Backbone Hydrocarbon-Constrained Nucleic Acids Modulate Hybridization Kinetics for RNA

Abstract

The binding affinity of therapeutic oligonucleotides (ONs) for their cognate RNA is determined by the rates of association (k a) and dissociation (k d). Single-stranded ONs are highly flexible and can adopt multiple conformations in solution, some of which may not be conducive for hybridization. We investigated if restricting rotation around the sugar–phosphate backbone, by tethering two adjacent backbone phosphonate esters using hydrocarbon bridges, can modulate hybridization kinetics of the modified ONs for complementary RNA. Given the large number of possible analogues with different tether lengths and configurations at the phosphorus atoms, we employed molecular dynamic simulations to optimize the size of the hydrocarbon bridge to guide the synthetic efforts. The backbone-constrained nucleotide trimers with stereodefined configurations at the contiguous backbone phosphorus atoms were assembled using a ring-closing metathesis reaction, then incorporated into oligonucleotides by an in situ synthesis of the phosphoramidites followed by coupling to solid supports. Evaluation of the modified oligonucleotides revealed that 15-membered macrocyclic-constrained analogues displayed similar or slightly improved on-rates but significantly increased off-rates compared to unmodified DNA ONs, resulting in reduced duplex stability. In contrast, LNA ONs with conformationally preorganized furanose rings showed similar on-rates to DNA ONs but very slow off-rates, resulting in net improvement in duplex stability. Furthermore, the experimental data generally supported the molecular dynamics simulation results, suggesting that this strategy can be used as a predictive tool for designing the next generation of constrained backbone ON analogues with improved hybridization properties.

The Importance of Charge Transfer and Solvent Screening in the Interactions of Backbones and Functional Groups in Amino Acid Residues and Nucleotides

“…Similarly, C:G interactions are kcal mol −1 in MM against kcal mol −1 in PA. The lack of screening may be a problem for nucleotide interactions beyond those of pairs in a double-helix [ 97 ], as occur in packed DNA, in non-canonical or transitional structures [ 98 , 99 , 100 , 101 ], or in complexes with ligands [ 102 , 103 , 104 ].…”

Systematic Investigation of Tether Length and Phosphorus Configuration in Backbone Constrained Macrocyclic Nucleic Acids to Modulate Binding Kinetics for RNA

“…Each of the hybrid DNA•RNA systems was modeled for at least 1 μs and with five independent replicas using the methods described previously. 19 Using the resulting trajectory files, we calculated the structural deviation from a reference structure that consisted of a DNA• RNA hybrid of the same sequence (10 μs sampling time). The normalized root-mean-square structural deviation (RMSD) of the inner base pairs is shown in Figure 2.…”

“…We recently described a chemical strategy to pre-organize a trinucleotide subunit in a conformation suitable for Watson–Crick base pairing, to modulate the binding kinetics of single-stranded ONs for complementary RNA. This strategy was conceptually similar but structurally distinct from the constrained nucleic acid analogues described by Nielsen. − Our initial prototypes consisted of bis-phosphonate esters spanning adjacent thymidine nucleotides and linked by bridging hydrocarbon tethers . Given the large number of possible macrocyclic analogues with different tether lengths and configurations at the bridging phosphonates, we used molecular dynamics simulation to optimize the size of the hydrocarbon bridge and tested all four phosphonate configurations of the prototypical 11- and 15-membered macrocyclic rings experimentally.…”

Design, Synthesis, and Characterization of Stapled Oligosaccharides

“…Cyclization is a common strategy employed by nature to reduce conformational space and bestow different biomolecules with specific features. , Synthetic chemists have used cyclization to endow synthetic peptides with superior pharmacological features, including receptor binding affinity, cell-membrane permeability, and metabolic stability. − The so-called “stapling”, originally referred to the cyclization of two amino acid residues in a peptide chain by ring-closing metathesis (RCM), is a straightforward approach to prepare short helical peptides. , Now, a variety of stapling techniques are available to generate synthetic cell-accessible miniproteins and peptide ligands that mimic protein–protein interactions. − Similarly, stapling of oligonucleotide backbones enhances their stability and hybridization properties …”

“…20−22 Similarly, stapling of oligonucleotide backbones enhances their stability and hybridization properties. 23 Cyclic carbohydrates, such as cyclodextrins, are based on repetitive monosaccharides connected by glycosidic linkages. 24 Cyclization methods that enable the generation of nonsugar cross-linkers have been utilized for the synthesis of natural products.…”