Antisense oligonucleotides can bind too strongly

Central to the development of RNase H-recruiting antisense oligonucleotides for therapeutics lies efforts in medicinal chemistry to improve oligonucleotide stability,

biodistribution, as well as RNA binding affinity. With the introduction of high-affinity locked nucleic acids (LNAs), oligonucleotides as short as 12nt in length have been reported to achieve sufficient binding affinity to potently silence their targets.

Relationship between calculated affinity and experimental knockdown of ApoB mRNA by a range of LNA-containing oligonucleotides (From Pedersen et al)

Indeed it has been generally observed that high-affinity modifications improve the potency of the oligonucleotide compared to the same sequence with lower-affinity modifications. With such an apparent proportionality between the affinity and potency of antisense oligonucleotides, it could be expected that longer oligonucleotides tended to have higher potency than shorter ones, since more nucleotides increase affinity by allowing more hydrogen bonds and additional base stackings.

But this simple expectation is contradicted by experimental observations.

Potency has been evaluated and reported for LNA-modified oligonucleotides between 12 and 20nt in length with between 2 and 5 LNAs in the flanks. When stratifying by length, it has in some cases been observed that shorter oligonucleotides targeting the same target site has increased potency compared with longer versions. So far, no mechanism for this seemingly counterintuitive increase in potency with decreases in length and affinity has been demonstrated. Suggested explanations include variations in gapsize, less tendency to self-complementarity or improved pharmacokinetics of shorter oligonucleotides.

Scientists from COAT and Santaris Pharma now provide an alternative explanation that invokes only the kinetics behind oligonucleotide-mediated cleavage of RNA targets. Recently, we published a kinetic model based

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on the law of mass action in Molecular Therapy – Nucleic Acids, a companion journal to the well-established high impact journal, Molecular Therapy (Pedersen et al., 2014), which predict the existence of an optimal binding affinity. This prediction was confirmed by experiments across multiple RNA targets. Intuitively put, too high affinity between the target RNA and the oligonucleotide decreases the rate that at which the oligonucleotide leaves the cleaved target, which limits the catalytic cycle and therefore the potency of target degradation.

Thus, exaggerated affinity, and not length per se, is detrimental to potency. This finding clarifies how to optimally apply high-affinity modifications in the discovery of potent antisense oligonucleotide drugs.

Posted in Oligoinformatics, Research

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