Synthesis and oligodeoxynucleotide binding properties of pyrrolidinyl peptide nucleic acids bearing prolyl-2-aminocyclopentanecarboxylic acid (ACPC) backbones

A series of novel conformationally rigid pyrrolidinyl peptide nucleic acids (PNA) based on d-prolyl-2-aminocyclopentanecarboxylic acid (ACPC) backbones has been synthesized. Investigation of the binding properties of four stereoisomeric PNAs possessing different stereochemistry at the ACPC part with DNA revealed that a precise stereochemistry of the backbone is very important in determining the binding properties. Only the PNA containing (1S,2S)-ACPC can form a very stable 1:1 complex with the complementary DNA in a sequence-specific manner.     

Metal binding to ligand-containing peptide nucleic acids

The substitution of nucleobases in nucleic acid duplexes with ligands that have high affinity for transition metal ions creates metal-binding sites at specific locations within the duplexes. Several studies on the incorporation of metal ions into DNA and peptide nucleic acid (PNA) duplexes have suggested that the stability constant of the metal complex formed within the duplexes is a primary determinant of the thermal stability of the duplexes. To understand this relationship, we have synthesized two PNA monomers that carry the same ligand, namely 8-hydroxyquinoline, but have this ligand attached differently to the PNA backbone. The PNA monomers have been incorporated into PNA duplexes. UV and CD spectroscopy and calorimetric studies of the 8-hydroxyquinoline-PNA duplexes showed that the effect of the stability of the metal complex on the PNA duplexes was significantly modulated by the steric relationship between the complex and the duplex. This information is useful for the construction of hybrid inorganic-nucleic acid nanostructures.     

Side chain homologation of alanyl peptide nucleic acids: pairing selectivity and stacking

Alanyl peptide nucleic acids (alanyl-PNAs) are oligomers based on a regular peptide backbone with alternating configuration of the amino acids. All side chains are modified by covalently linked nucleobases. Alanyl-PNAs form very rigid, well defined, and linear double strands based on hydrogen bonding of complementary strands, stacking, and solvation. Side chain homology was examined by comparing a methylene linker (alanyl-PNA) with an ethylene linker (homoalanyl-PNA), a trimethylene linker (norvalyl-PNA), and PNA sequences with mixed linker length between nucleobase and backbone. Side chain homology in combination with a linear double strand topology turned out to be valuable in order to selectively manipulate pairing selectivity (pairing mode) and base pair stacking.     

Backbone-extended pyrrolidine peptide nucleic acids (bepPNA): design, synthesis and DNA/RNA binding studies

One-carbon extended conformationally constrained pyrrolidine PNA monomer (bepPNA) has been synthesized, incorporated into PNA sequences at predefined positions, and showed selective RNA binding properties.     

Clicked polycyclic aromatic hydrocarbon as a hybridization-responsive fluorescent artificial nucleobase in pyrrolidinyl peptide nucleic acids

Pyrene as well as other aromatic hydrocarbons could be successfully incorporated into pyrrolidinyl peptide nucleic acid bearing a d-prolyl-2-aminocyclopentane carboxylic acid backbone (acpcPNA) as a base surrogate via a triazole linker employing Cu-catalyzed alkyne–azide cycloaddition (click chemistry). The labeling can be performed via a pre-clicked pyrene monomer or by post-synthetic modification of azide-containing acpcPNA on solid support. Thermal denaturation experiments suggested that the pyrene–triazole unit can behave as a universal base in the acpcPNA system. The mode of base-pairing has been proposed based on molecular dynamics simulations. Importantly, the fluorescence spectra of the pyrene-labeled single stranded acpcPNA and its hybrid with DNA are quite different. The ratio of emissions at 380 and 460 nm changed significantly (up to a factor of 7) upon hybrid formation with complementary DNA.     

Dual pyrene-labeled pyrrolidinyl peptide nucleic acid as an excimer-to-monomer switching probe for DNA sequence detection

The unique ability of pyrene to form excimers with distinct emission characteristic from monomer offers an attractive means to signal the interactions between biomolecules. In this work, dual pyrene-labeled pyrrolidinyl peptide nucleic acid probe with a d-prolyl-2-aminocyclopetanecarboxylic acid α,β-dipeptide backbone (acpcPNA) was designed as an excimer-to-monomer switching probe for DNA sequence detection. In single stranded state, the excimer emission at 470 nm was mainly observed in the fluorescence spectrum. In the presence of DNA target, the hybridization resulted in separation of the two pyrene units, therefore the spectrum displayed increased monomer emission at 380 nm with concomitant decreased excimer emission. Switching ratio, which is defined as the ratio of the monomer to excimer in the double stranded form [F₃₈₀/F₄₇₀(ds)] divided by the same value obtained from the single stranded form [F₃₈₀/F₄₇₀(ss)], was used to describe the performance of the probes. Switching ratios in the range of 5–30 were observed with various dual pyrene-labeled acpcPNA probes bearing pyrenebutyryl label attached five-base apart. Practically no excimer-to-monomer switching behavior was observed with DNA targets carrying a single mismatched base as shown by the small switching ratios of ∼1.     

Probing binding preferences of DNA and RNA: backbone chirality of thioacetamido-linked nucleic acids and iso-thioacetamido-linked nucleic acids to differentiate DNA versus RNA selective binding

Subtle differences in RNA and DNA duplex geometry could be sensed by the changed stereochemistry at 3'-amino function in the 5-atom thioacetamido linker of thioacetamido-linked nucleic acids and iso-thioacetamido-linked nucleic acids modified oligomers. In contrast to the preferred N-type sugar conformations for either 3'- ribo- or xylo amino nucleosides, predominant S-type sugar conformations were found in the dimers. Although the CD spectral differences for the dimer blocks were found to be identical for those found in phosphodiester linked ribo/xylo dimers, the 5-atom thioactamido linker could reverse the RNA binding selectivity to DNA binding selectivity by the change in configuration at the 3'-amino-substituted sugar.     

A novel pyrrolidinyl PNA showing high sequence specificity and preferential binding to DNA over RNA

A novel conformationally constrained pyrrolidinyl peptide nucleic acid (PNA) carrying an D-aminopyrrolidine carboxylic acid (D-Apc) spacer was synthesized, and its interactions with complementary oligo- and polynucleotides were studied by UV and CD spectroscopy. The decathymine PNA formed very stable PNA-DNA complexes with poly(dA) and (dA)(10) by a sequence-specific A-T pairing. The interaction with poly(rA) gave the corresponding PNA-RNA complex with much lower stability.     

Synthesis of gamma-substituted peptide nucleic acids: a new place to attach fluorophores without affecting DNA binding

Molecular beacon strategies using PNA are currently restricted to fluorophore attachment to the ends of the PNA. We report the synthesis of PNA oligomers wherein fluorophores can be attached to the PNA backbone from novel gamma-lysine PNA monomers. Oligomers incorporating the modified PNA showed comparable thermal stability to the corresponding aegPNA oligomer with DNA. When the modified PNA oligomer was annealed with complementary DNA, the fluorescence intensity increased 4-fold over the unbound PNA. [structure: see text]     

(S,S)-trans-cyclopentane-constrained peptide nucleic acids. a general backbone modification that improves binding affinity and sequence specificity

Replacing the ethylenediamine portion of aminoethylglycine peptide nucleic acids (aegPNAs) with one or more (S,S)-trans-cyclopentane diamine units significantly increases binding affinity and sequence specificity to complementary DNA, making these modified PNAs ideal for use as nucleic acid probes in genomic analysis. The synthesis and study of this new class of PNAs (tcypPNAs) is described in which trans-cyclopentane diamine has been incorporated into several positions, and in varying number, within PNA backbones of mixed-base sequences.     

DNA hybridization assays using temperature gradient focusing and peptide nucleic acids

Two types of DNA hybridization assays are demonstrated with temperature gradient focusing (TGF) and peptide nucleic acids (PNAs). In TGF, the application of a controlled temperature gradient along the length of a microchannel filled with an appropriate temperature-dependent buffer results in the formation of a gradient in both the electric field and electrophoretic velocity. Ionic species move in this gradient and concentrate at a unique point where the total velocity sums to zero. The first assay is a mixing assay in which PNA is allowed to flow through spatially focused DNA targets within a capillary. The second assay detects single base pair mutations (SBPM) by monitoring the fluorescence intensity of PNA/DNA duplexes as a function of temperature within the capillary. The SBPM analysis can be performed in less than 5 min with 100-fold more dilute analyte compared to conventional UV melting measurements.     

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.     

Novel binding and efficient cellular uptake of guanidine-based peptide nucleic acids (GPNA)

Incorporation of a guanidine functional group into the PNA backbone facilitates cellular uptake of PNA into mammalian cells with efficiency comparable to that of the TAT transduction domain. The modified PNA recognizes and binds to the complementary DNA strand in accordance with Watson-Crick recognition rules. However, unlike polypyrimidine PNA which binds to DNA in 2:1 stoichiometry, the modified PNA binds to complementary DNA in a 1:1 ratio to form a highly stable duplex.     

Chimeric peptide nucleic acids incorporating (2S,5R)-aminoethyl pipecolyl units: synthesis and DNA binding studies

The design and facile synthesis of a novel chiral six-membered PNA analogue (2S,5R )-1-(N-Boc-aminoethyl)-5-(thymin-1-yl)pipecolic acid, aepipPNA, that upon incorporation into PNA sequences effected stabilization of complexes with target complementary DNA. This is the first example where a six membered-PNA is shown to be capable of forming stable complexes with DNA and further expands the repertoire of cyclic PNA analogues.     

2-aza-2'-deoxyadenosine: synthesis, base-pairing selectivity, and stacking properties of oligonucleotides

2-Aza-2'-deoxyadenosine (2, z2Ad) is synthesized via its 1,N6-etheno derivative 7 and enzymatically deaminated to 2-aza-2'-deoxyinosine (3). Compound 2 is converted into the phosphoramidite building block 10b. This is employed in solid-phase oligonucleotide synthesis. The 2-azapurine base forms a strong base pair with guanine, but a much weaker one with adenine, thymine, and cytosine. Oligonucleotide duplexes with dangling nucleotide residues, such as 2-aza-2'-deoxyadenosine and 7-deaza-2'-deoxyadenosine (4, c7Ad), either on one or both termini, are synthesized, and the thermal stability of the duplexes is correlated with the hydrophobic properties of the dangling nucleotide residues.     

Application of peptide nucleic acids containing azobenzene self-assembled electrochemical biosensors in detecting DNA sequences

Hybridization of peptide nucleic acids probe containing azobenzene (NH₂-TNT₄, N-PNAs) with DNA was performed by covalently immobilizing of NH₂-TNT₄ in sequence on the 3-mercaptopropionic acid self-assembled monolayer modified gold electrode with the helps of N-(3-dimethylaminopropy1)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and the hybrid was coded as N-PNAs/DNA. Using [Fe(CN)₆]^4−/3− (1:1) as the electrochemical indicator, the electrochemical properties of the N-PNAs self-assembled monolayer (N-PNAs-SAMs) and N-PNAs/DNA hybridization system under the conditions of before and after UV light irradiation were characterized with cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectra (EIS). Results showed that the redox currents decreased with the increase of irradiation time, suggesting that the ability of the charge transfer on the electrode surface was weakened and the conformation of hybrid system had been changed, and the control of PNAs/DNA hybridization could be realized by UV light irradiation. Supported by the National Natural Science Foundation of China (Grant No. 50572107) and “Top Hundred Talents Program” of Chinese Academy of Science     

Model calculations for the base-pairing specificity of mutagenic exocyclic DNA adduct 1,N 6-ethenoadenine

1,N ⁶-ethenoadenine (εA) has been studied more extensively due to highly fluorescent and mutagenic nature. We report the model calculations for the base-pairing specificity of mutagenic 1,N ⁶-ethenoadenine adduct. To shed insight into mutagenic process of DNA damage based on geometrical characteristics, electronic properties, B3LYP, M06, B97D, and wB97XD methods have been employed for these model calculations. Single point energy calculation at MP2/6-311++G** levels on corresponding B3LYP, M06, B97D, wB97XD-optimized geometries have also been carried out to better estimate the hydrogen-bonding strengths. The polarisable conductor calculation model (CPCM-UAKS) that accounts for the overall polarizability of the solvent has also been employed. The computed reaction enthalpy values concluded that both guanine and thymine are most favorable candidates to be misincorporated to 1,N ⁶-ethenoadenine adduct, which also in good agreement to experimental report (Leonard, Biochemistry 33: 4755–4761, 79).     

Nanocrystals modified with peptide nucleic acids (PNAs) for selective self-assembly and DNA detection

Gold nanocrystals modified with peptide nucleic acids (PNAs) have been prepared and applied to self-assembly and DNA sensing. Experiments with different PNA structural motifs show that (1). the versatility in PNA synthetic design can be used to modulate the electrostatic surface properties of nanocrystals, presenting an opportunity to control assembly rate and aggregate size, (2). short (6 base) PNAs can hybridize effectively while attached to nanoparticles, providing a route to generating materials with small interparticle spacings, and (3). the superior base pair mismatch selectivity of PNAs is further enhanced on nanosurfaces, enabling PNA-modified nanoparticles to act as highly selective nanoscale sensors, as well as synthons for defect-free self-assembly. This last feature was coupled with a substantial change in colloidal stability upon DNA hybridization to develop a novel colorimetric DNA assay that detects the presence of single base imperfections within minutes. Various modes of PNA hybridization, including the first practical application of PNA-PNA interactions, were used to direct the assembly of nanoparticles into macroscopic arrangements. Shorter duplex interconnects and greater specificity in assembly were obtained compared to similar experiments with DNA-modified nanocrystals.     

Cyanuryl peptide nucleic acid: synthesis and DNA complexation properties

The synthesis of cyanuryl PNA monomer (CyaPNA) 6 was achieved by direct N-monoalkylation of cyanuric acid with N-(2-Boc-aminoethyl)-N′-(bromoacetyl)glycyl ethyl ester 4. Compound 6 was incorporated as a T-mimic into PNA oligomers and biophysical studies on their triplexes/duplex complexes with complementary DNA oligomers indicated unusual stabilization of PNA:DNA hybrids when the cyanuryl unit was located in the middle of the PNA oligomer.     

Incorporation of extra amino acids in peptide recognition probe to improve specificity and selectivity of an electrochemical peptide-based sensor

We investigated the effect of incorporating extra amino acids (AA) at the n-terminus of the thiolated and methylene blue-modified peptide probe on both specificity and selectivity of an electrochemical peptide-based (E-PB) HIV sensor. The addition of a flexible (SG)₃ hexapeptide is, in particular, useful in improving sensor selectivity, whereas the addition of a highly hydrophilic (EK)₃ hexapeptide has shown to be effective in enhancing sensor specificity. Overall, both E-PB sensors fabricated using peptide probes with the added AA (SG-EAA and EK-EAA) showed better specificity and selectivity, especially when compared to the sensor fabricated using a peptide probe without the extra AA (EAA). For example, the selectivity factor recorded in the 50% saliva was ∼2.5 for the EAA sensor, whereas the selectivity factor was 7.8 for both the SG-EAA and EK-EAA sensors. Other sensor properties such as the limit of detection and dynamic range were minimally affected by the addition of the six AA sequence. The limit of detection was 0.5 nM for the EAA sensor and 1 nM for both SG-EAA and EK-EAA sensors. The saturation target concentration was ∼200 nM for all three sensors. Unlike previously reported E-PB HIV sensors, the peptide probe functions as both the recognition element and antifouling passivating agent; this modification eliminates the need to include an additional antifouling diluent, which simplifies the sensor design and fabrication protocol.