Further Probing of Cu2+-Dependent PNAzymes Acting as Artificial RNA Restriction Enzymes.

Peptide nucleic acid (PNA)-neocuproine conjugates have been shown to efficiently catalyse the cleavage of RNA target sequences in the presence of Cu2+ ions in a site-specific manner. These artificial enzymes are designed to force the formation of a bulge in the RNA target, the sequence of which has been shown to be key to the catalytic activity. Here, we present a further investigation into the action of Cu2+-dependent PNAzymes with respect to the dependence on bulge composition in 3- and 4-nucleotide bulge systems. Cu2+-dependent PNAzymes were shown to have a clear preference for 4-nucleotide bulges, as the cleavage of 3-nucleotide bulge-forming RNA sequences was significantly slower, which is illustrated by a shift in the half-lives from approximately 30 min to 24 h. Nonetheless, the nucleotide preferences at different positions in the bulge displayed similar trends in both systems. Moreover, the cleavage site was probed by introducing critical chemical modifications to one of the cleavage site nucleotides of the fastest cleaved 4-nucleotide RNA bulge. Namely, the exclusion of the exocyclic amine of the central adenine and the replacement of the 2'-hydroxyl nucleophile with 2'-H or 2'-OMe substituents in the RNA severely diminished the rate of RNA cleavage by the Cu2+-dependent PNAzyme, giving insight into the mechanism of cleavage. Moreover, the shorter recognition arm of the RNA/PNAzyme complex was modified by extending the PNAzyme by two additional nucleobases. The new PNAzyme was able to efficiently promote the cleavage of RNA when fully hybridised to a longer RNA target and even outperform the previous fastest PNAzyme. The improvement was demonstrated in cleavage studies with stoichiometric amounts of either PNAzyme present, and the extended PNAzyme was also shown to give turnover with a 10-fold excess of the RNA target.

19 F†NMR Spectroscopic Analysis of the Binding Modes in Triple-Helical Peptide Nucleic Acid (PNA)/MicroRNA Complexes.

Triplex-forming peptide nucleic acids (TFPNAs) were targeted to double-helical regions of 19 F-labeled RNA hairpin models (a UA-rich duplex with a hexaethylene glycol (heg) loop and a microRNA model, miR-215). In addition to conventional UV- and circular dichroism (CD)-based detection, binding was monitored by 19 F NMR spectroscopy. Detailed information on the stoichiometry and transition between the triple-helical peptide nucleic acid (PNA)/RNA and (PNA)2 /RNA binding modes could be obtained. γ-(R)-Hydroxymethyl-modified thymine-1-yl- and 2-aminopyridin-3-yl-acetyl derivatives of TFPNAs were additionally synthesized, which were targeted to the same RNA models, and the effect of the γ-(R)-hydroxymethyl group on binding was studied. An appropriate pattern of γ-(R)-hydroxymethyl modifications reduced the stability of the ternary complex and preferred stoichiometric binding to the miR-215 model.

Facile functionalization of peptide nucleic acids (PNAs) for antisense and single nucleotide polymorphism detection.

In this report, we show how a convenient on-resin copper-click functionalization of azido-functionalized peptide nucleic acids (PNAs) allows various PNA-based detection strategies. Firstly, a thiazole orange (TO) clicked PNA probe facilitates a binary readout when combined with F/Q labeled DNA, giving increased sensitivity for antisense detection. Secondly, our TO-PNA conjugate also allows single nucleotide polymorphism detection. Since antisense detection is also possible in the absence of the TO label, our sensing platform based on azido-d-ornithine containing PNA even allows for additional and more advanced functionalization and sensing strategies.

Zinc Ion-Dependent Peptide Nucleic Acid-Based Artificial Enzyme that Cleaves RNA-Bulge Size and Sequence Dependence.

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.

Clamping of RNA with PNA enables targeting of microRNA.

To be able to target microRNAs also at stages where these are in a double stranded or hairpin form we have studied BisPNA designed to clamp the target and give sufficient affinity to allow for strand invasion. We show that BisPNA complexes are more stable with RNA than with DNA. In addition, 24-mer BisPNA (AntimiR) constructs form complexes with a hairpin RNA that is a model of the microRNA miR-376b, suggesting that PNA-clamping may be an effective way of targeting microRNAs.

Synthesis of PNA oligoether conjugates.

Several different approaches have been explored for conjugation of oligoethers to PNA with internally or N-terminal placed diaminopropionic acid residues. Single and double conjugation of 2-(2-(2-aminoethoxy)ethoxy)ethanol was obtained using carbonyldimidazole. Using a post PNA-assembly coupling procedure the building block 2-(2-(2-(benzoyloxy)ethoxy)ethoxy)acetic acid multiple attachment of 2-(2-(2-hydroxyethoxy)ethoxy)acetyl groups to both N-terminal and ß-amino groups of inserted diaminopropionic acids residues was achieved. Use of a new oligoether functionalized amino acid allows inclusion of oligoether conjugates during on-line machine assisted synthesis which also allowed combination of methods for attachment of different oligoethers and co-conjugation of neocuproine as well as conjugation of an aminosugar.

Synthesis of fluorine-labeled peptide nucleic acid building blocks as sensors for the 19F NMR spectroscopic detection of different hybridization modes.

Peptide nucleic acid (PNA) building blocks, bearing a fluorine sensor at C-5 of the uracil base [viz. trifluoromethyl and 3,3-bis(trifluoromethyl)-4,4,4-trifluorobut-1-ynyl], were synthesized and incorporated to a PNA strand, and their applicability for the monitoring of different hybridization modes by (19)F NMR spectroscopy was studied. Both sensors gave unique (19)F resonance shifts in NMR when the PNA was targeted to a complementary antiparallel DNA, antiparallel RNA, parallel DNA, and parallel RNA. The 5-trifluoromethyl-derived sensor was additionally applied for the monitoring of interconversions from a parallel DNA/PNA complex to an antiparallel RNA/PNA complex and from a PNA/PNA complex to two DNA/PNA complexes (i.e., double-duplex invasion).

An activated triple bond linker enables 'click' attachment of peptides to oligonucleotides on solid support.

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.

Diaminopropionic acid lipopeptides: characterization studies of polyplexes aimed at pDNA delivery.

Here we report a novel class of peptides-d-diaminopropionic acids (Dap)-for gene delivery. These peptides have attractive properties for gene delivery, and the advantage that they can be easily manipulated in relation to their composition, abiding with tailored-design. We characterized the toxicological and biophysical properties of DNA particles resulting from the interaction of the nucleic acid with a series of Dap(8) peptides conjugated to different alkyl groups. These peptides formed small and homogenous DNA particle populations that protected against DNase I degradation at non-toxic concentrations. However, despite the similarity between these peptides and others that are arginine-rich, and efficient vectors, functional studies suggest the need for additional modifications in the carriers to improve their DNA delivery efficiency. Taken together, these studies underscore the relevance of the overall structure of the carrier and the complexity of designing from scratch a carrier.

Influence of sequence variation on the RNA cleavage activity of Zn2+-dimethyl-dppz-PNA-based artificial enzymes.

The development of Zn2+-dependent dimethyl-dppz-PNA conjugates (PNAzymes) as efficient site-specific artificial ribonucleases enables rapid sequence-specific degradation of clinically relevant RNA target sequences, but the significance of the RNA/PNAzyme sequence and structural demands for the identification of novel RNA targets are not fully understood. In the present study, we investigated the influence of sequence variation in the recognition arms of the RNA/PNAzyme complex on the RNA cleavage activity of the artificial enzymes. The base pairs closing the 3-nucleotide bulge region on both sides of the bulge as well as the neighbouring nucleobases were shown to significantly influence the RNA cleavage activity. Elongation of the RNA/PNAzyme complex was shown to be tolerated, although potentially prohibitive for catalytic turnover. The specificity of PNAzyme action was clearly demonstrated by the significantly reduced or absent cleavage activity in complexes containing mismatches. Further investigation into 2- and 4-nucleotide RNA bulges indicated that formation of 3-nucleotide bulges in the target RNA gives the optimal cleavage rates, while some potential off-target cleavage of formed 4-nucleotide bulges of select sequences should be considered.

2'-O-(N-(Aminoethyl)carbamoyl)methyl Modification Allows for Lower Phosphorothioate Content in Splice-Switching Oligonucleotides with Retained Activity.

2'-O-(N-(Aminoethyl)carbamoyl)methyl (2'-O-AECM)-modified oligonucleotides (ONs) and their mixmers with 2'-O-methyl oligonucleotides (2'-OMe ONs) with phosphodiester linkers as well as with partial and full phosphorothioate (PS) inclusion were synthesized and functionally evaluated as splice-switching oligonucleotides in several different reporter cell lines originating from different tissues. This was enabled by first preparing the AECM-modified A, C, G and U, which required a different strategy for each building block. The AECM modification has previously been shown to provide high resistance to enzymatic degradation, even without PS linkages. It is therefore particularly interesting and unprecedented that the 2'-O-AECM ONs are shown to have efficient splice-switching activity even without inclusion of PS linkages and found to be as effective as 2'-OMe PS ONs. Importantly, the PS linkages can be partially included, without any significant reduction in splice-switching efficacy. This suggests that AECM modification has the potential to be used in balancing the PS content of ONs. Furthermore, conjugation of 2'-O-AECM ONs to an endosomal escape peptide significantly increased splice-switching suggesting that this effect could possibly be due to an increase in uptake of ON to the site of action.

Zn2+-Dependent peptide nucleic acid-based artificial ribonucleases with unprecedented efficiency and specificity.

We present Zn2+-dependent dimethyl-dipyridophenazine PNA conjugates as efficient RNA cleaving artificial enzymes. These PNAzymes display site-specific RNA cleavage with 10 minute half-lives and cleave clinically relevant RNA models.

PNAzymes that are artificial RNA restriction enzymes.

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.

Cationic peptides that increase the thermal stabilities of 2'-O-MeRNA/RNA duplexes but do not affect DNA/DNA melting.

Several different cationic nonapeptides have been synthesized and investigated with respect to how they can influence the thermal melting of 2'-O-methylRNA/RNA and DNA/DNA duplexes. Each peptide has a C-terminal L-phenylalanine unit and is otherwise uniformly composed of a sequence of a specific basic D-amino acid that in most cases will be largely charged at neutral pH. These N-terminal octamer stretches are composed variously of the amino acids D-lysine, D-diaminobutyric acid (D-Dab), D-diaminopropionic acid (D-Dap), or D-histidine. None of the peptides substantially affected the thermal melting of DNA/DNA duplexes, which was in sharp contrast with their effects on 2'-O-methylRNA/RNA duplexes. In particular, the peptides based on diaminopropionic and diaminobutyric acid units had strong positive effects on the melting temperatures of the 2'-O-methylRNA duplexes (up to 16 °C higher with 1 equivalent of peptide) at pH 7, whereas at pH 6 the effect was even more drastic (ΔT(m) up to +25 °C). The shorter R groups of the Dap and Dab groups appear to have a better length than lysine for enhancement of the thermal melting of the 2'-O-methylRNA/RNA duplex, an effect that is more pronounced at lower pH but substantial even at pH 7, although the Dap derivative is not likely to be fully protonated. The dramatic difference between the influence, or lack thereof, on the 2'-O-methylRNA/RNA and the DNA/DNA thermal meltings suggest that, although electrostatic interactions probably play a role, there is another major and structurally dependent component influencing the properties of the duplexes. This is also seen in the observation that the oligo-Dap and oligo-Dab peptides give greater melting point enhancements than both the lysine peptide (with a longer side chain) and a ß-linked Dap peptide with a shorter side chain and a longer backbone.

PNA based artificial nucleases displaying catalysis with turnover in the cleavage of a leukemia related RNA model.

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.

Solid support post-conjugation of amino acids and a phenanthroline derivative to a central position in peptide nucleic acids.

A solid phase synthesis strategy for post-conjugation of amino acids and a phenanthroline derivative to peptide nucleic acids is described. The peptide nucleic acids, synthesized by 9-fluorenylmethyloxycarbonyl chemistry on TentaGel S Rink Amide resin, have an internally placed unit carrying an amino linker with 4-methyltrityl protection. Methyltrityl removal by mild acidic conditions and conjugation of amino acids or a phenanthroline derivative, via an amide or urea linker, was performed on-resin after completion of the chain assembly. This solid phase methodology resulted in excellent purities of the crude conjugates.

RNA cleavage by 2,9-diamino-1,10-phenanthroline PNA conjugates.

We report on the synthesis of 2,9-diamino-1,10-phenanthroline PNA conjugates as well as on their action in cleavage of a target RNA. Synthesis of the PNA conjugates are performed on solid support and the phenanthroline derivative is conjugated either to the amino-end or to a centrally positioned diaminopropionic acid in the PNA via a urea linker. Cleavage of the target RNA is achieved and compared to cleavage with the corresponding 2,9-dimethyl-1,10-phenanthroline and glycine conjugates.

Development of 2'-o-methyloligoribonucleotide and peptide nucleic acid based artificial ribonucleases.

We have developed 2'-O-methyloligoribonucleotide and peptide nucleic acid (PNA) based artificial ribonucleases (OBAN's), which in presence of zinc or copper ions cleave a potential therapy target in leukemia, an M-BCR/ABL mRNA model. The OBAN's give turnover of substrate and are thus real enzymes. A copper ion based system even gives single site cleavage in the RNA-substrate.

Synthesis of 8-aminoadenosine 5'-(aminoalkyl phosphates), analogues of aminoacyl adenylates.

A short and efficient route for the synthesis of aminoalkyl 8-aminoadenylates, potential aminoacyl-tRNA synthetase inhibitors, is presented. Aminoalkyl 8-aminoadenylates were synthesized using a 5'-H-phosphonate strategy involving minimal protecting group manipulations and a single final deprotection step.

Hydrolytic reactions of 3'-N-phosphoramidate and 3'-N-thiophosphoramidate analogs of thymidylyl-3',5'-thymidine.

The diastereomeric thiophosphoramidate analogs [(R(P))- and (S(P))-3[prime or minute],5[prime or minute]-Tnp(s)T] and the phosphoramidate analog [3[prime or minute],5[prime or minute]-TnpT] of thymidylyl-3[prime or minute],5[prime or minute]-thymidine were prepared and their hydrolytic reactions over the pH-range 1-8 at 363.2 K were followed by RP HPLC. At pH < 6, an acid-catalyzed P-N3[prime or minute] bond cleavage (first-order in [H(+)]) takes place with both 3[prime or minute],5[prime or minute]-Tnp(s)T and 3[prime or minute],5[prime or minute]-TnpT, the former being about 12 fold more stable than the latter. At pH > 4, Tnp(s)T undergoes two competing pH-independent reactions, desulfurization (yielding TnpT) and depyrimidination (cleavage of the N-glycosidic bond) the rates of which are of the same order of magnitude. Also with 3[prime or minute],5[prime or minute]-TnpT the pH-independent depyrimidination competes with P-N3[prime or minute] cleavage at pH > 5.