Abstract
Oligonucleotide conjugates of tris(2-aminobenzimidazole) have been reported previously to cleave complementary RNA strands with high levels of sequence and site specificity. The RNA substrates used in these studies were oligonucleotides not longer than 29-mers. Here we show that ~150–400-mer model transcripts derived from the 3′-untranslated region of the PIM1 mRNA reacted with rates and specificities comparable to those of short oligonucleotide substrates. The replacement of DNA by DNA/LNA mixmers further increased the cleavage rate. Tris(2-aminobenzimidazoles) were designed to interact with phosphates and phosphate esters. A cell, however, contains large amounts of phosphorylated species that may cause competitive inhibition of RNA cleavage. It is thus important to note that no loss in reaction rates was observed in phosphate buffer. This opens the way to in-cell applications for this type of artificial nuclease. Furthermore, we disclose a new synthetic method giving access to tris(2-aminobenzimidazoles) in multigram amounts.
Highlights
Synthetic nucleases used to cleave RNA in a sequence-specific manner are normally conjugates of a catalytic subunit and an oligonucleotide part required for substrate recognition via Watson-Crick base pairing [1,2,3,4,5,6]
Over the last two decades, gene silencing by siRNAs [23] has become an important approach in gapmers, chimeric antisense oligonucleotides combining modified building blocks with a central functional genomics
Both methods are based on the recruitment of cellular gapmers, chimeric antisense oligonucleotides combining modified building blocks with a central enzymes by synthetic oligonucleotides
Summary
Synthetic nucleases used to cleave RNA in a sequence-specific manner are normally conjugates of a catalytic subunit and an oligonucleotide part required for substrate recognition via Watson-Crick base pairing [1,2,3,4,5,6]. Complexes of lanthanide ions have been most widely used as RNA cleavers [7,8,9,10]. When attached to DNA [12] or PNA oligonucleotides [13,14], they hybridize with complementary RNA strands and cut their substrates with half-lives in the range of 10 to 20 h. A notable exception has been the site-specific cleavage of long transcripts encoding the proto-oncogenic protein kinase c-Raf. A lanthanide complex attached to an oligonucleotide was shown to cleave 60–70%
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