June 12, 2008
In collaboration with AM Biotechnologies, the thioaptamer company, the Oligo Factory is now offering phosphorodithioate oligos (PS2-oligos) which provide for an achiral internucleotide phosphodiester group with two sulfurs at the two non-bridging positions.
What are the attributes and applications for phosphorodithioate oligonucleotides (PS2-oligo)?
PS2-oligos have potentially useful characteristics such as 1) high binding affinities to proteins and cell surfaces; 2) are nuclease resistant and therefore stable in biological preparations; 3) are easily prepared; and 4) as thioaptamers show excellent specificity to proteins (4). Significantly, the PS2-oligos, in contrast to the monothiophosphate oligos (S-oligo), are achiral about the dithiophosphate center, so problems associated with diastereomers are completely avoided.
Protein specificity and affinity: Oligos with high proportions of phosphorothio and dithio linkages appear to lose some of their specificity and are "stickier" toward proteins in general than oligos with normal phosphate esters, an affect often attributed to non-specific interactions. This can be quite important since the recognition of nucleic acid sequences by proteins involves specific side chain and backbone interactions with both the nucleic acid bases as well as the phosphate ester backbone. You can take advantage of this "stickiness" to enhance the affinity of PS2-oligos for a protein target but must optimize the total number of PS2 linkages to decrease non-specific binding to the protein target and only enhance the specific favorable interactions with the specific proteins.
Aptamers: PS2 analogues have been successfully used as aptamers for a variety of protein targets including activated protein 1 (AP-1) as well as transcription factor NF-κB for which the PS2-oligos demonstrated 150 picomolar Kd with a dissociation time >12 hours. PS2-ODNs demonstrate up to 300x greater binding affinity for proteins than oligos without PS2 linkages with no loss of aptamer specificity. For HIV-1 RT, dithioates bind 28 (vs. monothioate) or 600 (normal backbone) times more tightly than the normal aptamer oligonucleotide or the S-analogue. For aptamer applications it is recommended no more than six dithio linkages be included in any PS2-oligo in order to minimize non-specific protein interactions.
PS2 oligos have been used in a variety of assays, instruments and technologies offering a wide field of use. They have shown utility in gel shift assays, nanoparticle technologies, microarrays, BioPlex based applications as well as in diagnostic applications (5,6).
Attributes of PS2-oligos:
The Tm of PS2-oligos is lower than the equivalent PS-oligo or the normal phosphate ester. with the degree of Tm depression paralleling the percent phosphorodithioate composition of the oligomer. It is in the range of 0.5 ~ 1.5 
C per dithio linkage.
Toxicity has not been observed in cell culture assays with the PS2-oligos (1,3)
Phosphorodithioate and phosphoromonothioate anions preferentially bind to Cd2+ and Mn2+ while phosphate ester anions preferentially bind to Mg2+ which may provide a mechanism to demonstrate protein binding domains for specific oligo sequences (7,8).
References:
1. Marshall, W.S., Beaton, G., Stein, C.A., Matsukura, M. and Caruthers, M.H. (1992) Inhibition of human immunodeficiency virus activity by phosphorodithioate oligodeoxycytidine. Proc Natl Acad Sci U S A, 89, 6265-6269.
2. Vaughn, J.P., Stekler, J., Demirdji, S., Mills, J.K., Caruthers, M.H., Iglehart, J.D. and Marks, J.R. (1996) Inhibition of the erbB-2 tyrosine kinase receptor in breast cancer cells by phosphoromonothioate and phosphorodithioate antisense oligonucleotides. Nucleic Acids Res, 24, 4558-4564.
3. Marshall, W.S. and Caruthers, M.H. (1993) Phosphorodithioate DNA as a potential therapeutic drug. Science, 259, 1564-1570.
4. Yang, X. and Gorenstein, D.G. (2004) Progress in thioaptamer development. Current drug targets, 5, 705-715.
5. Yang, X., Wang, H., Beasley, D.W., Volk, D.E., Zhao, X., Luxon, B.A., Lomas, L.O., Herzog, N.K., Aronson, J.F., Barrett, A.D. et al. (2006) Selection of thioaptamers for diagnostics and therapeutics. Ann N Y Acad Sci, 1082, 116-119.
6. Wang, H., Yang, X., Bowick, G.C., Herzog, N.K., Luxon, B.A., Lomas, L.O. and Gorenstein, D.G. (2006) Identification of proteins bound to a thioaptamer probe on a proteomics array. Biochem Biophys Res Commun, 347, 586-593.
7. Nawrot, B., Widera, K., Wojcik, M., Rebowska, B., Nowak, G. and Stec, W.J. (2007) Mapping of the functional phosphate groups in the catalytic core of deoxyribozyme 10-23. The FEBS journal, 274, 1062-1072.
8. Basu, S. and Strobel, S.A. (1999) Thiophilic metal ion rescue of phosphorothioate interference within the Tetrahymena ribozyme P4-P6 domain. RNA (New York, N.Y, 5, 1399-1407.
9. Yang, X., Bassett, S.E., Li, X., Luxon, B.A., Herzog, N.K., Shope, R.E., Aronson, J., Prow, T.W., Leary, J.F., Kirby, R. et al. (2002) Construction and selection of bead-bound combinatorial oligonucleoside phosphorothioate and phosphorodithioate aptamer libraries designed for rapid PCR-based sequencing. Nucleic Acids Res, 30, e132.
10. Yang, X., Fennewald, S., Luxon, B.A., Aronson, J., Herzog, N.K. and Gorenstein, D.G. (1999) Aptamers containing thymidine 3'-O-phosphorodithioates: synthesis and binding to nuclear factor-kappaB. Bioorg Med Chem Lett, 9, 3357-3362.
11. Wiesler, W.T. and Caruthers, M.H. (1996) Synthesis of Phosphorodithioate DNA via Sulfur-Linked, Base-Labile Protecting Groups(1). J Org Chem, 61, 4272-4281.
12. Cummins, L., Graff, D., Beaton, G., Marshall, W.S. and Caruthers, M.H. (1996) Biochemical and physicochemical properties of phosphorodithioate DNA. Biochemistry, 35, 8734-8741.
13. Wiesler, W.T., Marshall, W.S. and Caruthers, M.H. (1993) Synthesis and purification of phosphorodithioate DNA. Methods Mol Biol, 20, 191-206.
14. Yang, X., Hodge, R.P., Luxon, B.A., Shope, R. and Gorenstein, D.G. (2002) Separation of synthetic oligonucleotide dithioates from monothiophosphate impurities by anion-exchange chromatography on a mono-q column. Anal Biochem, 306, 92-99.
15. Cho, Y., Zhu, F.C., Luxon, B.A. and Gorenstein, D.G. (1993) 2D 1H and 31P NMR spectra and distorted A-DNA-like duplex structure of a phosphorodithioate oligonucleotide. J Biomol Struct Dyn, 11, 685-702.
16. Piotte, M.E., Granger, J.N., Cho, Y. and Gorenstein, D.G. (1990) 1H 2D Nuclear Magnetic Resonance Spectra of Oligonucleotide Phosphorodithioate, d(CGCTps2Tps2AAGCG). An Unusual Hairpin Loop Structure. J Am Chem Soc, 112, 8632-8634.