synthesised via Scheme 1 and attached to the 5' and 3' terminal phosphate groups of 8-mer oligonucleotides. Synthesis of 5 via Sonogashira cross-coupling (4) afforded a versatile precursor to target compounds such as 1 and 2.

Scheme 1: Synthesis of alkynylpyrene exciplex partners.

The oligonucleotide system constructed using phenylalkynylpyrene probes 1 and 2 could be successfully excited at wavelengths from 380-400 nm with exciplex emission at 505 nm. Such probes labeled with the alkynylpyrene moiety have been used in tandem with N?-methyl-N?-naphthalen-1-yl-ethanediamine labelled probes to detect the target DNA sequence. As with the prototype system based on pyrene itself, exciplex emission, when detected, was only produced in the presence of the target. The effect on exciplex fluorescence of mismatches or inserts in the target has also been determined and indicates the potential for SNP detection. We are presently studying the utility of these alkynylpyrene-based exciplex detection systems for human Cytochrome P450 allelic discrimination.

References and Footnotes
  1. Bichenkova, E. V., Savage, H. E., Sardarian, A. R., and Douglas, K. T. Biochem Biophys Res Commun 332, 956-964 (2005).
  2. Paris, P. L., Langenhan, J. M., and Kool, E. T. Nucl Acids Res 26, 3789-3793 (1998).
  3. Ebata, K., Masuko, M., Ohtani, H., and Kashiwasake-Jibu, M. Photochem Photobiol 62, 836-839 (1995).
  4. Malakhov, A. D., Skorobogatyi, M. V., Prokhorenko, I. A., Gontarev, S. V., Kozhich, D. T., Stetsenko, D. A., Stepanova, I. A., Shenkarev, Z. O., Berlin, Y. A., and Korshun, V. A. Eur J Org Chem 1298-1307 (2004).

Laura L. Etchells
Elena V. Bichenkova
Abdul Gbaj
Lindsey Walsh
Kenneth T. Douglas*

Wolfson Centre for Rational Structure-Based Design of Molecular Diagnostics
School of Pharmacy and Pharmaceutical Sciences
University of Manchester
Manchester, M13 9PL, U.K.

*Email: Ken.Douglas@manchester.ac.uk