5′-CP™ & scpBNA™ phosphoramidites

5′-Cyclopropylene alters the local 5′ sugar environment of a DNA nucleoside while retaining RNA–DNA duplex formation. Model 12-mers containing a single 5′-CP substitution showed small, base-dependent decreases in melting temperature (0.9–2.4 °C). [R1]

Four-base RNA duplex data

Nuclease resistance and RNase H

5′-CP-T model constructs show increased nuclease resistance while retaining RNase H-mediated RNA cleavage. In a venom-phosphodiesterase assay, the terminal 5′-CP-T model retained approximately 70% uncleaved oligonucleotide at 40 minutes, versus approximately 30% for the terminal single-PS comparator. In a separate E. coli RNase H assay, constructs with two and three 5′-CP-T residues gave 46% and 27% RNA cleavage, respectively, versus 31% for the reference. [R1, Examples 11–12]

Published incorporation conditions

A single site and its adjacent backbone linkage

Kuroda et al. first walked single 5′-CP substitutions through ASO DNA gaps, then tested the substitutions with replacement of the immediately 5′-adjacent phosphorothioate (PS) linkage by a phosphodiester (PO) linkage. In the ASO1 paired walk, gap positions 1, 3, 4 and 5 reduced cytotoxicity and toxicity-associated transcripts without significantly reducing knockdown in that screen. [R2, Figure 6B]

At ASO1 gap position 3, the combined 5′-CP/PO design reduced late-onset neurotoxicity while maintaining Mapt knockdown in mouse hippocampus and lumbar cord. The study used a 38.4 nmol intracerebroventricular dose, four animals per group, and assessment at 21 days. Adding the PO linkage preserved knockdown more effectively than the corresponding 5′-CP/all-PS design in ASO1 and ASO4. [R2, Figure 6; supplementary Figure S24]

The result supports testing nucleoside placement and the neighboring linkage together, but remains sequence- and position-dependent; the authors did not observe mitigation of ASO2 acute-onset neurotoxicity from these gap modifications. [R2, Discussion]

A separate patent liver experiment

A useful comparison for research

A matched set of parent ASO, 5′-CP substitution, PO-only variant and combined change can help assess 5′-CP incorporation and separate the two design variables. A preclinical screen may measure duplex binding, knockdown and tolerability separately.

scpBNA is a 2′-O,4′-C-spirocyclopropylene bridged nucleic acid. Published model-oligonucleotide experiments assess RNA affinity and terminal nuclease resistance as distinct properties. [R3–R4]

RNA affinity in model constructs

Terminal stability

A technical report describes a terminal scpBNA-T model without PS linkages that retained more than 80% intact oligonucleotide after 60 minutes of exonuclease exposure, with resistance equal to or slightly better than a single-PS comparator. In the foundational patent’s separate assay, combined 10-mer and 9-mer material remained at 60% or more after 80 minutes, while the unmodified DNA-thymidine and LNA samples were nearly degraded by 20 minutes. [R6, Figure 2; R3, Example 5]

Published synthesis precedents

All-PS scpBNA and LNA wing comparison

In a patent Pten experiment, an all-PS scpBNA-wing gapmer and its LNA comparator gave approximately 60–70% target suppression. The patent describes AST/ALT values for the scpBNA design at approximately half the LNA values. Male mice received 35 mg/kg intraperitoneally, with four animals per group and assessment at 72 h. [R3, Example 7]

Kawanobe 2025: all-scpBNA wings and mixed-chemistry designs

Kawanobe et al. compared Scarb1 gapmers with a ten-nucleotide DNA gap. The all-scpBNA construct LX-A2007 has three T/MeC residues in each wing and an all-PS backbone. Figure 1 compares this design with the LNA reference LX-A1656 and the AmNA-containing reference LX-A2001. [R5, Figure 1B]

The all-scpBNA-wing construct LX-A2007 retained substantial Scarb1 knockdown with lower AST than the LNA reference LX-A1656 and the AmNA-containing reference LX-A2001. Further liver assessments examined mixed AmNA/scpBNA wings with two intra-wing PO linkages. The combined design reduced ALT, AST, total-bile-acid elevations and selected liver lesions, although residual lesions remained. [R5, Figures 1–3]

The combined AmNA/scpBNA/PO construct LX-A3928 showed approximately 1.5-fold weaker on-target knockdown activity and 2.5-fold improvement in the AST dose-response comparison versus the LNA/PS reference. These findings reflect the combined architecture. [R5, Results and supplementary Figure S3]

What this adds to the design toolkit

Evaluate T and MeC terminal and wing substitutions with selected PO and/or PS backbone linkages; assess RNA affinity, nuclease resistance, target knockdown and tolerability.