Light-directed synthesis of peptide nucleic acids (PNAs) chips

We report herein the light-directed synthesis of peptide nucleic acids (PNAs) microarray using PNA monomers protected by photolabile protecting groups and a maskless technique that uses a digital micromirror array system to form virtual masks. An ultraviolet image from the virtual mask was cast onto the active surface of a glass substrate, which was mounted in a flow cell reaction chamber connected to a peptide synthesizer. Light exposure was followed by automatic chemical coupling cycles and these steps were repeated with different virtual masks to grow the desired PNA probes in a selected pattern. In a preliminary experiment, an array of PNA probes with dimensions of 4.11 mm × 4.11 mm was generated on each slide. Each synthesis region in the final array measured 210 μm × 210 μm for a total of 256 sites. The center-to-center space was 260 μm. It was observed from the hybridization pattern of the fluorescently labeled oligonucleotide targets that the fluorescence intensities of the matched, and mismatched sequences showed substantial difference, demonstrating specificity in the identification of complementary sequences. This opens the way to exploit processes from the microelectronics industry for the fabrication of PNA microarrays with high densities.     

Solid-phase synthesis of peptide nucleic acid (PNA) monomers and their oligomerization using disulphide anchoring linkers

A new simple solid-phase method has been developed for synthesizing Boc-protected peptide nucleic acid (PNA) monomers. An immobilized backbone 3 was built on Expansin® resin using an ester disulphide handle: 2-hydroxypropyl-dithio-2′-isobutyric acid (HPDI). The base acetic acids of thymine 5 , Z-cytosine 9 , Z-adenine 12 , and 6-O-benzyl guanine 17 were prepared and coupled to the immoblized backbone. The HPDI handle was cleaved under mild conditions by cyanolysis or assisted hydrolysis with tris(2-carboxyethyl)phosphine (TCEP) to give undamaged PNA monomers. These monomers were coupled to form oligomers by solid-phase method with another disulphide linkage: aminoethyldithio-2-isobutyric acid (AEDI) grafted on an amino-functionalized TentaGel® resin, using in situ neutralization and TBTU as activating reagent. Final cleavage of the AEDI linker gave PNA bearing a cysteamide residue that could be useful for optimizing PNA properties. Oligomers of up to 16 residues long were assembled. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Insight into why pyrrolidinyl peptide nucleic acid binding to DNA is more stable than the DNA x DNA duplex

Molecular dynamics (MD) simulations and experimental measurements of the stability of a novel pyrrolidinyl PNA binding to DNA (PNA·DNA) in both parallel and antiparallel configurations were carried out. For comparison, simulations were also performed for the DNA·DNA duplex. The conformations of the three simulated systems were found to retain well-defined base pairing and base stacking as their starting B-like structure. A large gas-phase energy repulsion of the two negatively charged sugar-phosphate backbones of the DNA strands was found to reduce the stability of the DNA·DNA duplex significantly compared with that of the PNA·DNA complexes, especially in the antiparallel binding configuration. In addition, the antiparallel PNA·DNA was observed to be less solvated than that of the other two systems. The simulated binding free energies and the experimental melting temperatures for the three investigated systems are in good agreement, indicating that the antiparallel PNA·DNA is the most stable duplex.     

Investigating the kinetics of DNA–DNA and PNA–DNA interactions using surface plasmon resonance-enhanced fluorescence spectroscopy

Plasmon surface polaritons, resonantly excited in the Kretschmann format, are used to enhance the fluorescence emission of chromophore-labeled oligonucleotides (15mers) binding to surface-attached (via biotin–streptavidin linkages) complement catcher probes. A detailed analysis of the association and dissociation kinetics as well as the affinity constants is given for a mismatch 1 hybrid, emphasizing, in particular, the experimental conditions that are required to allow for an artifact-free determination of rate constants. A first comparison between DNA- and peptide nucleic acid (PNA-) probes shows similar affinities, however, significant deviations from single-exponential kinetics predicted by a simple Langmuir model for the PNA case are found.     

C3′-endo-puckered pyrrolidine containing PNA has favorable geometry for RNA binding: Novel ethano locked PNA (ethano-PNA)

A novel peptide nucleic acid (PNA) analogue is designed with a constraint in the aminoethyl segment of the aegPNA backbone so that the dihedral angle β is restricted within 60–80°, compatible to form PNA:RNA duplexes. The designed monomer is further functionalized with positively charged amino-/guanidino-groups. The appropriately protected monomers were synthesized and incorporated into aegPNA oligomers at predetermined positions and their binding abilities with cDNA and RNA were investigated. A single incorporation of the modified PNA monomer into a 12-mer PNA sequence resulted in stronger binding with complementary RNA over cDNA. No significant changes in the CD signatures of the derived duplexes of modified PNA with complementary RNA were observed.     

Synthesis of peptide nucleic acid oligomers carrying 5-methylcytosine derivatives by postsynthetic substitution

The preparation of the thymine peptide nucleicacid (PNA) monomer carrying a 2-nitrophenyl group in position4 is described. This monomer is incorporated into PNAoligomers and reacted with amines to yield PNA oligomerscarrying 5-methylcytosine derivatives. During thedeprotection-modification step two side reactions weredetected: degradation of PNA oligomer from the N-terminal residue and modification of N ⁴-tert-butylbenzoyl cytosine residue. Protection of the N-terminal position and the use of N ⁴-acetyl group for the protection of cytosine eliminate these side reactions.     

Synthesis of peptide nucleic acid oligomers carrying 5-methylcytosine derivatives by postsynthetic substitution

Summary The preparation of the thymine peptide nucleic acid (PNA) monomer carrying a 2-nitrophenyl group in position 4 is described. This monomer is incorporated into PNA oligomers and reacted with amines to yield PNA oligomers carrying 5-methylcytosine derivatives. During the deprotection-modification step two side reactions were detected: degradation of PNA oligomer from theN-terminal residue and modification ofN ⁴-tert-butylbenzoyl cytosine residue. Protection of theN-terminal position and the use ofN ⁴-acetyl group for the protection of cytosine eliminate these side reactions.     

Preparation of radioiodinated peptide nucleic acids with high specific activity

Peptide nucleic acids (PNAs) have stronger affinity and greater specificity than do oligonucleotides for binding to DNA and RNA and, as such, have potential utility as probes in molecular biology applications. In this study, a novel approach for labeling the PNA with radioiodine that avoided solubility issues and poor labeling encountered when trying to radioiodinate PNAs directly in solution was developed. For this approach, a purpose-designed prosthetic group that incorporated both a radioiodinatable tyrosine and a triphenylphosphonium (TPP) moiety was synthesized. The latter is an organic cation that combines the properties of good solubility in both aqueous and organic solvents with a strong retention by reverse phase HPLC. Following radioiodination of the TPP-based prosthetic group in phosphate buffer, the prosthetic group was purified and coupled to the terminal amine of 15-mer PNA on the solid phase resin. After cleavage and deprotection of the PNA from the resin, the presence of the TPP group resulted in a clean separation of radioiodinated PNA from unlabeled PNA, yielding a high-specific activity probe in a single HPLC run. As an example of a potential molecular biology application of the resultant (125)I-labeled PNA probe, it was used to detect mRNA for the Lcn2 gene in Northern blotting.     

Synthesis of peptide nucleic acid-peptide chimeras carrying the c-myc tag-sequence

The preparation of peptide nucleic acids (PNA)carrying a c-myc tag-peptide sequence isdescribed. These PNA-peptide chimeras have higheraffinity to complementary DNA than unmodifiedoligonucleotides. Moreover, they can be used asnonradioactive probes with sensitivity similar toother nonradioactive methods.     

Peptide nucleic acids specifically cause antigene effects in vivo by systemic injection

Peptide nucleic acids (PNAs) are uncharged DNA analogs that hybridize to complementary sequences with high affinity and stability. We previously showed that PNAs, after intraperitoneal injection into rats, are effective antisense compounds in vivo. The present study was designed to test whether PNAs also have antigene effects in vivo. The renin-angiotensin system is critical in the control of blood pressure. We designed and synthesized sense (antigene) PNAs to angiotensinogen, which is the precursor protein that leads to angiotensin I and II. Spontaneously hypertensive rats received intraperitoneal injections of either 20 mg/kg sense-angiotensinogen-PNA, mismatch-angiotensinogen PNA, or saline. Only the sense-angiotensinogen PNA treatment resulted in a significant decrease in plasma angiotensin I, systolic blood pressure, and liver and brain angiotensinogen mRNA levels. Thus, these results demonstrate on the molecular, protein, and physiological levels that antigene PNAs are effective in vivo upon systemic administration.     

Synthesis of peptide nucleic acid-peptide chimeras carrying the c-myc tag-sequence

Summary The preparation of peptide nucleic acids (PNA) carrying a c-myc tag-peptide sequence is described. These PNA-peptide chimeras have higher affinity to complementary DNA than unmodified oligonucleotides. Moreover, they can be used as nonradioactive probes with sensitivity similar to other nonradioactive methods.     

3-Aminopyrrolidine-4-carboxylic acid as versatile handle for internal labeling of pyrrolidinyl PNA

Conformationally restricted pyrrolidinyl PNAs with an α/β-dipeptide backbone consisting of a nucleobase-modified proline and a cyclic five-membered amino acid spacer such as (1S,2S)-2-aminocyclopentanecarboxylic acid (ACPC) (acpcPNA) can form very stable hybrids with DNA with high Watson-Crick base pairing specificity. This work aims to explore the effect of incorporating 3-aminopyrrolidine-4-carboxylic acid (APC), which is isosteric to the ACPC spacer, into the acpcPNA. It is expected that the modification should not negatively affect the DNA binding properties, and that the additional nitrogen atom in the APC should provide a handle for internal modification. Orthogonally-protected (N(3)-Fmoc/N(1)-Boc and N(3)-Fmoc/N(1)-Tfa) APC monomers have been successfully synthesized and incorporated into the acpcPNA by Fmoc-solid-phase peptide synthesis. T(m), UV and CD spectroscopy confirmed that the (3R,4S)-APC could substitute the (1S,2S)-ACPC spacer in the acpcPNA with only slightly decreasing the stability of the hybrids formed between the modified acpc/apcPNA and DNA. In contrast, the (3S,4R) enantiomer of APC caused substantial destabilization of the hybrids. Furthermore, a successful on-solid-support internal labeling of the acpc/apcPNA via amide bond formation between pyrene-1-carboxylic acid or 4-(pyrene-1-yl) butyric acid and the pyrrolidine nitrogen atom of the APC spacer has been demonstrated. Fluorescence properties of the pyrene-labeled acpc/apcPNAs are sensitive to their hybridization states and can readily distinguish between complementary and single-mismatched DNA targets.