19. Synthesis, Characterization, and Potential Applications of Biotinylated Energy Transfer Oligonucleotides 

Jin Xie¹, Richard A. Mathies², and Alexander N. Glazer¹ 
Departments of ¹Molecular and Cell Biology and ²Chemistry, University of California, Berkeley, CA 94720 
Glazer@uclink4.berkeley.edu 

Energy transfer (ET) fluorescent dye-labeled primers have provided a decadic improvement in the performance of DNA sequencers for high-throughput sequencing^1,2. The acceptor emissions of high spectral purity also make ET primers ideal for diagnostic applications, such as forensic identification and genetic typing of short tandem repeats³. Biotin has an extraordinarily high affinity for streptavidin with a reported dissociation constant of ~10-15. This very strong binding affinity has made the biotin-streptavidin system very attractive for a multitude of in vitro labeling applications. We describe here the synthesis and characterization of biotinylated fluorescent ET reagents. Hung et al. have shown that CYA-ROX primers with a donor-acceptor spacing of 8-10 nucleotides offer excellent acceptor emission intensities coupled with negligible donor emissions^4-7. We have synthesized oligonucleotides with the sequence 5'-CYA- NNNNNNNNNTROXNNTBNNNNNNN-3' with donor-acceptor fluorophore pairs separated by 10 intervening nucleobases, but varying in the location of TB, in this example introduced two bases 3' to the base carrying the acceptor ROX. Biotin-labeled T (TB) was introduced by the use of biotin-dT phosphoramidite at different locations in the oligonucleotides. CYA, 3-(e-carboxypentyl)- 3'-ethyl-5,5'-dimethyloxacarbo-cyanine, a dye with a high absorption cross-section but a low fluorescence quantum yield, was chosen as an energy donor at the 5'-end of the oligonucleotides, and ROX as an acceptor was attached to a modified thymidine (TROX). We have compared the quantitative spectroscopic properties of four biotinylated ET reagents differing in the spacing between donor-biotin pairs and acceptor-biotin pairs. CYA10ROX- 2-Biotin (where 2 is the number of nucleotides between the acceptor and biotin) reagent offers the best combination of acceptor fluorescence emission intensity and spectral purity. With 488-nm excitation, the fluorescence emission intensity of C10R-2-Biotin is 16-fold stronger than that of the corresponding oligonucleotide labeled with the acceptor ROX as the only dye. These biotinylated ET reagents have a broad range of potential applications, e.g., affinity purification and detection in DNA mapping applications on chips, and in cell sorting⁸. For such purposes, we have prepared and characterized ET-oligonucleotide-streptavidin conjugates for use in multiplexed assay systems. 

¹J. Ju, A.N. Glazer, and R.A. Mathies Nature Medicine 2, 246-249 (1996). 

²A.N. Glazer and R.A. Mathies Curr. Opinion Biotechnol. 8, 94-102 (1997). 

³Y. Wang, S-C. Hung, J.F. Linn, G. Steiner, A.N. Glazer, D. Sidransky, and R.A. Mathies Electrophoresis 18, 1742-1749 (1997). 

⁴S-C. Hung, J. Ju, R.A. Mathies, and A.N. Glazer Anal. Biochem. 243, 15-27 (1997). 

⁵S-C. Hung, J. Ju, R.A. Mathies, and A.N. Glazer Anal. Biochem. 238, 165-170 (1996) 

⁶S-C. Hung, R.A. Mathies, and A.N. Glazer Anal. Biochem. 252, 78-88 (1997). 

⁷S-C. Hung, R.A. Mathies, and A.N. Glazer Anal. Biochem. 255, 32-38 (1998). 

⁸See abstract "Integrated Sequencing Sample Preparation on CE Microplate" by Y. Shi, I. Kheterpal, J. Xie, A.N. Glazer and R.A. Mathies.