Oligo modifications that block nuclease degradation

Modification highlight: Are you working with your oligos in cells culture? Find out which modifications can be added to an oligo to limit nuclease degradation.

When oligonucleotides are used in cell culture experiments—such as in antisense and RNAi applications, and in ribozyme technology, degradation by nucleases is a concern. Oligonucleotide stability is typically crucial to these types of studies; yet unmodified DNA and RNA oligonucleotides are quickly digested in vitro by endogenous nucleases. Multiple endo- and exonucleases exist [1]. In serum, the bulk of biologically relevant nucleolytic activity occurs as 3′ exonuclease activity [2], while within the cell, nucleolytic activity is affected by both 5′ and 3′ exonucleases [3].

Modifications

To limit nuclease degradation, investigators have substituted many different modifications into native phosphodiester oligodeoxyribonucleotide and ribonucleotide polymers. IDT offers several such modifications that are incorporated during oligonucleotide synthesis:

Phosphorothioate (PS) bonds

The phosphorothioate (PS) bond substitutes a sulfur atom for a non-bridging oxygen in the phosphate backbone of an oligonucleotide. Approximately 50% of the time (due to the 2 resulting stereoisomers that can form), PS modification renders the internucleotide linkage more resistant to nuclease degradation. Therefore, IDT recommends including at least 3 PS bonds at the 5′ and 3′ oligonucleotide ends to inhibit exonuclease degradation. Including PS bonds throughout the entire oligonucleotide will help reduce attack by endonucleases as well but may also increase toxicity.

2'-O-Methyl (2'OMe)

A naturally occurring post-transcriptional modification of RNA, 2'OMe, is found in tRNA and other small RNAs. Oligonucleotides can be directly synthesized to contain 2'OMe. This modification increases the melting temperature (Tm) of RNA:RNA duplexes, but results in only small changes in RNA:DNA stability. It prevents attack by single-stranded endonucleases, but not exonuclease digestion. Therefore, it is important to end block these oligos as well. DNA oligonucleotides that include this modification are typically 5- to 10-fold less susceptible to DNases than unmodified DNA. The 2′OMe modification is commonly used in antisense oligonucleotides to increase stability and binding affinity to target transcripts [4].

2' Fluoro bases

2'-fluoro bases have a fluorine-modified ribose which increases binding affinity and also confers some relative nuclease resistance relative to native RNA. IDT recommends using this modification in conjunction with PS-modified bonds.

Inverted dT and ddT

Inverted dT can be incorporated at the 3′ end of an oligonucleotide, leading to a 3'-3' linkage that will inhibit degradation by 3' exonucleases and extension by DNA polymerases. In addition, placing an inverted, 2′,3′ dideoxy-dT base (5' Inverted ddT) at the 5′ end of an oligonucleotide prevents spurious ligations and may protect against some forms of enzymatic degradation.

Phosphorylation

Phosphorylation of the 3′ end of oligonucleotides will inhibit degradation by some 3′-exonucleases.

C3 Spacer

The phosphoramidite C3 Spacer can be incorporated internally, or at either end of an oligo to introduce a long hydrophilic spacer arm for the attachment of fluorophores or other pendent groups. The C3 spacer also can be used to inhibit degradation by 3' exonucleases.

Avoiding unanticipated effects

It is important to test any modified oligonucleotides to establish that they work in your specific experimental context. Other considerations include ensuring that the resulting modified oligos:

  • Are not physiologically toxic
  • Are not easily degraded
  • Do not disrupt normal Watson–Crick base pairing
  • Do not induce any unanticipated, sequence-independent biological effects; e.g., off-target effects, or triggering an innate immune response. Residue modifications can also affect the ability of an oligo to trigger RNase H–mediated degradation of RNA following hybrid formation [4].

If you have any questions or want to discuss your experimental design, you can always contact us.

References

  1. Fisher TL, Terhorst T, Cao X, et al. Intracellular disposition and metabolism of fluorescently-labeled unmodified and modified oligonucleotides microinjected into mammalian cells. Nucleic Acids Res. 1993;21(16):3857-3865.
  2. Eder PS, DeVine RJ, Dagle JM, et al. Substrate specificity a8d kinetics of degradation of antisense oligonucleotides by a 3' exonuclease in plasma. Antisense Res Dev. 1991;1(2):141-151.
  3. Dagle JM, Weeks DL, Walder JA. Pathways of degradation and mechanism of action of antisense oligonucleotides in Xenopus laevis embryos. Antisense Res Dev. 1991;1(1):11-20.
  4. Yoo BH, Bochkareva E, Bochkarev A, et al. 2'-O-methyl-modified phosphorothioate antisense oligonucleotides have reduced non-specific effects in vitro. Nucleic Acids Res. 2004;32(6):2008-2016.

For research use only. Not for use in diagnostic procedures. Unless otherwise agreed to in writing, IDT does not intend these products to be used in clinical applications and does not warrant their fitness or suitability for any clinical diagnostic use. Purchaser is solely responsible for all decisions regarding the use of these products and any associated regulatory or legal obligations. RUO22-1600_001

Published Jan 14, 2014
Revised/updated Nov 24, 2022