RNase T1 mimicking artificial ribonuclease

Abstract

Recently, artificial ribonucleases (aRNases)—conjugates of oligodeoxyribonucleotides and peptide (LR)4-G-amide—were designed and assessed in terms of the activity and specificity of RNA cleavage. The conjugates were shown to cleave RNA at Pyr-A and G–X sequences. Variations of oligonucleotide length and sequence, peptide and linker structure led to the development of conjugates exhibiting G–X cleavage specificity only. The most efficient catalyst is built of nonadeoxyribonucleotide of unique sequence and peptide (LR)4-G-NH2 connected by the linker of three abasic deoxyribonucleotides (conjugate pep-9). Investigation of the cleavage specificity of conjugate pep-9 showed that the compound is the first single-stranded guanine-specific aRNase, which mimics RNase T1. Rate enhancement of RNA cleavage at G–X linkages catalysed by pep-9 is 108 compared to non-catalysed reaction, pep-9 cleaves these linkages only 105-fold less efficiently than RNase T1 (kcat_RNase T1/kcat_pep-9 = 105).

miRNases: Novel peptide-oligonucleotide bioconjugates that silence miR-21 in lymphosarcoma cells

“…This suggests that, in the absence of complementarity, conjugate 5'-luc-h-9/14 behaves as a usual non-specific ribonuclease, which we observed in earlier works [76,77]. This could be attributed to some type of opportunistic cleavage during transient electrostatic interactions or imperfect hydrogen bonding between the ribonuclease and RNA substrate.…”

“…Our recent data [50,51,76,77] and 5'-h-9/16 POCs, the antisense part of the conjugate was elongated from 14 to 16 nucleotide residues thus leading to shortening of the target single-stranded region of miR-21 within the hybridized complex. It was shown earlier [51] that narrowing of the RNA single-stranded target region upon binding with the conjugate may result in substantial decrease or even complete loss of catalytic activity, presumably due to reduced conformational freedom for the catalytic peptide.…”

“…via peptide C-terminus) by incorporation of the flexible linker into the conjugate structure, the specificity of chemical nucleases is switched to G-X. Previously, G-X specificity of RNA cleavage was observed for specially designed non-site selective artificial ribonuclease pep-9 [76,77] and 'dual' conjugates targeted to tRNA, both contained long flexible linkers, and the attachment via C-and N-terminus of the peptide was used [51].…”

“…Based on the experimental evidence that any considerable changes in the conjugate structure (i.e. chemical modifications of the oligonucleotide part) may lead to a significant reduction of the cleavage activity [44,76,77], in this study we decided to preserve the 'natural' sugar-phosphate backbone of the oligonucleotide recognition moieties. Our previous studies of 'dual' conjugates [51] have illuminated an importance of the conformational flexibility on their cleavage efficiency.…”

A Novel Family of Sequence-specific Endoribonucleases Associated with the Clustered Regularly Interspaced Short Palindromic Repeats

“…The reaction products were resolved by electrophoresis in 15% PAA, 8 M urea gels using TBE (10 mM Tris borate (pH 8.3), and 2 mM EDTA) as a running buffer. As nucleotide size markers, an imidazole ladder or a G-ladder produced by partial RNA cleavage by 2 M imidazole or RNase T1, respectively, was used (22,23). For the analysis of the RNA product 5Ј-end, after RNase reaction, RNA products were precipitated by 2% LiClO 4 , washed by acetone, dried, dissolved in Milli-Q water, and phosphorylated with [␥-32 P]ATP and T4 PNK using conditions for forward or phosphate exchange reaction according to the manufacturer's protocol (Fermentas).…”