Ribonuclease Activity of the Peptides with Alternating Arginine and Leucine Residues Conjugated to Tetrathymidilate

Abstract

RNA cleaving conjugates have been prepared by attachment of oligodeoxyribonucleotide TTTT to peptides containing arginine, leucine, proline and serine residues. The highest activity was displayed by the conjugates containing peptides with alternating arginine and leucine residues (LR)4G-amide. Ribonuclease activity of the conjugates pep-T4 decreases in the order T4-(LR)4G > T4-(LR)2G > T4-(LLRR)2G > T4-(LR)2PRLRG > S2R3-Hmda-T4 > or = R5 double dagger (LR)3. According to CD spectra, the free peptide (LR)4G-amide in water solution at neutral pH and physiological ionic strength has no pronounced secondary structure whereas conjugated to oligonucleotide it acquires a folding similar to alpha-helix.

RNA knockdown by synthetic peptidyl-oligonucleotide ribonucleases: behavior of recognition and cleavage elements under physiological conditions

“…Our recent work (Patutina et al, 2017(Patutina et al, , 2019Staroseletz, Williams et al, 2017;Williams et al, 2015) and earlier studies from other groups (Mironova et al, 2002;Mironova, Boutorine et al 2004;Mironova et al, 2006Mironova et al, , 2007Pyshnyi et al, 1997) demonstrated that the biological activity of this class of bioconjugates can only be achieved through chemical conjugation of the catalytic peptide with the oligonucleotide component. This suggested that the oligonucleotide component may induce an 'active' conformation of the peptide to make it catalytically active by some unclear molecular interactions between the oligonucleotide and peptide components.…”

“…We have recently developed a new class of catalytic bioconjugates, as synthetic ribonucleases using amphipathic peptides, which only cleave RNA when conjugated to oligonucleotide recognition motifs (Mironova et al, 2002;Mironova, Boutorine et al, 2004;Mironova et al, 2006Mironova et al, , 2007Patutina et al, 2017Patutina et al, , 2019Pyshnyi et al, 1997;Staroseletz, Williams et al, 2017;Williams et al, 2015). These RNA-targeting synthetic nucleases are chemically-engineered by conjugation of short, catalytically-inactive peptides with oligonucleotide recognition motif components, which range from fully-through poorlyto non-complementary to the target RNA, in order to produce novel biologically-active molecules capable of recognizing and cleaving RNA sequences quantitatively.…”

“…These RNA-targeting synthetic nucleases are chemically-engineered by conjugation of short, catalytically-inactive peptides with oligonucleotide recognition motif components, which range from fully-through poorlyto non-complementary to the target RNA, in order to produce novel biologically-active molecules capable of recognizing and cleaving RNA sequences quantitatively. The most remarkable feature of these novel biocatalysts is that the conjugation of peptide and oligonucleotide seems to produce a new, hybrid type of molecule (Mironova, Boutorine et al, 2004;Mironova et al, 2006), with new properties, which are not found in nature. This synergistic combination of the individual properties of the two components yields a new catalytic entity, which is capable of catalyzing the cleavage of phosphodiester bonds in RNA under physiological conditions, with a rate enhancement factor of RNA cleavage reaching 10 8 in some cases, as compared to non-catalyzed reaction (Mironova et al, 2007).…”

“…Peptidyl-oligonucleotide conjugates (POCs) designed to date have the ability to cleave RNA at two different extremes: (i) either sequence-specifically (Mironova et al, 2002;Patutina et al, 2017Patutina et al, , 2019Pyshnyi et al, 1997;Staroseletz, Williams et al, 2017;Williams et al, 2015) by site-directed cleavage of the regions adjacent to the RNA hybridization sites, but in a non-catalytic manner; or (ii) non-specifically at non-complementary regions, which were located distantly from the major RNA binding region, but with a high level of catalytic turnover (Mironova, Boutorine et al, 2004;Mironova et al, 2006Mironova et al, , 2007. These data provided some experimental indications that peptidyl-oligonucleotide conjugates may interact differently with the complementary and non-complementary regions of RNA sequences.…”

Peptide Conjugates of Oligonucleotides: Synthesis and Applications

“…Mironova and co-workers have synthesized a series of POCs capable of cleaving RNAs in a sequence specific manner. − The oligonucleotides 171 , 172 , and 173 with different specificities were linked to the core (catalytic) peptide sequence, [Arg-Leu] 4 -Gly, using the SPFC method to obtain the conjugate shown in Figure . The oligonucleotide and peptide fragments of these conjugates (pep− 171 , pep− 172 , and pep− 173 in Figure ) were connected by N -alkylphosphoramidate linkages between the 5‘-terminal phosphate of the oligonucleotide and the N-terminal-α-amino group of the corresponding oligopeptide.…”