Peptide Nucleic Acid Analogues Research Articles

Nucleobase intrinsic quenchers: A fluorescence off switch

Molecular beacons are important bioanalytical probes most often constructed from a single-stranded oligonucleotide which has been labeled at opposite termini with a fluorophore and a quencher. When the fluorophore and quencher are in close proximity, ideally no fluorescence is observed due to Fluorescence Resonance Energy Transfer (FRET). 4-Dimethylaminoazobenzene-4′-carboxylic acid (DABCYL) is a commonly used quencher in molecular beacons; however, DABCYL is unable to form a base-pair and is conventionally installed as an overhanging residue. In this arrangement, the DABCYL moiety has substantial mobility which affects the accuracy of FRET-related distance/orientation measurements and also limits the types of other conjugates that can be prepared. To address these limitations, we have synthesized peptide nucleic acid (PNA) analogues possessing DMPAU (5-[(4-dimethylaminophenyl)diazenyl]uracil) and NPhpC (6-(4-nitrophenyl)pyrrolocytosine) nucleobases which act as dark fluorophores, i.e. quenchers, while retaining the ability to base pair by canonical hydrogen bonding. This will allow more flexible design and construction of molecular beacons based on the insertion of nucleobase fluorophores and quenchers into the stem sequence. We have quantified the quenching ability of DMPAU and NPhpC PNA analogues by determining the Stern-Volmer constants against the intrinsically fluorescent nucleobase, phenylpyrrolocytosine, and other blue emitting fluorophores (pyrene, acridone). The hydrogen bonding ability of DMPAU with adenine, as determined by 1H NMR titration, is relatively unperturbed compared to uracil and thymine.

Multicomponent Synthesis of Diaminopurine and Guanine PNA's Analogues Active against Influenza A Virus from Prebiotic Compounds.

Peptide nucleic acids (PNAs) play a key role in prebiotic chemistry as a chimera between RNA and proteins. We developed an alternative synthesis of bioactive PNA's diaminopurine and guanine analogues from prebiotic compounds, such as aminomalononitrile (AMN), urea, and guanidine, using a two-step multicomponent microwave-assisted and solvent-free approach in the presence of selected amino acids. The novel derivatives showed selective inhibitory activity against influenza virus A/Puerto Rico/8/34 H1N1 encompassing the range of nanomolar activity. Derivatives decorated with the tyrosine residue showed the highest inhibitory activity against the virus.

Electrostatics Favor PNA : DNA Stability over Stereochemistry in Pyrrolidine‐Based Cationic Dual‐Backbone PNA Analogues

AbstractModifications to the peptide nucleic acid (PNA) backbone has been well known to alter the thermodynamical parameters of PNA : DNA complexes to broaden their utility for different applications. Electrostatic interactions between a modified PNA having a positively charged backbone and the negatively charged DNA has been shown to enhance thermal stabilities of PNA : DNA complexes at various instances. On the other hand, chiral introduction in PNA backbone leads to stereochemical preference that affects binding properties. However, the interplay between electrostatics and stereochemistry has not been systematically studied so far. Herein, we report the synthesis and biophysical characterization of cationic PNA named dapPNA, first of its kind, having a dual PNA backbone constituting of a pyrrolidine ring having a β‐substitution. One of the aims of this study was to investigate the role of electrostatics over stereochemical preferences. The results show that electrostatic attraction between cationic dapPNA and negatively charged DNA overcomes the unfavorable stereochemical effects and enhances stability of PNA : DNA complexes. Moreover, two different PNA backbones derived from a single PNA monomer expands the repertoire of pyrrolidine based PNA analogues.

Cγ(S/R)-Bimodal Peptide Nucleic Acids (Cγ-bm-PNA) Form Coupled Double Duplexes by Synchronous Binding to Two Complementary DNA Strands.

Peptide nucleic acids (PNAs) are linear equivalents of DNA with a neutral acyclic polyamide backbone that has nucleobases attached via tert-amide link on repeating units of aminoethylglycine. They bind complementary DNA or RNA with sequence specificity to form hybrids that are more stable than the corresponding DNA/RNA self-duplexes. A new type of PNA termed bimodal PNA [Cγ(S/R)-bm-PNA] is designed to have a second nucleobase attached via amide spacer to a side chain at Cγ on the repeating aeg units of PNA oligomer. Cγ-bimodal PNA oligomers that have two nucleobases per aeg unit are demonstrated to concurrently bind two different complementary DNAs, to form duplexes from both tert-amide side and Cγ side. In such PNA:DNA ternary complexes, the two duplexes share a common PNA backbone. The ternary DNA 1:Cγ(S/R)-bm-PNA:DNA 2 complexes exhibit better thermal stability than the isolated duplexes, and the Cγ(S)-bm-PNA duplexes are more stable than Cγ(R)-bm-PNA duplexes. Bimodal PNAs are first examples of PNA analogues that can form DNA2:PNA:DNA1 double duplexes via recognition through natural bases. The conjoined duplexes of Cγ-bimodal PNAs can be used to generate novel higher-level assemblies.

Triplex-Forming Peptide Nucleic Acids with Extended Backbones.

Peptide nucleic acid (PNA) forms a triple helix with double-stranded RNA (dsRNA) stabilized by a hydrogen-bonding zipper formed by PNA's backbone amides (N-H) interacting with RNA phosphate oxygens. This hydrogen-bonding pattern is enabled by the matching ∼5.7 Å spacing (typical for A-form dsRNA) between PNA's backbone amides and RNA phosphate oxygens. We hypothesized that extending the PNA's backbone by one -CH2 - group might bring the distance between PNA amide groups closer to 7 Å, which is favourable for hydrogen bonding to the B-form dsDNA phosphate oxygens. Extension of the PNA backbone was expected to selectively stabilize PNA-DNA triplexes compared to PNA-RNA. To test this hypothesis, we synthesized triplex-forming PNAs that had the pseudopeptide backbones extended by an additional -CH2 - group in three different positions. Isothermal titration calorimetry measurements of the binding affinity of these extended PNA analogues for the matched dsDNA and dsRNA showed that, contrary to our structural reasoning, extending the PNA backbone at any position had a strong negative effect on triplex stability. Our results suggest that PNAs might have an inherent preference for A-form-like conformations when binding double-stranded nucleic acids. It appears that the original six-atom-long PNA backbone is an almost perfect fit for binding to A-form nucleic acids.

Stereochemical conversion of nucleic acid circuits via strand displacement

Remarkable accomplishments demonstrating the importance of nucleic acids in molecular engineering and computation have been made over the past two decades. However, much of the work in this area so far has been carried out in vitro, utilizing almost exclusively homochiral D-DNAs (or D-RNAs) as chemical building blocks. Such natural building blocks are prone to enzymatic degradation and cross-hybridization with the host’s genetic materials. Here we report the development of an orthogonal nucleic acid system that is made up of a left-handed and a right-handed conformer, and a non-helical peptide nucleic acid analogue. We show that the stereochemical information inherent in the right-handed and left-handed conformers can be interconverted from (R) to (S) and vice versa, along with their helical sense and recognition capability, through strand displacement. The genetic information encoded in these synthetic building blocks can be interfaced with DNA or RNA through a molecular converter.

Fluorinated Peptide Nucleic Acids with Fluoroacetyl Side Chain Bearing 5-(F/CF3)-Uracil: Synthesis and Cell Uptake Studies.

Fluorine incorporation into organic molecules imparts favorable physicochemical properties such as lipophilicity, solubility and metabolic stability necessary for drug action. Toward such applications using peptide nucleic acids (PNA), we herein report the chemical synthesis of fluorinated PNA monomers and biophysical studies of derived PNA oligomers containing fluorine in in the acetyl side chain (-CHF-CO-) bearing nucleobase uracil (5-F/5-CF3-U). The crystal structures of fluorinated racemic PNA monomers reveal interesting base pairing of enantiomers and packing arrangements directed by the chiral F substituent. Reverse phase HPLC show higher hydrophobicity of fluorinated PNA oligomers, dependent on the number and site of the fluorine substitution: fluorine on carbon adjacent to the carbonyl group induces higher lipophilicity than fluorine on nucleobase or in the backbone. The PNA oligomers containing fluorinated bases form hybrids with cDNA/RNA with slightly lower stability compared to that of unmodified aeg PNA, perhaps due to electronic effects. The uptake of fluorinated homooligomeric PNAs by HeLa cells was as facile as that of nonfluorinated PNA. In conjunction with our previous work on PNAs fluorinated in backbone and at N-terminus, it is evident that the fluorinated PNAs have potential to emerge as a new class of PNA analogues for applications in functional inhibition of RNA.

Responsive Fluorescent PNA Analogue as a Tool for Detecting G-quadruplex Motifs of Oncogenes and Activity of Toxic Ribosome-Inactivating Proteins.

Fluorescent oligomers that are resistant to enzymatic degradation and report their binding to target oligonucleotides (ONs) by changes in fluorescence properties are highly useful in developing nucleic-acid-based diagnostic tools and therapeutic strategies. Here, we describe the synthesis and photophysical characterization of fluorescent peptide nucleic acid (PNA) building blocks made of microenvironment-sensitive 5-(benzofuran-2-yl)- and 5-(benzothiophen-2-yl)-uracil cores. The emissive monomers, when incorporated into PNA oligomers and hybridized to complementary ONs, are minimally perturbing and are highly sensitive to their neighboring base environment. In particular, benzothiophene-modified PNA reports the hybridization process with significant enhancement in fluorescence intensity, even when placed in the vicinity of guanine residues, which often quench fluorescence. This feature was used in the turn-on detection of G-quadruplex-forming promoter DNA sequences of human proto-oncogenes (c-myc and c-kit). Furthermore, the ability of benzothiophene-modified PNA oligomer to report the presence of an abasic site in RNA enabled us to develop a simple fluorescence hybridization assay to detect and estimate the depurination activity of ribosome-inactivating protein toxins. Our results demonstrate that this approach with responsive PNA probes will provide new opportunities to develop robust tools to study nucleic acids.

Influence of Pendant Chiral Cγ-(Alkylideneamino/Guanidino) Cationic Side-chains of PNA Backbone on Hybridization with Complementary DNA/RNA and Cell Permeability

Intrinsically cationic and chiral C(γ)-substituted peptide nucleic acid (PNA) analogues have been synthesized in the form of γ(S)-ethyleneamino (eam)- and γ(S)-ethyleneguanidino (egd)-PNA with two carbon spacers from the backbone. The relative stabilization (ΔTm) of duplexes from modified cationic PNAs as compared to 2-aminoethylglycyl (aeg)-PNA is better with complementary DNA (PNA:DNA) than with complementary RNA (PNA:RNA). Inherently, PNA:RNA duplexes have higher stability than PNA:DNA duplexes, and the guanidino PNAs are superior to amino PNAs. The cationic PNAs were found to be specific toward their complementary DNA target as seen from their significantly lower binding with DNA having single base mismatch. The differential binding avidity of cationic PNAs was assessed by the displacement of DNA duplex intercalated ethidium bromide and gel electrophoresis. The live cell imaging of amino/guanidino PNAs demonstrated their ability to penetrate the cell membrane in 3T3 and MCF-7 cells, and cationic PNAs were found to be accumulated in the vicinity of the nuclear membrane in the cytoplasm. Fluorescence-activated cell sorter (FACS) analysis of cell permeability showed the efficiency to be dependent upon the nature of cationic functional group, with guanidino PNAs being better than the amino PNAs in both cell lines. The results are useful to design new biofunctional cationic PNA analogues that not only bind RNA better but also show improved cell permeability.

ChemInform Abstract: Synthesis and Antihepatitis B Virus Activity of New Pyrimidine Peptide Nucleic Acid Analogues.

AbstractA series of novel peptide nucleic acid analogues (III) is synthesized and tested for activity against hepatitis B virus.

γ sulphate PNA (PNA S): Highly Selective DNA Binding Molecule Showing Promising Antigene Activity

Peptide Nucleic Acids (PNAs), nucleic acid analogues showing high stability to enzyme degradation and strong affinity and specificity of binding toward DNA and RNA are widely investigated as tools to interfere in gene expression. Several studies have been focused on PNA analogues with modifications on the backbone and bases in the attempt to overcome solubility, uptake and aggregation issues. γ PNAs, PNA derivatives having a substituent in the γ position of the backbone show interesting properties in terms of secondary structure and affinity of binding toward complementary nucleic acids. In this paper we illustrate our results obtained on new analogues, bearing a sulphate in the γ position of the backbone, developed to be more DNA-like in terms of polarity and charge. The synthesis of monomers and oligomers is described. NMR studies on the conformational properties of monomers and studies on the secondary structure of single strands and triplexes are reported. Furthermore the hybrid stability and the effect of mismatches on the stability have also been investigated. Finally, the ability of the new analogue to work as antigene, interfering with the transcription of the ErbB2 gene on a human cell line overexpressing ErbB2 (SKBR3), assessed by FACS and qPCR, is described.

Preparation and determination of optical purity of γ-lysine modified peptide nucleic acid analogues

Peptide nucleic acids (PNAs) are DNA analogues in which the nucleic acid backbone is replaced by a pseudopeptide backbone and nucleobases are attached to the backbone by methylene carbonyl linkers. γ-Carbon modification of the PNA structure allows monomers, and subsequently oligomers, with improved properties to be obtained. In this study, we report the convenient synthesis of γ-lysine-modified PNA monomers for pyrimidine bases (thymine and cytosine) with high optical purity (> 99.5%) and direct enantiomer separation of γ-lysine-modified PNA analogs, using chiral HPLC to determine the optical purity.

Synthesis and anti-hepatitis B virus activity of new pyrimidine peptide nucleic acid analogs

A series of 4-methylsulfanylpyrimidin-2(1H)-one peptide nucleic acid analogs were synthesized and tested for their antiviral activity against hepatitis B virus. Plaque reduction infectivity assay was used to determine the virus count reduction as a result of treatment with tested compounds.

Chemical structure requirements and cellular targeting of microRNA-122 by peptide nucleic acids anti-miRs

Anti-miRs are oligonucleotide inhibitors complementary to miRNAs that have been used extensively as tools to gain understanding of specific miRNA functions and as potential therapeutics. We showed previously that peptide nucleic acid (PNA) anti-miRs containing a few attached Lys residues were potent miRNA inhibitors. Using miR-122 as an example, we report here the PNA sequence and attached amino acid requirements for efficient miRNA targeting and show that anti-miR activity is enhanced substantially by the presence of a terminal-free thiol group, such as a Cys residue, primarily due to better cellular uptake. We show that anti-miR activity of a Cys-containing PNA is achieved by cell uptake through both clathrin-dependent and independent routes. With the aid of two PNA analogues having intrinsic fluorescence, thiazole orange (TO)-PNA and [bis-o-(aminoethoxy)phenyl]pyrrolocytosine (BoPhpC)-PNA, we explored the subcellular localization of PNA anti-miRs and our data suggest that anti-miR targeting of miR-122 may take place in or associated with endosomal compartments. Our findings are valuable for further design of PNAs and other oligonucleotides as potent anti-miR agents.

Synthesis of N-propynyl analogues of peptide nucleic acid (PNA) monomers and their use in the click reaction to prepare N-functionalized PNAs

Application of the click reaction for coupling a 2-(2-aminoethoxy)ethoxy (AEE) function to thyminyl, cytosinyl and adeninyl peptide nucleic acid (PNA) monomer analogues bearing a N-propynyl group, in place of the original N-2-aminoethyl moiety, has been explored. The N-propynyl PNA analogues were prepared from glycine ethyl ester hydrochloride and the structure of the thyminyl derivative was confirmed by X-ray diffraction. These monomers were employed in click reactions with Boc-protected AEE azide to afford the corresponding triazolyl-linked PNA–AEE conjugates in yields ranging from 64 to 76%. [1,4]-Regiochemistry was verified from a NOESY correlation experiment.

Molecular Dynamics of Potential rRNA Binders: Single-Stranded Nucleic Acids and Some Analogues

By hindering or "silencing" protein translation in vivo, antisense nucleic acid analogues that hybridize to bacterial rRNA could serve as a promising class of antibacterial compounds. Thus, we performed a comparative analysis of the dynamical properties of modified oligonucleotides based upon a sequence (5')r(UGUUACGACU)(3') that is complementary to bacterial ribosomal A-site RNA. In particular, 25 ns explicit solvent molecular dynamics simulations were computed for the following six single-stranded decamers: (1) the above RNA in unmodified form; (2) the 2'-O-methyl-modified RNA; (3) peptide nucleic acid (PNA) analogues of the above sequence, containing either (a) T or (b) U; and (4) two serine-substituted PNAs. Our results show that 2'-O-methylation attenuates RNA backbone dynamics, thereby preventing interconversion between stacked and unstacked conformations. The PNA analogue is rendered less flexible by replacing uracil with thymine; in addition, we found that derivatizing the PNA backbone with serine leads to enhanced base-stacking interactions. Consistent with known solubility properties of these classes of molecules, both RNAs exhibited greater localization of water molecules than did PNA. In terms of counterions, the initially helical conformation of the 2'-O-methyl RNA exhibits the highest Na(+) density among all the simulated decamers, while Na(+) build-up was most negligible for the neutral PNA systems. Further studies of the conformational and physicochemical properties of such modified single-stranded oligomers may facilitate better design of nucleic acid analogues, particularly those capable of serving as specific, high-affinity ribosomal A-site binders.

DNA mimics based on pyrrolidine and hydroxyproline

In order to improve physicochemical and biological properties of natural oligonucleotides in particular increasing their affinity for nucleic acids, the selectivity of action and biological sustainability, several types of DNA mimics were designed. The survey collected data on the synthesis and properties of the DNA mimics - peptide-nucleic acids analogues, which are derivatives of pyrrolidine and hydroxyproline. We examine some physicochemical and biological properties of negatively charged mimics of this type, containing phosphonate residues, and possessing a high affinity for DNA and RNA, selective binding with nucleic acids and stability in various biological systems. Examples of the use of these mimics as tools for molecular biological research, particularly in functional genomics are given. The prospects for their use in diagnostics and medicine are discussed.

Synthesis of Chiral Heteronucleotide ONA Sequences by a Fmoc/Boc-Based Submonomeric Strategy

Peptide nucleic acid (PNA) is a DNA analog able to form hybridization complexes with complementary DNA or RNA strands. Many PNAs have been described in recent years, particularly chiral PNA analogs. Chiral heteronucleotide ONA (Orn backbone PNA) is an important tool in the antisensing field, but was not been fully explored yet. In the present work, we performed studies toward the synthesis of chiral heteronucleotide ONA sequences by utilizing a Fmoc/Boc-based submonomer approach on solid support. The desired oligomers with different nucleic content and length were obtained in very good yields and high purity. Specific binding to the complimentary ssDNA oligomers was demonstrated.

Synthesis of a peptide nucleic acid with a novel 1‐methyl‐6‐mercaptopurine base

Abstractmagnified imageA novel peptide nucleic acid (PNA) monomer 16 containing a novel 1‐methyl‐6‐mercaptopurine base was synthesized by coupling the in situ generated acid chloride of (1‐methyl‐6‐mercaptopurin‐9‐yl)acetic acid (6) into an L‐lysine backbone (13) using 10% CCl4 in pyridine and Ph3P. Compound 6 was synthesized from 6‐mercapto‐1‐methylpurine and ethylbromoacetate in the presence of NaH followed by alkaline hydrolysis and subsequent neutralization with a cation exchange resin. The L‐lysine backbone (13) was obtained by the reaction of Nϵ‐CBZ‐L‐lysine allyl ester with Boc‐aminoactaldehyde in the presence of NaBH3CN under reductive amination conditions. Oligomerization of the monomer 16 to PNA analogues was achieved using BOC‐BHA‐PEG‐PS resin as a solid support and the in situ generated acid chloride of 16 by 10% CCl4 in DCM in the presence of Ph3P.

New peptide nucleic acid analogues: synthesis and applications

Peptide nucleic acids are oligonucleotide mimics characterised by high chemical and enzymatic stability, high specificity and affinity toward complementary DNA/RNA. The lack of charge and polar groups in the backbone decrease their solubility in aqueous environment and their ability to cross cell membranes, reducing their performance in in vivo applications. To improve solubility, increase affinity and specificity of binding and to control recognition between nucleic acids, several analogues bearing modifications on the nucleobase, nucleobase-backbone linker and on the backbone were synthesised. This paper describes the synthesis and applications of Peptide nucleic acid analogues and discusses the potential of analogues for which no application is reported.