Pyrrolidinyl Peptide Nucleic Acid Research Articles

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.

Reductive Alkylation and Sequential Reductive Alkylation-Click Chemistry for On-Solid-Support Modification of Pyrrolidinyl Peptide Nucleic Acid

A methodology for the site-specific attachment of fluorophores to the backbone of pyrrolidinyl peptide nucleic acids (PNAs) with an α/β-backbone derived from D-prolyl-(1S,2S)-2-aminocyclopentanecarboxylic acid (acpcPNA) has been developed. The strategy involves a postsynthetic reductive alkylation of the aldehyde-containing labels onto the acpcPNA that was previously modified with (3R,4S)-3-aminopyrrolidine-4-carboxylic acid on the solid support. The reductive alkylation reaction is remarkably efficient and compatible with a range of reactive functional groups including Fmoc-protected amino, azide, and alkynes. This allows further attachment of readily accessible carboxyl-, alkyne-, or azide-containing labels via amide bond formation or Cu-catalyzed azide-alkyne cycloaddition (CuAAC, also known as click chemistry). The label attached in this way does not negatively affect the affinity and specificity of the pairing of the acpcPNA to its DNA target. Applications of this methodology in creating self-reporting pyrene- and thiazole orange-labeled acpcPNA probes that can yield a change in fluorescence in response to the presence of the correct DNA target have also been explored. A strong fluorescence enhancement was observed with thiazole orange-labeled acpcPNA in the presence of DNA. The specificity could be further improved by enzymatic digestion with S1 nuclease, providing a 9- to 60-fold fluorescence enhancement with fully complementary DNA and a less than 3.5-fold enhancement with mismatched DNA targets.

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/β-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.

Positively charged polymer brush-functionalized filter paper for DNA sequence determination following Dot blot hybridization employing a pyrrolidinyl peptide nucleic acid probe

As inspired by the Dot blot analysis, a well known technique in molecular biology and genetics for detecting biomolecules, a new paper-based platform for colorimetric detection of specific DNA sequences employing peptide nucleic acid (PNA) as a probe has been developed. In this particular study, a pyrrolidinyl PNA bearing a conformationally rigid d-prolyl-2-aminocyclopentanecarboxylic acid backbone (acpcPNA) was used as a probe. The filter paper was modified to be positively charged with grafted polymer brushes of quaternized poly(dimethylamino)ethyl methacrylate (QPDMAEMA) prepared by surface-initiated polymerization of 2-(dimethylamino)ethyl methacrylate from the filter paper via ARGET ATRP followed by quaternization with methyl iodide. Following the Dot blot format, a DNA target was first immobilized via electrostatic interactions between the positive charges of the QPDMAEMA brushes and negative charges of the phosphate backbone of DNA. Upon hybridization with the biotinylated pyrrolidinyl peptide nucleic acid (b-PNA) probe, the immobilized DNA can be detected by naked eye observation of the yellow product generated by the enzymatic reaction employing HRP-labeled streptavidin. It has been demonstrated that this newly developed assay was capable of discriminating between complementary and single base mismatch targets at a detection limit of at least 10 fmol. In addition, the QPDMAEMA-grafted filter paper exhibited a superior performance to the commercial membranes, namely Nylon 66 and nitrocellulose.

A comparative study of a label-free DNA capacitive sensor using a pyrrolidinyl peptide nucleic acid probe immobilized through polyphenylenediamine and polytyramine non-conducting polymers

This work has, for the first time, reported a comparative study on the performances of a label-free capacitive DNA sensor based on an immobilized pyrrolidinyl peptide nucleic acid with a d-prolyl-2-aminocyclopentanecarboxylic acid backbone (acpcPNA) attached to three easy to prepare non-conducting polymers. Two phenylenediamine (PD) isomers (ortho- and para-) and one tyramine monomer were electropolymerized to form polymer films. The hybridization between the acpcPNA probes, immobilized on the polymer layer, and the target DNA was detected directly by measuring the capacitance change. Using the optimal electropolymerized conditions, the highest sensitivity of 20.9 ± 0.6-nF cm−2 (log M)−1 was obtained from poly-para-PD (PpPD) with a lowest detection limit of 0.2 pM. A wide linear range, 1.0 × 10−12 to 1.0 × 10−8 M, was obtained for both the PpPD and polytyramine (Pty). A narrower linear range of 1.0 × 10−11 to 1.0 × 10−8 M was obtained from the poly-ortho-PD (PoPD). The surface morphology was observed using scanning electron microscopy and atomic force microscopy. The polymer with the better surface morphology, i.e., smoother, denser and more homogeneous was the one with more immobilized probes and a better overall performance. All modified polymers gave a high % signal suppression (%SS) for both the single and double mismatched target DNAs, and indicated its high specificity. The electrodes can be reused for at least 68 analytical cycles. The effects of the target length and probe concentrations were also investigated. Under the operating conditions of this work the system would be very useful for the cost-effective analysis of ultra-trace levels of DNA in samples.

Label-free capacitive DNA sensor using immobilized pyrrolidinyl PNA probe: Effect of the length and terminating head group of the blocking thiols

This paper reports, for the first time, the influence of the length and the terminating head group of blocking thiols on the sensitivity and specificity of a label-free capacitive DNA detection system using immobilized pyrrolidinyl peptide nucleic acid (acpcPNA) probes. A C-terminal lysine-modified acpcPNA was immobilized through four different alkanethiol self-assembled monolayers (SAMs), i.e., 3-mercaptopropionic acid (MPA), thioctic acid (TA), thiourea (TU) and mercaptosuccinic acid (MSA). The hybridization between the acpcPNA probes and the target DNA was directly measured using the capacitive system. Five blocking thiols of various lengths (C=3, 6, 8, 9 and 11), with the –OH terminating head group, i.e., 3-mercapto-1-propanol (3-MPL), 6-mercapto-1-hexanol (6-MHL), 8-mercapto-1-octanol (8-MOL), 9-mercapto-1-nonanol (9-MNL), 11-mercapto-1-undecanol (11-MUL) and another blocking thiol (C=11) with a –CH3 terminating head group, and 1-dodecanethiol (1-DDT) were investigated. The blocking thiol with the same length as the total spacer of the immobilized acpcPNA gave the highest sensitivity and specificity with the –OH terminating head group providing a slightly better signal than the –CH3 group. Under the optimized conditions, the immobilized acpcPNA probes provided a wide linear range for DNA detection (1.0×10–11–1.0×10−8M) with a very low detection limit in the picomolar range. The modified acpcPNA electrode could be reused through at least 58 cycles. The high sensitivity and very low detection limits are potentially useful for the analysis of ultra-trace levels of DNA in samples. Preliminary studies were also performed to see the effect of probe concentration and target length.

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.

Pyrrolidinyl Peptide Nucleic Acid Homologues: Effect of Ring Size on Hybridization Properties

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

FRET detection of DNA sequence via electrostatic interaction of polycationic phenyleneethynylene dendrimer with DNA/PNA hybrid

Interaction between a polycationic compound and DNA is a useful phenomenon for development of a new DNA sensing system. In this work, dendritic polycationic phenyleneethynylene fluorophores are investigated as a Förster resonance energy transfer (FRET) donor for the detection of DNA hybridization in conjunction with a fluorescein-labeled pyrrolidinyl peptide nucleic acid (Fl-acpcPNA) probe. The first generation dendrimer is an efficient energy donor for the fluorescein acceptor but also shows non-specific FRET signal with Fl-acpcPNA. The addition of N-methyl 2-pyrrolidone can virtually completely remove the non-specific interaction between Fl-acpcPNA and the dendrimer. Under the optimal condition, the complementary DNA gives a distinctively high FRET ratio (1.42) comparing with those of the non-complementary (0.26) and singly mismatched (0.51) DNAs. The FRET ratio responses linearly with the DNA concentration with the detection limit lower than 1nM. The FRET ratio is even higher for the complementary target DNAs with extra hanging nucleotide sequences, which is a more frequently encountered scenario in real applications.

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.

DNA-, RNA- and self-pairing properties of a pyrrolidinyl peptide nucleic acid with a (2′ R,4′ S)-prolyl-(1 S,2 S)-2-aminocyclopentanecarboxylic acid backbone

A new pyrrolidinyl peptide nucleic acid (PNA) comprising of an alternate sequence of 4′-nucleobase-modified proline with (2′ R,4′ S) configuration and a (1 S,2 S)-2-aminocyclopentanecarboxylic acid [(2′ R,4′ S)-acpcPNA] backbone was synthesized and its DNA-, RNA- and self-pairing properties studied. T m and CD studies suggested that the (2′ R,4′ S)-acpcPNA forms antiparallel hybrids to DNA and RNA with high sequence and direction specificity. The stability of these hybrids is comparable to those of the (2′ R,4′ R)-acpcPNA hybrids previously reported by our group. On the other hand, experiments with a self-complementary sequence indicated that the new (2′ R,4′ S)-acpcPNA forms a more stable antiparallel self-hybrid than (2′ R,4′ R)-acpcPNA.

Hydrogen-Bonding Strengths in Pyrrolidinyl Peptide Nucleic Acid and DNA Base Pairs: A Density Functional Theory (DFT) Study

The H-bond strengths of the single base pair formed from Pyrrolidinyl Peptide Nucleic Acid (PNA) and charged as well as neutral Deoxyribonucleic Acid (DNA) were studied using the density functional theory. The B3LYP/6-31+G(d,p) level of theory was employed for evaluating the binding energies and structural parameters of heterogeneous and homogeneous base pairs. The strongest H-bond strengths were obtained from the heterogeneous base pairs, yielding the binding energies of −29.9 and −18.9 kcal/mol for the PNA-GC-DNA and PNA-AT-DNA base pairs, respectively. In contrast, a dramatic change on the H-bond strengths was observed from the charged homogenous base pairs with the binding energies of −6.2 and +10.2 kcal/mol for the DNA-GC-DNA and DNA-AT-DNA base pairs, respectively. With the neutralization of negative charges in the DNA backbone, the corresponding values of −29.1 and −11.7 kcal/mol were elucidated from the Na-DNA-GC-DNA-Na and Na-DNA-AT-DNA-Na base pairs, respectively, proving that the repulsion between two negative charges in the phosphate backbone plays a significant role to the H-bond interactions in base pairs. In addition, a high specificity and preferential binding between the pyrrolidinyl PNA and DNA base pairs were also observed.

Comparison of DNA, aminoethylglycyl PNA and pyrrolidinyl PNA as probes for detection of DNA hybridization using surface plasmon resonance technique

Pyrrolidinyl peptide nucleic acid bearing a d-prolyl-2-aminocyclopentanecarboxylic acid backbone (acpcPNA) has been evaluated as a new sensing probe for detection of DNA hybridization. In this study, the biotinylated acpcPNA was immobilized on surface plasmon resonance (SPR) sensor chips via biotin–streptavidin interactions for solid-phase DNA hybridization. A critical comparison between acpcPNA, DNA and conventional peptide nucleic acid (aegPNA) probes of the same sequence was made by means of SPR on various important aspects. These include the effect of ionic strength on hybridization efficiency, the specificity to detect the mismatch(es) in target DNAs, the direction of binding (parallel or antiparallel) to target DNAs, and the effect of target DNA concentration on hybridization efficiency. Results indicated that the immobilized acpcPNA probe possesses distinct hybridization properties relative to aegPNA (and/or DNA) counterparts, including a higher single-base mismatch sensitivity, antiparallel selectivity and low ionic strength dependence of target hybridization. These properties substantiate the acpcPNA applicability as sensor probes for clinical and diagnostic applications. With a proper selection of regeneration conditions (10 mM NaOH, 2 min exposure), the sensor can be reused for multiple cycles of hybridization with as little as 1.3% loss in hybridization activity per regeneration cycle.

Thiolated pyrrolidinyl peptide nucleic acids for the detection of DNA hybridization using surface plasmon resonance

Thiolated pyrrolidinyl peptide nucleic acids (HS-PNAs) bearing d-prolyl-2-aminocyclopentanecarboxylic acid (ACPC) backbones with different lengths and types of thiol modifiers were synthesized and then characterized by MALDI–TOF mass spectrometry. These HS-PNAs were immobilized on gold-coated glass by self-assembled monolayer (SAM) formation via S atom linkage for the detection of DNA hybridization using surface plasmon resonance (SPR). The amount and the stability of the immobilized HS-PNAs, as well as the effects of spacer and blocking thiol on DNA hybridization efficiency, were determined. SPR results indicated that the hybridization efficiency was enhanced when the distance between the PNA portion and the thiol terminal was increased and/or when blocking thiol was used following the HS-PNA immobilization. The immobilized HS-PNA could discriminate between fully complementary DNA from one or two base mismatched DNA with a relatively high degree of mismatch discrimination (>45%) in PBS buffer at 25 °C. The lowest DNA concentration at which reliable discrimination between fully complementary and single mismatched DNA could still occur was at about 0.2 μM, which is equivalent to 10 pmol of DNA. This research demonstrates that using these novel thiolated PNAs in combination with the SPR technique offers a direct, rapid and non-label based method that could potentially be applied for the analysis of genomic or PCR-amplified DNA in the future.

Synthesis and immobilization of thiolated pyrrolidinyl peptide nucleic acids on gold-coated piezoelectric quartz crystals for the detection of DNA hybridization

Two novel thiolated pyrrolidinyl peptide nucleic acids (HS-PNA) carrying d-prolyl-2-aminocyclopentane-carboxylic acid (ACPC) backbones were synthesized and directly immobilized on gold-coated quartz crystals by self-assembled monolayer (SAM) formation (via S atom linkage) for the detection of DNA hybridization. The amount of immobilized HS-PNA was determined by quartz crystal microbalance (QCM), whilst the surface functionality and wettability of the gold surfaces before and after immobilization of the HS-PNA were characterized by reflection absorption infrared spectroscopy (RAIRS) and water contact angle analysis, respectively. Three different procedures for immobilization of HS-PNA were investigated; (I) HS-PNA without blocking thiol, (II) HS-PNA with subsequent blocking thiol and (III) co-immobilization with mixed HS-PNA and blocking thiol. The amount of immobilized HS-PNA on the gold electrode increased asymptotically with the HS-PNA concentration. The sequential blocking step (procedure II) was critical to the success of subsequent DNA hybridization. For hybridization, 0.5 mM phosphate buffer at pH 7.0 with no added NaCl was found to be optimal. Under these conditions, discrimination between complementary and single or multiple base mismatched DNA targets was achieved using a combination of pyrrolidinyl HS-PNA with QCM.

Multiplex Mass Spectrometric Genotyping of Single Nucleotide Polymorphisms Employing Pyrrolidinyl Peptide Nucleic Acid in Combination with Ion-Exchange Capture

A new ion-exchange capture technique is introduced for label-free sample preparation in single nucleotide polymorphism (SNP) genotyping. The DNA sample is hybridized with a new pyrrolidinyl peptide nucleic acid (PNA) probe and treated with a strong anion exchanger. The complementary PNA.DNA hybrid is selectively captured by the anion exchanger in the presence of noncomplementary or unhybridized PNA, allowing direct detection of the hybridization event on the anion exchanger by MALDI-TOF mass spectrometry after simple washing. The high specificity of the pyrrolidinyl PNA allows simultaneous multiplex SNP typing to be carried out at room temperature without the need for enzyme treatment or heating. Exemplary applications of this technique, in the identification of meat species in feedstuffs and in multiplex SNP typing of the human IL-10 gene promoter region are demonstrated, clearly suggesting the potential for much broader applications.

Insight into why pyrrolidinyl peptide nucleic acid binding to DNA is more stable than the DNA·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.

Hybridization of Pyrrolidinyl Peptide Nucleic Acids and DNA: Selectivity, Base-Pairing Specificity, and Direction of Binding

[structure: see text] A mixed-base, beta-amino acid containing, pyrrolidinyl peptide nucleic acid (PNA) binds strongly and selectively to complementary DNA in an exclusively antiparallel fashion. The PNA-DNA binding specificity strictly follows the Watson-Crick base-pairing rules.

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 (1 S,2 S)-ACPC can form a very stable 1:1 complex with the complementary DNA in a sequence-specific manner.

Synthesis and nucleic acid binding studies of novel pyrrolidinyl PNA carrying an N-amino- N-methylglycine spacer

Two novel pyrrolidinyl peptide nucleic acids comprising alternating sequences of thymine-modified d- or l-proline and an N-amino- N-methylglycine spacer were synthesized using solid-phase methodology. UV and CD titrations together with a gel-binding shift assay revealed that neither of the homothymine PNA decamers bind to their complementary DNA or RNA. This was considered to be due to an unfavorable secondary structure which could not be alleviated by the presence of the positively charged protonated amine in the PNA backbone.