Azidopeptide nucleic acid. An alternative strategy for solid-phase peptide nucleic acid (PNA) synthesis

[reaction: see text] A practical and efficient method for PNA synthesis using an azide group to mask the N-terminus is reported. The deprotection was carried out in 5 min, while couplings were complete within 60 min. The near neutral conditions of the phosphine deprotection combined with the base-free coupling using hydroxybenzotriazole-activated monomers make this approach very mild.     

Reaching into the major groove of B-DNA: synthesis and nucleic acid binding of a neomycin-hoechst 33258 conjugate

Synthesis of a neomycin-Hoechst 33258 conjugate is reported. The conjugate combines the ligands known to selectively bind in the duplex and the triplex groove. The conjugate stabilizes DNA duplex over DNA triplex. The conjugate selectively stabilizes the DNA duplex (in the presence of salt), with as little as 2 muM of the ligand leading to a DeltaTm of 25 degrees C.     

Parallel nucleic acid recognition by the LNA (locked nucleic acid) stereoisomers beta-L-LNA and alpha-D-LNA; studies in the mirror image world

Two LNA (locked nucleic acid) stereoisomers (beta-L-LNA and alpha-D-LNA) are evaluated in the mirror-image world, that is by the study of two mixed sequences of LNA and alpha-L-LNA and their L-DNA and L-RNA complements. Both are found to display high-affinity RNA-recognition by the formation of duplexes with parallel strand orientation.     

Food analysis and food authentication by peptide nucleic acid (PNA)-based technologies

This tutorial review will address the issue of DNA determination in food by using Peptide Nucleic Acid (PNA) probes with different technological platforms, with a particular emphasis on the applications devoted to food authentication. After an introduction aimed at describing PNAs structure, binding properties and their use as genetic probes, the review will then focus specifically on the use of PNAs in the field of food analysis. In particular, the following issues will be considered: detection of genetically modified organisms (GMOs), of hidden allergens, of microbial pathogens and determination of ingredient authenticity. Finally, the future perspectives for the use of PNAs in food analysis will be briefly discussed according to the most recent developments.     

Triplex formation with alpha-L-LNA (alpha-L-ribo-configured locked nucleic acid)

Using UV melting and CD spectroscopy, we show that alpha-l-LNA-modified oligonucleotides possess the ability to form triplexes at pH 6.8 with significantly increased thermostability relative to DNA triplexes.     

Peptide nucleic acid and amino acid modified peptide nucleic acid analysis by capillary zone electrophoresis

A rapid, high resolution, and low sample consumption CZE method is developed for peptide nucleic acid (PNA) analysis for the first time. 30% v/v acetonitrile in PNA sample and 20% v/v acetonitrile in 50 mM borax‐boric acid (pH 8.7) as BGE were employed after optimization. The calibration curves were linear for PNA concentration ranging from 1 to 50 μmol/L. LOD and LOQ of PNA were 0.2 and 1.0 μmol/L, respectively. Since the commercially available reagent gives rise to huge PNA peak and an apparent impurity peak, the purity of PNA was evaluated to be about 81.4% by CZE method, obviously lower than the supplier's purity value of 99.9% evaluated by RP–HPLC, and also lower than 94.8% determined with RP–HPLC by our research group. The CZE method takes only 5 min, needs only 90 nL PNA, much less than 20 min and 20 μL PNA in RP–HPLC method. Moreover, the CZE method is applicable for the analysis of glutamic acid modified and lysine modified PNAs, they show different migration time with their corresponding complementary PNAs. Our results show CZE provides a new choice for PNA and modified PNA analysis, also their purity or quality evaluation.     

Highly fluorescent conjugated pyrenes in nucleic acid probes: (phenylethynyl)pyrenecarbonyl-functionalized locked nucleic acids

In recent years, fluorescently labeled oligonucleotides have become a widely used tool in diagnostics, DNA sequencing, and nanotechnology. The recently developed (phenylethynyl)pyrenes are attractive dyes for nucleic acid labeling, with the advantages of long-wave emission relative to the parent pyrene, high fluorescence quantum yields, and the ability to form excimers. Herein, the synthesis of six (phenylethynyl)pyrene-functionalized locked nucleic acid (LNA) monomers M(1)-M(6) and their incorporation into DNA oligomers is described. Multilabeled duplexes display higher thermal stabilities than singly modified analogues. An increase in the number of phenylethynyl substituents attached to the pyrene results in decreased binding affinity towards complementary DNA and RNA and remarkable bathochromic shifts of absorption/emission maxima relative to the parent pyrene fluorochrome. This bathochromic shift leads to the bright fluorescence colors of the probes, which differ drastically from the blue emission of unsubstituted pyrene. The formation of intra- and interstrand excimers was observed for duplexes that have monomers M(1)-M(6) in both complementary strands and in numerous single-stranded probes. If more phenylethynyl groups are inserted, the detected excimer signals become more intense. In addition, (phenylethynyl)pyrenecarbonyl-LNA monomers M(4), M(5), and M(6) proved highly useful for the detection of single mismatches in DNA/RNA targets.     

Modular nucleic acid surrogates. Solid phase synthesis of alpha-helical peptide nucleic acids (alpha PNAs)

[formula: see text] The synthesis and characterization of prototype alpha-helical peptide nucleic acid (alpha PNA) modules 1-3 as well as disulfide dimers 4 and 5 are reported. These molecules combine an alpha-helical peptidyl scaffold with well-defined nucleobase molecular recognition patterns and could serve as a basis for novel antisense and/or antigene agents. Structure assignments for these alpha PNAs were supported by MALDI-TOF mass spectrometry, and the alpha-helical nature of 4 in water was confirmed by circular dichroism (CD) spectroscopy.     

LNA (locked nucleic acid) and analogs as triplex-forming oligonucleotides

The triplex-forming abilities of some conformationally restricted nucleotide analogs are disclosed and compared herein. 2'-Amino-LNA monomers proved to be less stabilising to triplexes than LNA monomers when incorporated into a triplex-forming third strand. N2'-functionalisation of 2'-amino-LNA monomers with a glycyl unit induced the formation of exceptionally stable triplexes. Nucleotide analogs containing a C2',C3'-oxymethylene linker (E-type furanose conformation) or a C2',C4'-propylene linker (N-type furanose conformation) had no significant effect on triplex stability proving that conformational restriction per se is insufficient to stabilise triplexes.     

Chiral peptide nucleic acid monomers (PNAM) with modified backbones

Convenient high yielding syntheses of optically pure PNAMs comprising l- or d-serine, l-lysine and l-arginine units linked to thymine or Cbz-cytosine are described. Simple workup and inexpensive reagents are employed and free amino acids are used as coupling components.     

alpha-L-ribo-configured locked nucleic acid (alpha-L-LNA): synthesis and properties

The syntheses of monomeric nucleosides and 3'-O-phosphoramidite building blocks en route to alpha-L-ribo-configured locked nucleic acids (alpha-L-LNA), composed entirely of alpha-L-LNA monomers (alpha-L-ribo configuration) or of a mixture of alpha-L-LNA and DNA monomers (beta-D-ribo configuration), are described and the alpha-L-LNA oligomers are studied. Bicyclic 5-methylcytosin-1-yl and adenine-9-yl nucleoside derivatives have been prepared and the phosphoramidite approach has been used for the automated oligomerization leading to alpha-L-LNA oligomers. Binding studies revealed very efficient recognition of single-stranded DNA and RNA target oligonucleotide strands. Thus, stereoirregular alpha-L-LNA 11-mers containing a mixture of alpha-L-LNA monomers and DNA monomers ("mix-mer alpha-L-LNA") were shown to display DeltaT(m) values of +1 to +3 degrees C per modification toward DNA and +4 to +5 degrees C toward RNA when compared with the corresponding unmodified DNA x DNA and DNA x RNA reference duplexes. The corresponding DeltaT(m) values per modification for the stereoregular fully modified alpha-L-LNA were determined to be +4 degrees C (against DNA) and +5 degrees C (against RNA). 11-Mer alpha-L-LNAs (mix-mer alpha- L-LNA or fully modified alpha- L-LNA) were shown in vitro to be significantly stabilized toward 3'-exonucleolytic degradation. A duplex formed between RNA and either mix-mer alpha-L-LNA or fully modified alpha-L-LNA induced in vitro Escherichia coli RNase H-mediated cleavage, albeit very slow, of the RNA targets at high enzyme concentrations.     

Design of a new oligotriazole peptide nucleic acid analogue (oT-PNA)

We describe in this Letter the synthesis of an original thymine azido-heterotrimer generated by Click Chemistry. This trimer has been obtained from an azido-thymidine and a new chloroethyl-propargylated PNA monomer analogue, after two azidation/click-reaction cycles. Conformational preferences of a rotameric intermediate have also been studied     

Superior duplex DNA strand invasion by acridine conjugated peptide nucleic acids

DNA helix invasion by P-loop forming peptide nucleic acids (PNAs) is extremely sensitive to increased ionic strength as this stabilizes the DNA duplex. To address this, the DNA intercalator 9-aminoacridine was conjugated to helix invading PNAs, and the duplex DNA binding efficiency of such constructs was measured at different ionic strength conditions by electrophoretic mobility shift analysis. Remarkably, at physiogically relevant ionic strength (140 mM K+/10 mM Na+, 2 mM Mg2+), acridine conjugated PNAs showed 20-150-fold superior binding to a cognate sequence target as compared to the conventional PNAs. This enhancement occurred without compromising the sequence specificity of binding. Thus, simply conjugating the DNA intercalator 9-aminoacridine to PNA represents a major step toward the development of helix invading constructs for in vivo applications such as gene targeting.     

Design, synthesis, conformational analysis and nucleic acid hybridisation properties of thymidyl pyrrolidine-amide oligonucleotide mimics (POM)

Pyrrolidine-amide oligonucleotide mimics (POM) 1 were designed to be stereochemically and conformationally similar to natural nucleic acids, but with an oppositely charged, cationic backbone. Molecular modelling reveals that the lowest energy conformation of a thymidyl-POM monomer is similar to the conformation adopted by ribonucleosides. An efficient solution phase synthesis of the thymidyl POM oligomers has been developed, using both N-alkylation and acylation coupling strategies. 1H NMR spectroscopy confirmed that the highly water soluble thymidyl-dimer, T2-POM, preferentially adopts both a configuration about the pyrrolidine N-atom and an overall conformation in D2O that are very similar to a typical C3'-endo nucleotide in RNA. In addition the nucleic acid hybridisation properties of a thymidyl-pentamer, T5-POM, with an N-terminal phthalimide group were evaluated using both UV spectroscopy and surface plasmon resonance (SPR). It was found that T5-POM exhibits very high affinity for complementary ssDNA and RNA, similar to that of a T5-PNA oligomer. SPR experiments also showed that T5-POM binds with high sequence fidelity to ssDNA under near physiological conditions. In addition, it was found possible to attenuate the binding affinity of T5-POM to ssDNA and RNA by varying both the ionic strength and pH. However, the most striking feature exhibited by T5-POM is an unprecedented kinetic binding selectivity for ssRNA over DNA.     

Electrochemical analysis of the self-complementary B-DNA decamer d(CCAGGCCTGG)

The self-complementary decamer d(CCAGGCCTGG), native and denatured calf thymus DNAs were studied by means of differential pulse polarography with the DME and by cyclic voltammetry with the HMDE. The decamer (which represents one turn of the DNA double helix in the B form) produced cathodic and anodic signals similar to those yielded by high-molar mass DNAs. By measuring the anodic peak (due to guanine residues), it was possible to detect the decamer at subnanomolar concentrations by adsorptive stripping cyclic voltammetry at relatively short waiting times. The high sensitivity of the electrochemical analysis for small changes in the DNA double helix observed earlier in high-molar mass DNAs, together with the low requirements for the amount of the analyzed decamer sample, suggest that electrochemical techniques may become useful also in oligonucleotide studies.