Abstract
Antisense oligonucleotides conjugated with boron clusters (B-ASOs) have been described as potential gene expression inhibitors and carriers of boron for boron neutron capture therapy (BNCT), providing a dual-action therapeutic platform. In this study, we tested the nucleolytic stability of DNA oligonucleotides labeled with metallacarborane [(3,3’-iron-1,2,1’,2’-dicarbollide)(−1)]ate [Fe(C2B9H11)2] (FESAN) against snake venom phosphodiesterase (svPDE, 3’→5’-exonuclease). Contrary to the previously observed protective effect of carborane (C2B10H12) modifications, the B-ASOs containing a metallacarborane moiety at the 5’-end of the oligonucleotide chain were hydrolyzed faster than their parent nonmodified oligomers. Interestingly, an enhancement in the hydrolysis rate was also observed in the presence of free metallacarborane, and this reaction was dependent on the concentration of the metallacarborane. Microscale thermophoresis (MST) analysis confirmed the high affinity (Kd nM range) of the binding of the metallacarborane to the proteins of crude snake venom and the moderate affinity (Kd µM range) between the metallacarborane and the short single-stranded DNA. We hypothesize that the metallacarborane complex covalently bound to B-ASO holds DNA molecules close to the protein surface, facilitating enzymatic cleavage. The addition of metallacarborane alone to the ASO/svPDE reaction mixture provides the interface to attract freely floating DNA molecules. In both cases, the local DNA concentration around the enzymes increases, giving rise to faster hydrolysis. It was experimentally shown that an allosteric effect, possibly attributable to the observed boost in the 3’→5’-exonucleolytic activity of snake venom phosphodiesterase, is much less plausible.
Highlights
The inhibition of gene expression via the antisense approach involves the use of a DNA oligonucleotide to “arrest” the target RNA by hybridization according to the Watson-Crick mode followed by the cleavage of mRNA in the resultant heteroduplex by RNase H [1,2]
Resulting oligonucleotides 1a, 1b, 1c, 2a and 2b containing a uridine unit labeled with ferra(III) bis(dicarbollide) were isolated by RP-HPLC and characterized by MALDI-TOF or electrospray ionization (ESI) mass spectrometry and electrophoresis in 20% polyacrylamide/7 M gel (PAGE) (Table 1, Figures S1 and S2)
PAGE analysis (20% polyacrylamide/7 M urea) of the resultant mixtures showed that 1a and 2a were almost completely degraded in 15 min (Figure 2). These results show that the presence of a single units bearing the cage (UB) unit at the 5′-end significantly accelerated the enzymatic hydrolysis of B-antisense oligonucleotides (ASOs)
Summary
The inhibition of gene expression via the antisense approach involves the use of a DNA oligonucleotide (typically 13–25 mers) to “arrest” the target RNA by hybridization according to the Watson-Crick mode followed by the cleavage of mRNA in the resultant heteroduplex by RNase H [1,2]. Some antisense oligonucleotides (ASOs) directed towards the 5’-end of the target mRNA or to the region of the AUG start codon are designed to inhibit mRNA function by sterically blocking the translational machinery [3], but other mechanisms may be used to interfere with the mRNA maturation process [4], including inhibition of the 5’-cap formation [5], inhibition of the RNA splicing [6], blocking. IRo-aladbioe-led labeled adenosine 5’-triphosphate ([γ-32P]-ATP) was purchased from Hartmann-Analytik (Braunschweig, Germany), and T4 polynucleotide kinase and kinase buffer were purchased from. Biomolecules 2020, 10, 718 adenosine 5’-triphosphate ([γ-32P]-ATP) was purchased from Hartmann-Analytik (Braunschweig, Germany), and T4 polynucleotide kinase and kinase buffer were purchased from BioLabs (New England, MA, USA). The snake venom phosphodiesterase I from Crotalus atrox Western Diamondback Rattlesnake was purchased from Sigma-Aldrich (USA)
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