Authors:
Ajaya R. Shrestha, Ryohei Kajino
Luxna Biotech Co., Ltd.
2-8 Yamadaoka, Suita, Osaka, JAPAN
Introduction
2′-O,4′-C-Spirocyclopropylene bridged nucleic acid (scpBNA™)1,2 is an analogue of 2′-O,4′-C-methylene bridged nucleic acid (2′,4′-BNA/LNA), which is characterized by a cyclopropane ring at the 6′-position of its sugar moiety (Figure 1). The scpBNA™-modified oligonucleotides can show excellent duplex-forming ability with complementary single-stranded RNA. They can also exhibit highly increased enzymatic stability of the oligonucleotide as compared to that of the phosphorothioate-, 2′-methoxylethyl-RNA- (2′-MOE), and LNA-containing oligonucleotides.
Figure 1. scpBNA™ monomer structure
1. scpBNA™ exhibits high binding affinity towards complementary strands with high RNA selectivity
Compared to the natural DNA-oligonucleotides, the scpBNA™-modified oligonucleotides show excellent binding affinity toward complementary ssRNA with a ∆Tm 4.8 – 5.1 °C per modification, which is comparable to those of LNA-modified oligonucleotides. As for the ssDNA complements, scpBNA™ displayed similar but slightly lower duplex-forming ability (∆Tm 1 – 2 °C per mismatch) relative to the LNA counterpart, exhibiting its RNA selectivity. This reduced binding affinity of scpBNA™ toward ssDNA is possibly due to the conformational change of the sugar moiety upon incorporation of the highly strained cyclopropane unit. It is also possible that the bulky bridge structure of scpBNA™ produces large steric perturbations in the narrow minor groove
of the duplex.
2. scpBNA™ modification is useful to reduce the number of phosphorothioate bonds of therapeutic oligonucleotides
One of the important applications of scpBNA™ is that it reduces the number of phosphorothioate bonds from an oligonucleotide without compromising metabolic stability. The phosphorothioate bonds are generally introduced to enhance the metabolic stability of an oligonucleotide. The phosphorothioate modification, however, is also known to cause toxicity due to their non-specific binding towards proteins. Therefore, reducing the number of phosphorothioates is beneficial to reduce toxicity of an oligonucleotide.3 Since scpBNA™ can exhibit excellent nuclease resistance, it can be incorporated at the 3′-end or both the 3′-and 5′-ends of an oligonucleotide while removing phosphorothioate bonds and retaining metabolic stability. In a model sequence with scpBNA™ at the 3′-end without phosphorothioate bonds, the exonuclease resistance was found to be equal or slightly better than that of a single phosphorothioate modification. More than 80% of the scpBNA™-modified oligonucleotide remained intact even after 60 min of incubation with snake venom phosphodiesterase (Figure 2). Other modifications such as LNA and MOE at the same position without the phosphorothioate modification did not resist enzymatic digestion, and the oligonucleotides were rapidly degraded within 30 min.
Figure 2. Exonuclease stability of oligonucleotide containing scpBNA™.
GCGTTTTTTGCT; where T = DNA, DNA-PS, LNA, MOE, scpBNA™
Conditions: Each oligonucleotide (1 nmol) was incubated with 200 μL of 0.004 unit Crotalus adamanteus venom phosphodiesterase (CAVP) in 50 mM Tris-HCl buffer (pH 8.0) containing 10 mM MgCl² at 37 ºC for up to 60 min.
The remaining intact oligonucleotide was calculated from the area under the peaks analyzed by HPLC.
3. scpBNA™ can be easily incorporated into an oligonucleotide
The scpBNA™-modified oligonucleotides can be synthesized in an automated DNA synthesizer following the standard phosphoramidite protocol. scpBNA™ is compatible with other modified monomers, modifiers, and labels. The phosphoramidites can be dissolved in anhydrous acetonitrile to standard concentrations, except for the 5-methylcytosine, which requires an acetonitrile-THF (3:1, v/v) or acetonitrile-dichloromethane (1:1, v/v) solution. Similar to LNA-phosphoramidites, scpBNA™-phosphoramidites are sterically hindered and require a prolonged coupling time of 8 – 12 min for their incorporation into an oligonucleotide. The solid-supported scpBNA™-oligonucleotides can be treated with concentrated ammonium hydroxide at 55 °C, 12 h for cleavage and deprotection. Although scpBNA™-modifications introduce hydrophobicity, scpBNA™-oligonucleotides with a few scpBNA™ monomers are soluble in water and can be purified and analyzed using the same methods used for other modified oligonucleotides.
References
Product Information
5-Me-C-scpBNA™-CE Phosphoramidite (10-2211-xx)
T-scpBNA™-CE Phosphoramidite (10-2230-xx)
