Structural Determinants for the Interactions of Chemically Modified Nucleic Acids with the Stabilin‑2 Clearance Receptor

The Stabilin receptors are systemic clearance receptors for some classes of chemically modified nucleic acid therapeutics. In this study, the recombinant human secreted ecto-domain of the small isoform of Stabilin-2 (s190) was purified from cell culture and evaluated for direct binding with a multitude of antisense oligonucleotides (ASOs) using a fluorescence polarization-based assay. The tested ASOs varied in their backbone composition, modification of the ribose 2′ position, overall length of the oligo, and sequence of the nucleotide bases. A fully phosphorothioate (PS) ASO with a 5–10–5 pattern of flanking 2′-<i>O</i>-methoxyethyl modifications was then used to test the effects of pH and salt concentration on receptor binding. These tests concluded that the PS backbone was the primary determinant for ASO binding and that decreasing pH and increasing salt generally increased the rate of ligand dissociation and fit within the biological parameters expected of a constitutive recycling receptor. These results will be useful in the rational design of therapeutic oligonucleotides for enhancing their affinity or avoidance of the Stabilin receptors

Structural Determinants for the Interactions of Chemically Modified Nucleic Acids with the Stabilin‑2 Clearance Receptor

The Stabilin receptors are systemic clearance receptors for some classes of chemically modified nucleic acid therapeutics. In this study, the recombinant human secreted ecto-domain of the small isoform of Stabilin-2 (s190) was purified from cell culture and evaluated for direct binding with a multitude of antisense oligonucleotides (ASOs) using a fluorescence polarization-based assay. The tested ASOs varied in their backbone composition, modification of the ribose 2′ position, overall length of the oligo, and sequence of the nucleotide bases. A fully phosphorothioate (PS) ASO with a 5–10–5 pattern of flanking 2′-<i>O</i>-methoxyethyl modifications was then used to test the effects of pH and salt concentration on receptor binding. These tests concluded that the PS backbone was the primary determinant for ASO binding and that decreasing pH and increasing salt generally increased the rate of ligand dissociation and fit within the biological parameters expected of a constitutive recycling receptor. These results will be useful in the rational design of therapeutic oligonucleotides for enhancing their affinity or avoidance of the Stabilin receptors

Synthesis and Biophysical Properties of Constrained d‑Altritol Nucleic Acids (cANA)

The first synthesis of constrained altritol nucleic acids (cANA) containing antisense oligonucleotides (ASOs) was carried out to ascertain how conformationally restricting the d-altritol backbone-containing ASO (Me-ANA) would affect their ability to form duplexes with RNA. It was found that the thermal stability was reduced (cANA/RNA −1.1 °C/modification) compared to DNA/RNA, suggesting the constrained system results in a small destabilizing perturbation in the duplex structure

Synthesis and Biophysical Properties of Constrained d‑Altritol Nucleic Acids (cANA)

The first synthesis of constrained altritol nucleic acids (cANA) containing antisense oligonucleotides (ASOs) was carried out to ascertain how conformationally restricting the d-altritol backbone-containing ASO (Me-ANA) would affect their ability to form duplexes with RNA. It was found that the thermal stability was reduced (cANA/RNA −1.1 °C/modification) compared to DNA/RNA, suggesting the constrained system results in a small destabilizing perturbation in the duplex structure

Differential Effects on Allele Selective Silencing of Mutant Huntingtin by Two Stereoisomers of α,β-Constrained Nucleic Acid

We describe the effects of introducing two epimers of neutral backbone α,β-constrained nucleic acid (CNA) on the activity and allele selectivity profile of RNase H active antisense oligonucleotides (ASOs) targeting a single nucleotide polymorphism (SNP) for the treatment of Huntington’s disease (HD). ASOs modified with both isomers of α,β-CNA in the gap region showed good activity versus the mutant allele, but one isomer showed improved selectivity versus the wild-type allele. Analysis of the human RNase H cleavage patterns of α,β-CNA modified ASOs versus matched and mismatched RNA revealed that both isomers support RNase H cleavage on the RNA strand across from the site of incorporation in the ASOan unusual observation for a neutral linkage oligonucleotide modification. Interestingly, ASOs modified with (<i>R</i>)- and (<i>S</i>)-5′-hydroxyethyl DNA (RHE and SHE respectively) formed by partial hydrolysis of the dioxaphosphorinane ring system in α,β-CNA also showed good activity versus the mutant allele but an improved selectivity profile was observed for the RHE modified ASO. Our observations further support the profiling of neutral and 5′-modified nucleic acid analogs as tools for gene silencing applications

Biophysical and Biological Characterization of Hairpin and Molecular Beacon RNase H Active Antisense Oligonucleotides

Antisense oligonucleotides (ASOs) are single stranded, backbone modified nucleic acids, which mediate cleavage of complementary RNA by directing RNase H cleavage in cell culture and in animals. It has generally been accepted that the single stranded state in conjunction with the phosphorothioate modified backbone is necessary for cellular uptake and transport to the active compartment. Herein, we examine the effect of using hairpin structured ASOs to (1) determine if an ASO agent requires a single stranded conformation for efficient RNA knock down, (2) use a fluorophore-quencher labeled ASO to evaluate which moieties the ASO interacts with in cells and examine if cellular distribution can be determined with such probes, and (3) evaluate if self-structured ASOs can improve allele selective silencing between closely related huntingtin alleles. We show that hairpin shaped ASOs can efficiently down-regulate RNA <i>in vitro</i>, but potency correlates strongly negatively with increasing stability of the hairpin structure. Furthermore, self-structured ASOs can efficiently reduce huntingtin mRNA in the central nervous system of mice

Synthesis of <i>cis</i>- and <i>trans</i>-α‑l‑[4.3.0]Bicyclo-DNA Monomers for Antisense Technology: Methods for the Diastereoselective Formation of Bicyclic Nucleosides

Two α-l-<i>ribo</i>-configured bicyclic nucleic acid modifications, represented by analogues <b>12</b> and <b>13</b>, which are epimeric at C₃′ and C₅′ have been synthesized using a carbohydrate-based approach to build the bicyclic core structure. An intramolecular l-proline-mediated aldol reaction was employed to generate the <i>cis</i>-configured ring junction of analogue <b>12</b> and represents a rare application of this venerable organocatalytic reaction to a carbohydrate system. In the case of analogue <b>13</b>, where a <i>trans</i>-ring junction was desired, an intermolecular diastereoselective Grignard reaction followed by ring-closing metathesis was used. In order to set the desired stereochemistry at the C₅′ positions of both nucleoside targets, a study of diastereoselective Lewis acid mediated allylation reactions on a common bicyclic aldehyde precursor was carried out. Analogue <b>12</b> was incorporated in oligonucleotide sequences, and thermal denaturation experiments indicate that it is destabilizing when paired with complementary DNA and RNA. However, this construct shows a significant improvement in nuclease stability relative to a DNA oligonucleotide

Synthesis of <i>cis</i>- and <i>trans</i>-α‑l‑[4.3.0]Bicyclo-DNA Monomers for Antisense Technology: Methods for the Diastereoselective Formation of Bicyclic Nucleosides

Two α-l-<i>ribo</i>-configured bicyclic nucleic acid modifications, represented by analogues <b>12</b> and <b>13</b>, which are epimeric at C₃′ and C₅′ have been synthesized using a carbohydrate-based approach to build the bicyclic core structure. An intramolecular l-proline-mediated aldol reaction was employed to generate the <i>cis</i>-configured ring junction of analogue <b>12</b> and represents a rare application of this venerable organocatalytic reaction to a carbohydrate system. In the case of analogue <b>13</b>, where a <i>trans</i>-ring junction was desired, an intermolecular diastereoselective Grignard reaction followed by ring-closing metathesis was used. In order to set the desired stereochemistry at the C₅′ positions of both nucleoside targets, a study of diastereoselective Lewis acid mediated allylation reactions on a common bicyclic aldehyde precursor was carried out. Analogue <b>12</b> was incorporated in oligonucleotide sequences, and thermal denaturation experiments indicate that it is destabilizing when paired with complementary DNA and RNA. However, this construct shows a significant improvement in nuclease stability relative to a DNA oligonucleotide

A Constrained Tricyclic Nucleic Acid Analogue of α‑l‑LNA: Investigating the Effects of Dual Conformational Restriction on Duplex Thermal Stability

A constrained tricyclic analogue of α-l-LNA (<b>2</b>), which contains dual modes of conformational restriction about the ribose sugar moiety, has been synthesized and characterized by X-ray crystallography. Thermal denaturation experiments of oligonucleotide sequences containing this tricyclic α-l-LNA analogue (α-l-TriNA 2, <b>5</b>) indicate that this modification is moderately stabilizing when paired with complementary DNA and RNA, but less stabilizing than both α-l-LNA (<b>2</b>) and α-l-TriNA 1 (<b>4</b>)

A Constrained Tricyclic Nucleic Acid Analogue of α‑l‑LNA: Investigating the Effects of Dual Conformational Restriction on Duplex Thermal Stability

A constrained tricyclic analogue of α-l-LNA (<b>2</b>), which contains dual modes of conformational restriction about the ribose sugar moiety, has been synthesized and characterized by X-ray crystallography. Thermal denaturation experiments of oligonucleotide sequences containing this tricyclic α-l-LNA analogue (α-l-TriNA 2, <b>5</b>) indicate that this modification is moderately stabilizing when paired with complementary DNA and RNA, but less stabilizing than both α-l-LNA (<b>2</b>) and α-l-TriNA 1 (<b>4</b>)