Glen Report 36-12: Technical Brief — Backbone Modifications

Previously, we discussed nucleobase and sugar conformations and modifications.1, 2 In this issue, we are highlighting the third component of nucleic acids: its backbone. The natural nucleic acid backbone is comprised of 3′-5′-phosphodiester linkages. As is the case of nucleobase and sugar components, researchers have studied the effects of non-natural modifications of this phosphate backbone. There are numerous ways to modify the natural phosphate backbone, including the overall stereochemistry via L-DNA monomers, 2′-5′-linkages, methyl phosphonate linkages, and replacement of non-bridged oxygen atom(s) with sulfur (Figure 1). This, of course, is not a comprehensive list of all modifications studied, but for the purposes of this article, we will be focusing on backbone modifications offered by Glen Research.

Figure 1. Backbone modifications
Figure 1. Backbone modifications 

Out of the five modifications, L-DNA stands apart from the rest as it does not involve a chemical substitution. Instead, an oligonucleotide made up of L-DNA monomers is the mirror image of naturally occurring D-DNA. These L-DNA oligonucleotides have been discussed in previous Glen Reports.3 Due to its structure, L-DNA is not recognized by naturally occurring DNA-binding proteins. This allows L-DNA oligonucleotides to evade degradation by nucleases. L-DNA oligonucleotides have been used in various applications, including aptamers, molecular beacons, and drug delivery nanostructures.3

Oligonucleotides containing 2′-5′-phosphate linkages selectively bind to complementary, single stranded RNA sequences. These duplexes do not activate RNase H and also exhibit less nonspecific binding to cellular proteins. It has been observed that 2′-5′-linkages reduce the duplex melting temperature by about 0.5 °C per insertion.4 

These 2′-5′-linkages have been utilized in various applications. Despite poor binding with complementary DNA, 2′-5′-linkages have a stabilizing effect for triplex forming oligonucleotides (TFO). Addition of this modified backbone into a homopyrimidine oligonucleotide enhanced triplex stability under physiological conditions.5 A combination of 3′-deoxy-2′-phosphoramidites and 2′-deoxy-3′-phosphoramidites have been used to produce a gapmer with 2'-5'-linked ends and 3′-5′-linked central regions.  A 3′-deoxynucleoside at the 3′-terminus of an otherwise normal oligonucleotide effectively blocks polymerase extension.

Methyl Phosphonamidites are used to produce oligonucleotides containing methyl phosphonates. These backbones are neutral (not charged) and decrease the overall polarity compared to a negatively charged phosphodiester backbone. While oligonucleotides bearing methyl phosphonate linkages are still taken up into cells,  they do so through a different mechanism and to a lesser extent relative to other backbone modifications.6, 7 Methyl phosphonates confer resistance to nucleases. Each methyl phosphonate linkage introduces a chiral center into the oligonucleotide. Methyl phosphonolated oligonucleotides have been evaluated in antisense therapeutics.8

Introducing phosphorothioate (PS) linkages are unique from the others in this list because no special phosphoramidite is required. A standard phosphoramidite is used on a synthesizer and the oxidizer is replaced with sulfurizing reagent. While several sulfurizing reagents have been used and evaluated in the literature, such as the Beaucage Reagent, we recommend DDTT ((dimethylamino-methylidene)amino)-3H-1,2,4-dithiazole-3-thione) (Figure 2) for sulfurization. Sulfur atoms are larger than oxygen and better at dispersing the negative charge in a PS linkage. This provides resistance to nuclease degradation. Phosphorothioates are often used in antisense oligonucleotides (ASOs). In fact, PS linkages have been utilized in 10 of the 18 FDA-approved therapeutic oligonucleotides.9 Similar to methyl phosphonates, phosphorothioates are chiral and will produce two diastereomers per insertion.


Figure 2. Sulfurizing Reagent II (DDTT)

Figure 2. Sulfurizing Reagent II (DDTT)


Phosphorodithioate linkages are prepared by combining thiophosphoramidites with the above mentioned, sulfurization reagent. Phosphorodithioates reduce oligonucleotide complexity because each insertion is achiral. Phosphorodithoates and their use in oligonucleotide therapeutics have recently been discussed.10 Glen Research also offers 2′-OMe thiophosphoramidites.

Modifications of the phosphate backbone lead to differences in nucleic acid activity. With various substitutions, nuclease resistance and thermal stability can be altered to best fit one’s needs (Table 1).


Nuclease Resistance 


Thermal Stability 











Methyl Phosphonates 











Table 1. Overview of standard and modified backbones available at Glen Research
∇ = decreased, * = depends on stereopurity11, 12 



  1. The Glen Report, 2021, 33.1, 8.
  2. The Glen Report, 2023, 35.1, 10-11.
  3. The Glen Report, 2019, 31.2, 4-6.
  4. P. Bhan, et. al., Nucleic Acids Res, 1997, 25, 40-41.
  5. S. Obika, et. al., Nucleosides, Nucleotides, and Nucleic Acids, 2005, 24, 1005-1058.
  6. G.D. Gray, S. Basu, and E. Wickstrom, Biochem Pharmacol, 1997, 53, 1465-76.
  7. Y. Shoji, S. Akhtar, A. Periasamy, B. Herman, and R.L. Juliano, Nucleic Acids Res, 1991, 19, 5543-50.
  8. P. Sarin, et. al., Proc. Natl. Acad. Sci., 1988, 85, 7448-7451.
  9. M. Egli, and M. Manoharan, Nucleic Acids Res, 2023, 51, 2529-2573.
  10. The Glen Report, 2022, 34.1, 5-6.
  11. N. Iwamoto, et al., Nat Biotechnol, 2017, 35, 845-851.
  12. A. Pontarelli, and C.J. Wilds, Chembiochem, 2023, 24, e202300068.

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