Hydrophilic and Cell-Penetrable Pyrrolidinyl Peptide Nucleic Acid via Post-synthetic Modification with Hydrophilic Side Chains.

Peptide nucleic acid (PNA) is a nucleic acid mimic in which the deoxyribose-phosphate was replaced by a peptide-like backbone. The absence of negative charge in the PNA backbone leads to several unique behaviors including a stronger binding and salt independency of the PNA-DNA duplex stability. However, PNA possesses poor aqueous solubility and cannot directly penetrate cell membranes. These are major obstacles that limit in vivo applications of PNA. In previous strategies, the PNA can be conjugated to macromolecular carriers or modified with positively charged side chains such as guanidinium groups to improve the aqueous solubility and cell permeability. In general, a preformed modified PNA monomer was required. In this study, a new approach for post-synthetic modification of PNA backbone with one or more hydrophilic groups was proposed. The PNA used in this study was the conformationally constrained pyrrolidinyl PNA with prolyl-2-aminocyclopentanecarboxylic acid dipeptide backbone (acpcPNA) that shows several advantages over the conventional PNA. The aldehyde modifiers carrying different linkers (alkylene and oligo(ethylene glycol)) and end groups (-OH, -NH2, and guanidinium) were synthesized and attached to the backbone of modified acpcPNA by reductive alkylation. The hybrids between the modified acpcPNAs and DNA exhibited comparable or superior thermal stability with base-pairing specificity similar to those of unmodified acpcPNA. Moreover, the modified apcPNAs also showed the improvement of aqueous solubility (10-20 folds compared to unmodified PNA) and readily penetrate cell membranes without requiring any special delivery agents. This study not only demonstrates the practicality of the proposed post-synthetic modification approach for PNA modification, which could be readily applied to other systems, but also opens up opportunities for using pyrrolidinyl PNA in various applications such as intracellular RNA sensing, specific gene detection, and antisense and antigene therapy.

Bringing macromolecules into cells and evading endosomes by oxidized carbon nanoparticles.

A great challenge exists in finding safe, simple, and effective delivery strategies to bring matters across cell membrane. Popular methods such as viral vectors, positively charged particles and cell penetrating peptides possess some of the following drawbacks: safety issues, lysosome trapping, limited loading capacity, and toxicity, whereas electroporation produces severe damages on both cargoes and cells. Here, we show that a serendipitously discovered, relatively nontoxic, water dispersible, stable, negatively charged, oxidized carbon nanoparticle, prepared from graphite, could deliver macromolecules into cells, without getting trapped in a lysosome. The ability of the particles to induce transient pores on lipid bilayer membranes of cell-sized liposomes was demonstrated. Delivering 12-base-long pyrrolidinyl peptide nucleic acids with d-prolyl-(1S,2S)-2-aminocyclopentanecarboxylic acid backbone (acpcPNA) complementary to the antisense strand of the NF-κB binding site in the promoter region of the Il6 gene into the macrophage cell line, RAW 264.7, by our particles resulted in an obvious accumulation of the acpcPNAs in the nucleus and decreased Il6 mRNA and IL-6 protein levels upon stimulation. We anticipate this work to be a starting point in a new drug delivery strategy, which involves the nanoparticle that can induce a transient pore on the lipid bilayer membrane.

Specific recognition of cytosine by hypoxanthine in pyrrolidinyl peptide nucleic acid.

Hypoxanthine is an unnatural base that can potentially pair with all natural nucleobases. While hypoxanthine in DNA exhibits marginal preference for pairing with cytosine (C), little is known about its pairing behavior in other DNA analogues. In this study, we synthesized a hypoxanthine-containing monomer and incorporated it into pyrrolidinyl peptide nucleic acid with α/ß-peptide backbone derived from D-prolyl-(1S,2S)-2-aminocyclopentanecarboxylic acid (acpcPNA). DNA binding studies clearly revealed that hypoxanthine in acpcPNA behaves like G-analogue because it can specifically form a stable base pair with dC in DNA. The ability to replace G by hypoxanthine without affecting the base pairing properties of acpcPNA can solve a number of problems associated with G-rich acpcPNA including difficult synthesis, formation of secondary structures and fluorescence quenching.

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

Conformationally restricted pyrrolidinyl PNAs with an α/ß-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.

New calix[4]arene derivatives as ionophores in polymeric membrane electrodes for Ag(I): comparative selectivity studies and detection of DNA hybridization.

Four calix[4]arene derivatives containing various donor atoms and different topology (L1-L4) have been synthesized and used as neutral ionophores to fabricate silver ion selective electrodes (Ag-ISEs) which were characterized in terms of their potentiometric selectivities and complex formation constants. The ionophore L2 having two nitrogen and two sulfur donors showed stronger interactions with Ag(+) and the highest selectivity coefficient towards Ag(+). The best membrane electrode was prepared from L2 and used to fabricate silver ion selective microelectrodes (Ag-ISµEs) which could detect silver ions in 1000 µL samples with detection limit around 1 µM using sodium ion microelectrodes as a pseudo reference electrode. Such potentiometric measurement was then applied to detect DNA hybridization on a gold substrate, employing immobilized lipoic acid-modified pyrrolidinyl PNA (Lip-acpcPNA) as a probe. The hybridization between the neutral Lip-acpcPNA probe and DNA target led to a negatively charged surface that could bind positively charged silver nanoparticles (AgNPs(+)) via electrostatic interactions. The hybridization signal was observed by dissolution of the electrostatically adsorbed AgNPs(+) with hydrogen peroxide. Excellent discrimination of complementary from single mismatched and non-complementary DNA targets was achieved under non-stringent conditions. The detection limit of DNA was 0.2 µM in 1000 µL samples.

Pyrrolidinyl peptide nucleic acid homologues: effect of ring size on hybridization properties.

The effect of ring size of four- to six-membered cyclic ß-amino acid on the hybridization properties of pyrrolidinyl peptide nucleic acid with an alternating α/ß peptide backbone is reported. The cyclobutane derivatives (acbcPNA) show the highest T(m) and excellent specificity with cDNA and RNA.

5-(Pyren-1-yl)uracil as a base-discriminating fluorescent nucleobase in pyrrolidinyl peptide nucleic acids.

A pyrene-labeled uridine (U(Py)) monomer for a pyrrolidinyl peptide nucleic acid with an alternating proline/2-aminocyclopentanecarboxylic acid backbone (acpcPNA) was synthesized and incorporated into the PNA. The U(Py) base in acpcPNA could specifically recognize the base A in its complementary DNA strand as determined by thermal denaturation (T(m)) experiments. The fluorescence of the U(Py)-containing single-stranded acpcPNA was very weak in aqueous buffer. In the presence of a complementary DNA target, the fluorescence was enhanced significantly (2.7-41.9 folds, depending on sequences). The fluorescence enhancement was specific to the pairing between U(Py) and dA, making the U(Py)-modified acpcPNA useful as a hybridization-responsive fluorescence probe for DNA-sequence determination.

Pyrrolidinyl peptide nucleic acid with α/ß-peptide backbone: A conformationally constrained PNA with unusual hybridization properties.

We describe herein a new conformationally constrained analog of PNA carrying an alternating α/ß amino acid backbone consisting of (2'R,4'R)-nucleobase-subtituted proline and (1S,2S)-2-aminocyclopentanecarboxylic acid (acpcPNA). The acpcPNA has been synthesized and evaluated for DNA, RNA and self-pairing properties by thermal denaturation experiments. It can form antiparallel hybrids with complementary DNA with high affinity and sequence specificity. Unlike other PNA systems, the thermal stability of acpcPNA·DNA hybrid is largely independent of G+C contents, and is generally higher than that of acpcPNA·RNA hybrid with the same sequence. Thermodynamic parameters analysis suggest that the A·T base pairs in the acpcPNA·DNA hybrids are enthalpically stabilized over G·C pairs. The acpcPNA also shows a hitherto unreported behavior, namely the inability to form self-pairing hybrids. These unusual properties should make the new acpcPNA a potentially useful candidate for various applications including microarray probes and antigene agents.