DNA-cleaving restriction enzymes are well-known tools in biomedical and biotechnological research. There are, however, no corresponding enzymes known for RNA cleavage. There has been an ongoing development of artificial ribonucleases, including some attempts at sequence selectivity. However, so far these systems have displayed modest rates of cleavage, and in most cases, the cleaver has been used in excess or in stoichiometric amounts. In the current work, we present PNA-based systems (PNAzymes) that carry a Cu(II)-2,9-dimethylphenanthroline group and that act as site and sequence specific RNases. The general basis for the systems is that the target is cleaved at a nonbase paired region (RNA bulge) which is formed in the substrate upon binding of the PNAzyme. With this copper based system, cleavage takes place at virtually only one site and with a half-life of down to 30 min under stoichiometric conditions. Efficient turnover of RNA-substrate is shown with a 100-fold excess of substrate, thus, demonstrating true enzyme behavior. In addition, alteration of the sequence in the RNA bulge or a mismatch in the base-pairing region leads to substantial decreases in rate showing both kinetic resolution and binding discrimination in the substrate selectivity. The selectivity is further demonstrated by the substrates, with two potential cleavage sites differing in only one base, are cleaved only at the site that either does not have a mismatch or is kinetically preferred. We suggest that these systems can serve as a basis for construction of RNA restriction enzymes for in vitro manipulations.
A general procedure, based on a new activated alkyne linker, for the preparation of peptide–oligonucleotide conjugates (POCs) on solid support has been developed. With this linker, conjugation is effective at room temperature (RT) in millimolar concentration and submicromolar amounts. This is made possible since the use of a readily attachable activated triple bond linker enhances the Cu(I) catalyzed 1,3-dipolar cycloaddition (‘click’ reaction). The preferred scheme for conjugate preparation involves sequential conjugation to oligonucleotides on solid support of (i) an H-phosphonate-based aminolinker; (ii) the triple bond donor p-(N-propynoylamino)toluic acid (PATA); and (iii) azido-functionalized peptides. The method gives conversion of oligonucleotide to the POC on solid support, and only involves a single purification step after complete assembly. The synthesis is flexible and can be carried out without the need for specific automated synthesizers since it has been designed to utilize commercially available oligonucleotide and peptide derivatives on solid support or in solution. Methodology for the ready conversion of peptides into ‘clickable’ azidopeptides with the possibility of selecting either N-terminus or C-terminus connection also adds to the flexibility and usability of the method. Examples of synthesis of POCs include conjugates of oligonucleotides with peptides known to be membrane penetrating and nuclear localization signals.
Several peptide nucleic acid based artificial nucleases (PNAzymes) are designed to create a bulge in the target RNA, which is a short model of the leukemia related bcr/abl mRNA. The target RNA is cleaved by the PNAzymes with a half-life of down to 11 h (using a 1 : 1 ratio of PNA-conjugate to target) and only upon base-pairing with the substrate. The PNA based systems are also shown to act in a catalytic fashion with turnover of substrate and are thus the first reported peptide nucleic acid based artificial RNA-cleaving enzymes.
In this report, we investigate the efficiency and selectivity of a Zn2+-dependent peptide nucleic acid-based artificial ribonuclease (PNAzyme) that cleaves RNA target sequences. The target RNAs are varied to form different sizes (3 and 4 nucleotides, nt) and sequences in the bulge formed upon binding to the PNAzyme. PNAzyme-promoted cleavage of the target RNAs was observed and variation of the substrate showed a clear dependence on the sequence and size of the bulge. For targets that form 4-nt bulges, we identified systems with an improved efficacy (an estimated half-life of ca 7–8 h as compared to 11–12 h for sequences studied earlier) as well as systems with an improved site selectivity (up to over 70% cleavage at a single site as compared to 50–60% with previous targets sequences). For targets forming 3-nt bulges, the enhancement compared to previous systems was even more pronounced. Compared to a starting point of targets forming 3-nt AAA bulges (half-lives of ca 21–24 h), we could identify target sequences that were cleaved with half-lives three times lower (ca 7–8 h), i.e., at rates similar to those found for the fastest 4-nt bulge system. In addition, with the 3-nt bulge RNA target site selectivity was improved even further to reach well over 80% cleavage at a specific site.
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