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

The development of Zn-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.

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

2'--(-(Aminoethyl)carbamoyl)methyl (2'--AECM)-modified oligonucleotides (ONs) and their mixmers with 2'--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.

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

We present Zn-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.

Further Probing of Cu-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 Cu 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 Cu-dependent PNAzymes with respect to the dependence on bulge composition in 3- and 4-nucleotide bulge systems.

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 Zn-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.

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.

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 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 F NMR spectroscopy. Detailed information on the stoichiometry and transition between the triple-helical peptide nucleic acid (PNA)/RNA and (PNA) /RNA binding modes could be obtained.

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 β-amino groups of inserted diaminopropionic acids residues was achieved.

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.

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.

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).

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.

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.

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.

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.