Evaluation of multiple-turnover capability of locked nucleic acid antisense oligonucleotides in cell-free RNase H-mediated antisense reaction and in mice

Abstract

The multiple-turnover ability of a series of locked nucleic acid (LNA)-based antisense oligonucleotides (AONs) in the RNase H-mediated scission reaction was estimated using a newly developed cell-free reaction system. We determined the initial reaction rates of AONs under multiple-turnover conditions and found that among 24 AONs tested, AONs with melting temperatures (Tm) of 40°C-60°C efficiently elicit multiple rounds of RNA scission. On the other hand, by measuring Tm with two 10-mer RNAs partially complementary to AONs as models of cleaved 5' and 3' fragments of mRNA, we found that AONs require adequate binding affinity for efficient turnover activities. We further demonstrated that the efficacy of a set of 13-mer AONs in mice correlated with their turnover efficiency, indicating that the intracellular situation where AONs function is similar to multiple-turnover conditions. Our methodology and findings may provide an opportunity to shed light on a previously unknown antisense mechanism, leading to further improvement of the activity and safety profiles of AONs.

FIG. 1.

Recycling of antisense oligonucleotides in antisense reaction. (A) Structures of bridged nucleic acids (BNAs). (B) Schematic illustration of cell-free Förster resonance energy transfer (FRET)-based turnover monitoring system used in this study. Color images available online at www.liebertpub.com/nat

FIG. 2.

(A) Relationship between turnover rate and gross or local affinity. Initial rates (v₀, bar graph) of turnover reaction rearranged in ascending order of melting temperature (T_m) values (black line) of corresponding antisense oligonucleotides (AONs). Data are means±standard deviation (SD). (B) Melting temperatures with MRNA-1 (solid) and MRNA-2 (dotted). All experiments here were repeated at least three times. Color images available online at www.liebertpub.com/nat

FIG. 3.

Reduction of apoB mRNA in the livers (bar graph) of mice (n=3/group) receiving a single subcutaneous dose of 0.75 mg kg^–1 of a series of 13-mer AONs (A) rearranged in ascending order of T_m values along with melting temperatures versus MRNA-1 (solid) and MRNA-2 (dashed). Dunnett's multiple comparison test, ***p<0.001; **p<0.01; N.S., not significant. (B) The mouse liver content of a series of 13-mer AONs. Bonferroni multiple comparison tests did not reveal any of arms to be significantly different across groups, p<0.05. Error bars represent group means+SD n=3.