A Trisbenzimidazole Phosphoramidite Building Block Enables High-Yielding Syntheses of RNA-Cleaving Oligonucleotide Conjugates

Abstract

The RNA cleaving catalyst tris(2-aminobenzimidazole) when attached to the 5’ terminus of oligonucleotides cuts complementary RNA strands in a highly site-specific manner. Conjugation was previously achieved by the acylation of an amino linker by an active ester of the catalyst. However, this procedure was low yielding and not reliable. Here, a phosphoramidite building block is described that can be coupled to oligonucleotides by manual solid phase synthesis in total yields around 85%. Based on this chemistry, we have now studied the impact of LNA (locked nucleic acids) nucleotides on the rates and the site-specificities of RNA cleaving conjugates. The highest reaction rates and the most precise cuts can be expected when the catalyst is attached to a strong 5’ closing base pair and when the oligonucleotide contains several LNA units that are equally distributed in the strand. However, when placed in the 5’ position, LNA building blocks tend to diminish the specificity of RNA cleavage.

Site-Selective Artificial Ribonucleases: Renaissance of Oligonucleotide Conjugates for Irreversible Cleavage of RNA Sequences

“…M. Göbel et al designed “linear” ss-aRNAses consisting of tris(2-aminobenzimidazole) and 15-mer DNA, PNA or DNA-LNA mixmer oligonucleotides targeted to 22–29-mer fragments of 3′-UTR PIM1 mRNA [ 26 , 30 , 32 , 72 ]. The half-lives (τ½) of the cleavage of DNA- or PNA-ss-aRNases vary from 11.2 to 16.5 h [ 26 , 30 , 32 , 72 ]. In turn, conjugates of DNA-LNA mixmers exhibit significantly faster cleavage of RNA with τ½ 3.5 h [ 26 , 30 , 32 , 72 ].…”

“…The half-lives (τ½) of the cleavage of DNA- or PNA-ss-aRNases vary from 11.2 to 16.5 h [ 26 , 30 , 32 , 72 ]. In turn, conjugates of DNA-LNA mixmers exhibit significantly faster cleavage of RNA with τ½ 3.5 h [ 26 , 30 , 32 , 72 ]. It should also be noted that another conjugate developed by this group, the “bulge-inducing” PNAzyme targeted to 15-mer synthetic RNA, is able to degrade up to 70% of the 5-fold RNA excess within 84 h in a catalytic manner, which shows a significant achievement for metal-independent PNA-based ss-aRNAses [ 31 ].…”

“…Such ss-aRNases, as shown in [ 33 , 80 ], exhibited Pyr-A specificity during tRNA cleavage. In turn, the specificity of conjugates comprised of more sophisticated imidazole-based catalytic domains, such as tris(2-aminobenzimidazole), was not limited by pyrimidine-A cleavage specificity, but also include degradation at purine-X motifs [ 26 , 30 , 31 , 32 , 72 ]. Established in our group, conjugates of the peptide [(ArgLeu) 2 Gly] 2 attached to the hairpin oligonucleotide via an aminohexyl linker cleaved miR-21 exclusively at G-X bonds, representing mimics of RNase T1 [ 35 ].…”

“…The catalytic properties of tris(2-aminobenzimidazole)-based ss-aRNases, which contain different patterns of LNA modifications of oligonucleotide domain, were also investigated [ 72 ]. It was shown that the introduction of one LNA modification does not influence the ribonuclease activity of conjugate: τ½ of DNA and LNA-modified ss-aRNase were 14 ± 0.9 h and 13.3 ± 0.8 h, respectively [ 72 ]. The addition of two or three LNA units promoted a 2-fold increase in the rate of cleavage, resulting in τ ½ equal to 6.4 ± 0.2 h [ 72 ].…”

“…It was shown that the introduction of one LNA modification does not influence the ribonuclease activity of conjugate: τ½ of DNA and LNA-modified ss-aRNase were 14 ± 0.9 h and 13.3 ± 0.8 h, respectively [ 72 ]. The addition of two or three LNA units promoted a 2-fold increase in the rate of cleavage, resulting in τ ½ equal to 6.4 ± 0.2 h [ 72 ]. The introduction of 6 LNA units leads to the additional enhancement of ribonuclease activity of ss-aRNase, lowering its τ ½ to 3.5 ± 0.4 h [ 72 ].…”