Know your oligo mod: phosphorothioate bonds

Updated : Wed, March 29, 2023 @ 10:56 AM

This post in our continuing series, “Know your oligo mod” will cover phosphorothioate bonds, which are available for incorporation into custom oligonucleotides through Biosearch Technologies. This modification is interesting as it is not an addition of a chemical moiety per se, such as a nonstandard base or a quencher, but rather it is a special linkage between the bases. While the canonical DNA backbone found in nature includes phosphodiester linkages between bases, this bond can be altered to a phosphorothioate linkage along the backbone of the oligonucleotide (Fig 1).

Fig. 1 - On the left, we see a schematic of a standard oligo linkage with a phosphodiester bond linking the two 5-carbon sugars through the phosphate bond forming the prototypical backbone of the oligo. On the right, we can see the phosphorothioate linkage in which a sulfur atom replaces one of the oxygens in the phosphate linkage between bases in the oligo.

The phosphorothioate bond oligo modification alters the phosphate linkage by replacing one of the non-bridging oxygens with a sulfur atom. This alteration of the bonds actually changes the overall chemical properties of the oligo making it more suitable for some molecular and cell biology research applications. In particular, one outcome after adding phosphorothioate bonds is the stabilization of the oligo backbone against nuclease degradation, effectively enhancing the oligo’s half-life in the cellular milieu^1,2. This makes the modified bonds incredibly useful in the creation of antisense oligonucleotides, which when introduced into cells or biological matrices can interact with target nucleic acids to silence the expression of a particular transcript. Oligos containing phosphorothioate bonds accomplish this feat either through direct blockage of translation or enable enzymatic degradation of the target transcript typically through a RNase H mediated mechanism³.

Although these specialised bonds confer stability against indiscriminate nuclease digestion, the repeated introduction of these bonds can also create limitations to their function as antisense oligonucleotides. Each phosphorothioate bond introduced into the oligo creates a chiral center at each bond which is designated as either a “Sp” or “Rp” conformation. This ultimately leads to multiple isomers of the oligo generated during synthesis⁴. These individual isomers have been shown to have differential characteristics and functional properties. Fortunately, much of the isomer effects on antisense oligos are mitigated through careful positioning of the modifications or by using additional modifications in conjunction with the phosphorothioate bonds; significantly diminishing concerns about non-racemic mixtures^5,6,7.

Ultimately, the design* of oligos using phosphorothioate bonds can require significant trial and error to achieve the desired results as the effect of modified bond incorporations can be unpredictable⁸. However, there are some general recommendations for the creation of these oligos, which should be considered in the design process. For oligos requiring protection against exonucleases, it is indicated to incorporate the phosphorothioate bonds near the 5’ and 3’ ends of the oligo. However, for protection against endonucleases, it is recommended that these bonds are included throughout the oligo sequence. In either case, it is important to realize that the phosphorothioate bond modification should not be incorporated at every available position in the oligonucleotide. Rather, they should be used sparingly and with careful consideration to the impact each bond might contribute to the chirality issue mentioned above, and also because this bond has a slightly destabilizing effect on the oligo hybridization kinetics. Increasing the number of phosphorothioate bonds in an oligo tends to lower the melting temperature (Tm) of the oligonucleotide for its intended target. Therefore, when designing these oligos, the desired nuclease resistance properties must carefully balance the need for effective and specific hybridization to the target sequence.

LGC Biosearch Technologies offers the synthesis of oligonucleotides incorporated with phosphorothioate bonds through our Custom Oligo Service. The standard notation for this modification is simply designated on the order form as a bracketed asterisk between bases like so, [*]. For example, an oligonucleotide sequence of ACTGACTG containing three phosphorothioate bonds would be notated as follows during the ordering process: A[*]CTG[*]ACT[*]G.

For more information about custom oligonucleotides visit Build Your Oligo to order unlabeled, single labeled or non-standard oligos and primers online. Simply enter your sequence, select from the industry's largest selection of modifications, and choose a preferred purification option.

*LGC Biosearch Technologies does not currently offer design services of antisense oligos or specific guidance in their use, rather we provide the custom synthesis and purification of these specialty modified oligos. If interested in additional information or resources about antisense oligonucleotides, please contact our technical support team: techsupport@biosearchtech.com

If you are thinking about bringing oligo synthesis in-house, there are many considerations to account for beyond instrument selection. But the benefits of synthesizing your own oligos are clear.  Download this guide that summarises all the practical and technical information in a single place, to ensure that your synthesizers run at full capacity and you maximise your return on investment.

References

1. Effect of deoxynucleoside phosphorothioates incorporated in DNA on cleavage by restriction enzymes. Vosberg, H. P., and F. Eckstein. Journal of Biological Chemistry 257.11 (1982): 6595-6599.

2. Antisense research and applications. Crooke, Stanley T., and Bernard Lebleu. CRC Press, 1993.

3. Antisense technologies. Kurreck, Jens. European Journal of Biochemistry270.8 (2003): 1628-1644.

4. Bond order and charge localization in nucleoside phosphorothioates. Frey, Perry A., and R. Douglas Sammons. Science 228.4699 (1985): 541-545.

5. Stability of Stereoregular Oligo (nucleoside Phosphorothioate) s in Human Plasma: Diastereoselectivity of Plasma 3‵-Exonuclease. Koziolkiewicz, Maria, et al. Antisense and Nucleic Acid Drug Development 7.1 (1997): 43-48.

6. Stereodifferentiation—the effect of P chirality of oligo (nucleoside phosphorothioates) on the activity of bacterial RNase H. Koziolkiewicz, Maria, et al. Nucleic acids research 23.24 (1995): 5000-5005.

7. Synthesis, biophysical properties and biological activity of second generation antisense oligonucleotides containing chiral phosphorothioate linkages. Wan, W. Brad, et al. Nucleic acids research (2014): gku1115. doi: 10.1093/nar/gku1115.

8. Antisense oligonucleotides: from design to therapeutic application. Chan, Jasmine HP, Shuhui Lim, and W. S. Wong. Clinical and experimental pharmacology and physiology 33.5‐6 (2006): 533-540.