Glen Report 35-13: New Product — dSpacer-5’-CE Phosphoramidite (Reverse dSpacer)

When researchers are synthesizing oligonucleotides for their therapeutic studies, they will often have to place blocking groups on the 3’-end and sometimes at the 5’-end as well. An inverted dT is commonly used to protect oligonucleotides from exonucleases, and a Spacer C3 is often used to prevent polymerase extension. A third option that one might consider is the use of an inverted dSpacer. It shares the same three carbon skeleton of Spacer C3 while omitting the thymine base from the inverted dT (Figure 1).

Figure 1. Blocking groups at the 3’-end

Figure 1

There are many examples of researchers using inverted dSpacer modifications in the literature. This has been particularly true for siRNA.1-3 Both ends of the passenger strand and the 3’-end of the guide strand can all be capped by the addition of an inverted dSpacer to enhance nuclease stability. Incorporating an inverted dSpacer at the 5’-end of the passenger strand also serves to block loading into Ago2, which minimizes competition between the guide and passenger strands. All three incorporations do not reduce the gene silencing effect. Similarly, the passenger strand of small activating RNAs (saRNAs) can also be modified with 5’/3’ inverted dSpacers.4 Finally, the hammerhead ribozyme has been modified with a 3’ inverted dSpacer.5, 6 Studies using inverted dT have demonstrated greater than 30-fold increase in serum half-times with no reduction in catalysis,7 and the inverted dSpacer should yield similar stability enhancement. 

There are two approaches to introducing an inverted dSpacer into oligonucleotides. The first approach would be to use our dSpacer CE Phosphoramidite (Figure 2) along with reverse phosphoramidites. The standard dSpacer is typically used to give stable abasic sites for the study of structure as well as DNA damage. When paired with a reverse 5’-3’ synthesis direction, inverted dSpacer linkages are obtained. The second, and probably more logical approach would be to use a reverse dSpacer phosphoramidite in the standard 3’-5’ synthesis direction. This way, only one reverse phosphoramidite is required. 

To give our customers more access to the inverted dSpacer modification as well as blocking groups in general, we are introducing dSpacer-5’-CE Phosphoramidite (Figure 2). Like our standard version, our reverse dSpacer does not require any extended coupling times and is compatible with all standard deprotection conditions.

Figure 2. dSpacer Phosphoramidites

Figure 2


  1. H.J. Haringsma, et al., Nucleic Acids Res, 2012, 40, 4125-36.
  2. Y. Pei, et al., RNA, 2010, 16, 2553-63.
  3. F. Czauderna, et al., Nucleic Acids Res, 2003, 31, 2705-16.
  4. S. Yoon, and J.J. Rossi, Curr Pharm Biotechnol, 2018, 19, 604-610.
  5. T.C. Jarvis, et al., J Biol Chem, 1996, 271, 29107-12.
  6. D.G. Macejak, et al., Hepatology, 2000, 31, 769-76.
  7. L. Beigelman, et al., J Biol Chem, 1995, 270, 25702-8.

Product Information

dSpacer CE Phosphoramidite (10-1914)

dSpacer-5’-CE Phosphoramidite (10-4191)