Controlled sulfur-based engineering confers mouldability to phosphorothioate antisense oligonucleotides

Abstract

Phosphorothioates (PS) have proven their effectiveness in the area of therapeutic oligonucleotides with applications spanning from cancer treatment to neurodegenerative disorders. Initially, PS substitution was introduced for the antisense oligonucleotides (PS ASOs) because it confers an increased nuclease resistance meanwhile ameliorates cellular uptake and in-vivo bioavailability. Thus, PS oligonucleotides have been elevated to a fundamental asset in the realm of gene silencing therapeutic methodologies. But, despite their wide use, little is known on the possibly different structural changes PS-substitutions may provoke in DNA·RNA hybrids. Additionally, scarce information and significant controversy exists on the role of phosphorothioate chirality in modulating PS properties. Here, through comprehensive computational investigations and experimental measurements, we shed light on the impact of PS chirality in DNA-based antisense oligonucleotides; how the different phosphorothioate diastereomers impact DNA topology, stability and flexibility to ultimately disclose pro-Sp S and pro-Rp S roles at the catalytic core of DNA Exonuclease and Human Ribonuclease H; two major obstacles in ASOs-based therapies. Altogether, our results provide full-atom and mechanistic insights on the structural aberrations PS-substitutions provoke and explain the origin of nuclease resistance PS-linkages confer to DNA·RNA hybrids; crucial information to improve current ASOs-based therapies.

Graphical Abstract

Phosphorothioate antisense oligonucleotide showing how proper stereoisomer configuration can lead to a selected XNA:RNA topology for RNase H recognition, binding and processing.

Figure 1.

Circular dichroism spectra of the different DNA·RNA duplexes here investigated. Reference conformations are reported by dashed lines: the red dashed one indicates the canonical B-conformation of DNA·DNA duplex, the purple dashed one describes the canonical A-conformation typical of the RNA·RNA duplex while the dashed green line represents the non-A/non-B conformation associated with the hybrid DNA·RNA duplexes. RpDNA·RNA is shown as pink line, SpDNA·RNA in blue, SpRpDNA·RNA in yellow and RpSpDNA·RNA in black. See Methods for the experimental conditions.

Figure 2.

Representation of chemical shift differences between the RpSp (left), SpRp (center) and Sp (right) with respect to the Rp hybrid duplex. Nucleobase protons are shown in blue and sugar protons in cyan, light orange and light green. The size of the spheres represents the chemical shift differences (small, medium or large mean differences of 0.04–0.1, 0.1–0.2 or >0.2 ppm, respectively). RNA phosphates are shown in dark red; PS DNA linkages are shown in magenta (Rp) or pink (Sp).

Figure 3.

(A) Thermodynamic cycle calculated to investigate energetically Sp to Rp (ad vice-versa) interconversion. (B) panels show progressive substitution while tables on the right report detailed free energy values (in kJ/mol). The standard errors associated to values in panel A are always ≤0.004 kJ/mol. Those at the right have associated standard errors which are typically around 1–5% of the estimated value.

Figure 4.

Thermodynamic cycle calculated to investigate energetically Rp → O alchemical transformation and relative free energy values. Averages shown in panel A have an associated standard error ≤0.004 kJ/mol. Those at the right have associated standard errors which are typically around 2–6% of the estimated value.

Figure 5.

Active site of Klenow fragment exonuclease complexed with DNA·DNA (A) SpDNA·DNA (B) and RpDNA·DNA (C). The two-Mg²⁺ centered active site is shown. The nucleophilic water molecule (WAT_Nuc) is coordinated to one of the Mg²⁺ ion (in pink). The reactive distance (i.e. d_Nuc) between nucleophile (i.e. WAT_Nuc) and electrophile (i.e. P atom) is reported and taken as a key geometrical description to evaluate the correct geometry of Exonuclease reactive active site, a well-known arrangement (25). (D) Frequency Distribution of d_Nuc length calculated for the different systems under investigation. Three additional replicas have been performed to explore the reproducibility of these findings. The corresponding histograms nearly overlap and are not shown for clarity reasons, but are available upon request.

Figure 6.

(A) Ternary complex of RNAse H in complex with SpDNA:RNA and catalytic Mg²⁺ ions. The highlighted green area refers to the interaction interface between the RNase H enzyme and the hybrid duplex. (B) Detail on the triad-substrate interactions and reported free energy data and relative standard errors in parenthesis. Motifs are reported in 3′-5′ direction.