A Direct Comparison of Anti-microRNA Oligonucleotide Potency

Abstract

Incorporating high binding affinity modifications, such as LNA and 2'F bases, increases AMO potency while maintaining specificity; nevertheless, use of low dose is preferred when using high potency reagents to minimize the potential for cross reactivity. 2'OMe/LNA chimeras with PS modifications were the most potent constructs tested for miRNA inhibition in vitro.

Chemical modification and design of anti-miRNA oligonucleotides

“…Interestingly, the effect of mutations on AMO function did not track this way. Instead, mutations placed at the center of the AMOs (corresponding to bases 9-12 of the miRNA) and the 5¢-ends (bases [19][20][21][22][23] showed relatively little impact on AMO potency whereas mutations placed in the 3¢-end (bases 3-8) and off-center to the 5¢-side (bases [13][14][15][16][17][18] showed much greater effects. The 3¢-domain of the AMO pairs with the 5¢-end of the miRNA where the seed region is located; bases 2-8 are most critical for determining miRNA specificity.…”

“…For example, DNA oligonucleotides are rapidly degraded in liver microsomal extracts (which mimics an intracellular environment) whereas 2¢OMe oligonucleotides survive this treatment for at least 24 h, yet both 2¢OMe and DNA oligonucleotides are rapidly degraded in serum. 18 The primary nuclease in serum that degrades synthetic oligonucleotides is a 3¢-exonuclease. 19 These patterns of relative nuclease resistance/sensitivity suggest that 2¢OMe AMOs should show improved performance if measures were taken to block exonuclease activity.…”

“…Although secondary structure and self-dimerization predictions suggest that this is not the case for the AMO sequences in these experiments, there could potentially be something in the chemical composition of the LNA/2¢MOE-PS AMO that is prohibitive to miRISC invasion or annealing with the seed region of the targeted miRNA. 18 In anti-mRNA antisense applications, a 'gapmer' ASO design is routinely used, which triggers RNase H degradation of the target mRNA. This design employs a central domain of B10 PS-modified Table 1 Continued DNA residues flanked by high binding affinity 2¢-modified bases, with the DNA domain functioning as the RNase H activating region.…”

“…33,34 Typically, the probes used in these methods employ a repeating pattern of two DNAs followed by one LNA nucleotide and incorporate seven LNAs in a 21-23mer probe. The relative potency of a variety of AMO designs using a miR-21 reporter system were directly compared by Lennox and Behlke 18 and LNA mixmers were among the most potent designs tested (Table 1). LNA/DNA mixmers with a PO backbone were found to degrade in serum, whereas PS end-blocked chimeras or fully PS-modified versions were stable for at least 24 h. The most potent design tested was an LNA/2¢OMe mixmer (repeating unit of two 2¢OMe and one LNA nucleotide), however this design also showed decreased specificity relative to the less potent, lower binding-affinity compounds (specificity will be discussed in greater detail later in this review).…”

“…We similarly observed that this dumbbell design increases potency well above that seen with otherwise unmodified linear 2¢OMe AMOs. 18 Non-nucleotide chemical modifiers can also be used to increase AMO potency. Lennox et al 20 have developed a napthyl-based chemical modifying group that increases binding affinity and blocks exonuclease attack when placed between terminal nucleotides; the greatest benefit is seen when one modifier is placed near each end (KA Lennox and MA Behlke, manuscript in preparation).…”