What are Backbone Modifications in Oligonucleotide Synthesis?

Chemically modified nucleotides are widely used to increase the stability of oligonucleotides or oligonucleotide conjugates. Many modifications of the oligonucleotide backbone have been used as antisense reagents or in synthetic siRNA for the control of gene expression. The backbone of oligonucleotides refers to the internucleotide linkage and/or the sugar moiety.

Recent advancements made in oligonucleotide therapeutics depended on the progress achieved in the chemistry of these molecules. For example, phosphorothioate (PS) backbone modifications have been the key to the development and success of antisense oligonucleotides (ASOs) and splice switching oligonucleotides (SSOs).

Structures of the phosphodiester (PO) backbone, the phosphororthioate (PS) backbone and the phosphorodithioate (PdiS) backbone or linkage.


The phosphorothioate (PS) backbone modification has been used for ASOs and SSOs. The replacement of one oxygen atom with a sulfur atom results in a mixture of two diastereoisomers which are designated as Rp and Sp. The CIP convention defines the stereochemistry with the priority S > O3’ > O5’ >O(=P).

PS slightly reduces binding affinity but improves stability to nucleases in blood and tissues. It also promotes protein binding and supports interactions with albumin and other blood protein to retard renal clearance. They are more resistant to exo- and endonuclease and are therefore used to enhance the stability of oligonucleotides.

Unfortunately, significant toxicities exist. However, the modification is fully consistent with RNase H activity.

Phosphorothioates can also be modified via a post-synthetic reaction with α-haloacetamide reagents to form phosphotriesters.


Here both non-bridging oxygen atoms are replaced by sulfur. These linkages are non-chiral and completely resistant to cleavage by all known nucleases. However, phosphorodithioates are used less often because they bind to complementary oligonucleotides with reduced discrimination and also bind to various proteins as well.


Methylphosphonates are uncharged analogs of phosphodiesters. In these molecules, a non-bridging oxygen atom of the phosphate group has been replaced with a methyl group. Other alkyl or aryl groups can also be used. Again, methylphosphonates are chiral at the phosphate group, and a mixture or isomers usually occur. This linkage has enhanced stability to exo- and endonucleases. Duplexes that contain phosphonates have increased Tms. Because of the neutral charge oligonucleotides containing multiple phsophonates are less soluble in aqueous solvents and tend to aggregate.


In phosphoramidates, an oxygen atom is replaced with an amino group either at the 3’- or 5’-oxygen. These type of linkages have increased resistance to snake venom phosphodiesterases and higher Tms during duplex formation with complementary DNA and RNA as well as enhanced resistance to nucleases.  

Other Neutral backbone

Neutral backbones use the phosphorodiamidate morpholino oligomer (PMO) and the peptide nucleic acid (PNA) modifications. These modifications provid neutral backbones and high resistance to nucleases but do not support RNase H activity. Therefore their use appears to be limited to SSOs as therapeutic agents.


Modifications of the 2’-sugar position with methyl (Me) and methoxyethyl (MOE) groups are the most widely used modifications. Both modifications promote the A-form or RNA –like conformation in oligonucleotides, considerably increase binding affinity to RNA, and have enhanced nuclease resistance. Because the 2’-O-methyl sugar adopts a C3’-endo ribose conformation these oligonucleotides are more stable when binding complementary DN or RNA than oligodeoxyribonucleotides. The MOE modification shows a similar characteristic.

Also, oligonucleotides fully modified at the 2’-position do not support RNase H activity. A gapmer design on the other hand allows for the design and production of RNase H dependent antisense oligonucleotides. A classical gapmer consists of a central section of seven (7) unmodified residues flanked by 2’-modified regions. 2’-O-Me and 2’-F are often used in combination with the PS backbone modification for ASOs, SSOs, and siRNAs. In siRNAs, the 2’-modification can reduce immunostimulatory and off-target effects.

The fluorine containing 2’-deoxy-2-‘-fluoro-β-D-ribofuranoside is an analog of the natural β-D-ribose found in RNA. Here the sugar ring favors a C3’-endo pucker and a A-type conformation when hybridizing with RNA.

Modifications at the 2’-position are well tolerated in oligonucleotide duplexes. Therefore the 2’-position has been widely used for the attachment of a variety of functional groups, e.g. fluorophores.  

Bridged Rings or Bridged Nucleic Acids

In bridged nucleic acids (BNAs) such as in BNA-NC[Me], 2’,4’-BNAs (LNAs),  ethyl (cEt) constrained as well as in tricycle-DNA (tc-DNA) the sugar ring is modified via a bridge or third ring structure. Each of these modifications promotes RNA-like structures, display nuclease resistance and a dramatic increase in binding affinity to both DNA and RNA. Furthermore, they do not support RNase H activity. Therefore they can be effectively used as antisense gapmers or as SSOs as well as in a variety of molecular probes and conjugates.  

Other approaches

Charge-neutralizing phosphotriester backbone modifications can also be used for siRNA. These are called short interfering ribonucleic neutrals (siRNNs). Apparently siRNNs can be delivered into cells more effectively than siRNAs but will be converted into siRNAs ones inside the cell by cytoplasmic thioesterases. 

Another newly developed approach is the use of alternative genetic polymers (XNAs) that contain polymers formed from building blocks not found in nature mimicking properties of RNA and DNA for the synthesis of oligonucleotide mimics. 

XNAs are nucleic acids in which the ribofuranose ring of DNA and RNA is replaced by five- or six-membered  modified ribose molecules such as 1,5 anhydrohexitol nucleic acids (HNAs), cyclohexenyl nucleic acids (CeNAs), and 2’4’-C-(N-methylaminomethylene) bridged nucleic acids (BNAs), 2′-O,4′-C-methylene-β-D-ribonucleic acids or locked nucleic acids (LNAs), ANA (arabinonucleic acids), 2′-fluoro-arabinonucleic acid (FANAs) and α-L-threofuranosyl nucleic acids (TNAs).

Nucleic Acid Delivery

Recently, a number of clinical trial using different types of oligonucleotides have already shown promising results. Examples are the use of a  receptor targeted siRNA conjugate, the use of modified antisense constructs to treat or prevent disease, anticancer effects of miRNA, as well as the treatment of neurodegenerative diseases via SSOs. 

However, a few major obstacles when using modified oligonucleotides as therapeutic drugs still have to be addressed. For example, oligonucleotide based therapeutics will need to bypass or penetrate various tissue barriers to allow for cellular uptake and intracellular trafficking of oligonucleotides and to reach their target. Therefore medical researchers are investigating a variety of approaches for enhancing the delivery of oligonucleotides.

Presently new molecular systems that include ligand-oligonucleotide conjugates, lipid- and polymer-based nanoparticles, antibody conjugates as well as other selected small molecules are investigated for their ability to improve oligonucleotide delivery.


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