Peptidyl–Oligonucleotide Conjugates Demonstrate Efficient Cleavage of RNA in a Sequence-Specific Manner

Abstract

Described here is a new class of peptidyl-oligonucleotide conjugates (POCs) which show efficient cleavage of a target RNA in a sequence-specific manner. Through phosphoramidate attachment of a 17-mer TΨC-targeting oligonucleotide to amphiphilic peptide sequences containing leucine, arginine, and glycine, zero-linker conjugates are created which exhibit targeted phosphodiester cleavage under physiological conditions. tRNA(Phe) from brewer's yeast was used as a model target sequence in order to probe different structural variants of POCs in terms of selective TΨC-arm directed cleavage. Almost quantitative (97-100%) sequence-specific tRNA cleavage is observed for several POCs over a 24 h period with a reaction half-life of less than 1 h. Nontargeted cleavage of tRNA(Phe) or HIV-1 RNA is absent. Structure-activity relationships reveal that removal of the peptide's central glycine residue significantly decreases tRNA cleavage activity; however, this can be entirely restored through replacement of the peptide's C-terminal carboxylic acid group with the carboxamide functionality. Truncation of the catalytic peptide also has a detrimental effect on POC activity. Based on the encouraging results presented, POCs could be further developed with the aim of creating useful tools for molecular biology or novel therapeutics targeting specific messenger, miRNA, and genomic viral RNA sequences.

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

“…Over the last couple of decades, some progress has been achieved in the field of designing siteselective artificial ribonucleases [42][43][44][45][46][47][48][49][50][51][52][53][54]. It was shown that short peptides, containing either alternating leucine and arginine residues or imidazole-based catalytic groups, conjugated to antisense oligonucleotides targeting tRNA, were able to hydrolyze linkages adjacent to an oligonucleotide-binding site without involvement of exogenous species such as metal ions, enzymes or cofactors (e.g., RISC, RNase H) [42,44,45,47,50,51].…”

“…It was shown that short peptides, containing either alternating leucine and arginine residues or imidazole-based catalytic groups, conjugated to antisense oligonucleotides targeting tRNA, were able to hydrolyze linkages adjacent to an oligonucleotide-binding site without involvement of exogenous species such as metal ions, enzymes or cofactors (e.g., RISC, RNase H) [42,44,45,47,50,51]. Effective cleavage of complementary substrates was also demonstrated for tris(2-aminobenzimidazole) ribonuclease conjugated to PNA oligomers [49].…”

“…The details of chemicals, reagents and facilities used in this research have already been provided in our previous publications [50,51]. The software Topspin 3.2 was used to analyze the NMR data obtained.…”

“…Catalytic peptide Acetyl-(LeuArg) 2 -Gly-(LeuArg) 2 -Gly-COOH was synthesized by manual solid-phase methodology on Fmoc-Gly-Wang resin using the Fmoc/ t Bu strategy as described in [50]. Following completion of the peptide sequence, the N-terminus was acetylated by shaking with acetic anhydride (10 mmol) and DIPEA (10 mmol) in DMF (10 mL) for 30 min, followed by resin washing with DMF (2 × 10 mL), DCM (2 × 10 mL and DMF (2 × 10 mL).…”

“…After acetylation of the sequence, the peptide was cleaved from the resin using the same protocol reported in [50]. Crude lyophilized peptide was solubilized in 30% acetic acid and purified using RP-HPLC as described earlier [50].…”