Aminoethylprolyl peptide nucleic acids (aepPNA): chiral PNA analogues that form highly stable DNA:aepPNA2 triplexes

[formula: see text] The replacement of the glycyl component in the peptide nucleic acid (PNA) backbone by a prolyl unit bearing a nucleobase leads to the aminoethylprolyl (aep) PNAs, which are chiral and cationic. The homooligomeric aepPNA binds to complementary DNA sequences with high affinity and sequence specificity, forming highly stable triplexes.     

PNA-DNA duplexes, triplexes, and quadruplexes are stabilized with trans-cyclopentane units

Peptide nucleic acids (PNAs) are non-natural nucleic acid mimics that bind to complementary DNA and RNA with high affinity and selectivity. PNA can bind to nucleic acids in a number of different ways. Currently, the formation of PNA-oligonucleotide duplex, triplex, and quadruplex structures have been reported. PNAs have been used in numerous biomedicial applications, but there are few strategies to predictably improve the binding properties of PNAs by backbone modification. We have been studying the benefits of incorporating (S,S)-trans-cyclopentane diamine units (tcyp) into the PNA backbone. In this Communication, we report the improvement in stability associated with tcyp incorporation into PNA-DNA duplexes, triplexes, and quadruplexes. The broad utility of this modification across multiple types of PNA structures is unique and should prove useful in the development of applications that rely on PNA.     

Cyclohexanyl peptide nucleic acids (chPNAs) for preferential RNA binding: effective tuning of dihedral angle beta in PNAs for DNA/RNA discrimination

[structures: see text] A serious drawback of peptide nucleic acids (PNAs) from an application perspective that has not been adequately dealt with is nondiscrimination of identical DNA and RNA sequences. An analysis of the available X-ray and NMR solution structures of PNA complexes with DNA and RNA suggested that it might be possible to rationally impart DNA/RNA duplex binding selectivity by tuning the dihedral angle beta of the flexible ethylenediamine part of the PNA backbone (II) via suitable chemical modifications. Cyclohexanyl PNAs (chPNAs) with beta approximately = 65 degrees were designed on the basis of this rationale. The chPNAs introduced remarkable differences in duplex stabilities among their DNA and RNA complexes, with melting temperatures (deltaTm(RNA-DNA) = +16-50 degrees C) depending on the number of modifications and the stereochemistry. This is a highly significant, exceptional binding selectivity of a mix sequence of PNA to RNA over the same DNA sequence as that seen to date. In contrast, cyclopentanyl PNAs (cpPNAs) with beta approximately = 25 degrees hybridize to DNA/RNA strongly without discrimination because of the ring puckering of the cyclopentane ring. The high affinity of chPNAs to bind to RNA without losing base specificity will have immediate implications in designing improved PNAs for therapeutic and diagnostic applications.     

Aminomethylene peptide nucleic acid (am-PNA): synthesis, regio-/stereospecific DNA binding, and differential cell uptake of (α/γ,R/S)am-PNA analogues

Inherently chiral, cationic am-PNAs having pendant aminomethylene groups at α(R/S) or γ(S) sites on PNA backbone have been synthesized. The modified PNAs are shown to stabilize duplexes with complementary cDNA in a regio- and stereo-preferred manner with γ(S)-am PNA superior to α(R/S)-am PNAs and α(R)-am PNA better than the α(S) isomer. The enhanced stabilization of am-PNA:DNA duplexes is accompanied by a greater discrimination of mismatched bases. This seems to be a combined result of both electrostatic interactions and conformational preorganization of backbone favoring the cDNA binding. The am-PNAs are demonstrated to effectively traverse the cell membrane, localize in the nucleus of HeLa cells, and exhibit low toxicity to cells.     

New Synthetic Route to γ-Mercaptomethyl PNA Monomers

Peptide nucleic acids (PNAs) are oligonucleotide mimics widely used as antisense, antigene molecules, and biotechnological tools. Recently, several microarrays and other biosensors based on PNAs have been developed. The construction of PNA molecular beacons or light-up probes for DNA detection requires the labelling of the PNA moiety. Labels are usually attached at the C or N terminal end by a flexible linker or in the middle of a PNA sequence, substituting one PNA base with an artificial base or by attaching fluorophores to a modified PNA backbone. The need to develop simple protocols to label PNAs encouraged us to design a new procedure for the synthesis of γ-mercaptomethyl-modified PNA. Here we propose a new strategy for the synthesis of modified PNAs, bearing amino acid side chains. The synthesis is straightforward and is an improvement to the procedures reported so far, as it uses stable intermediates and proceeds with better yields.     

Backbone extended pyrrolidine PNA (bepPNA): a chiral PNA for selective RNA recognition

Synthesis of cationic, chiral PNA analogues with an extra atom in the backbone (bepPNA) is reported. The (2S,4S) geometry of the pyrrolidine ring, and an additional carbon atom in the backbone of homopyrimidine-bepPNAs resulted in the optimization of the inter-nucleobase distance, such that selective binding to complementary RNA over DNA was observed in the triplex mode. It was evident from circular dichroism studies that oligomers with mixed aminoethylglycyl-bep (aeg-bep) repeating units, and also bepPNA with homogeneous backbone attained structures quite different from those of aegPNA₂:RNA/DNA complexes. The bepPNA, when incorporated in a duplex forming mixed purine-pyrimidine sequence, also showed a preference for binding complementary RNA over DNA.     

Calcium ions effectively enhance the effect of antisense peptide nucleic acids conjugated to cationic tat and oligoarginine peptides

Cell-penetrating peptides have been widely used to improve cellular delivery of a variety of proteins and antisense agents. However, recent studies indicate that such cationic peptides are predominantly entering cells via an endosomal pathway. We now show that the nuclear antisense effect in HeLa cells of a variety of peptide nucleic acid (PNA) peptide conjugates is significantly enhanced by addition of 6 mM Ca(2+) (as well as by the lysosomotrophic agent chloroquine). In particular, the antisense activities of Tat(48-60) and heptaarginine-conjugated PNAs were increased 44-fold and 8.5-fold, respectively. Evidence is presented that the mechanism involves endosomal release. The present results show that Ca(2+) can be used as an effective enhancer for in vitro cellular delivery of cationic peptide-conjugated PNA oligomers, and also emphasize the significance of the endosomal escape route for such peptides.     

Synthesis of a C-linked glycosylated thymine-based PNA monomer and its incorporation into a PNA oligomer

Backbone modification of peptide nucleic acids (PNAs) by glycosylation has been shown to enhance selective biodistribution and cellular targeting of PNA oligomers based on sugar and cell surface lectin interactions. Here we report the synthesis of a new backbone-glycosylated thymine-based PNA monomer (T(gal)). The sugar residue was attached to the backbone of PNA via a stable carbon-carbon linkage between the sugar and the PNA monomers. Also, incorporation of the modified monomer into a PNA decamer (H-Ala(gal)-G-G-G-T(gal)-C-A-G-C-T(gal)-T-Lys-NH2) was successfully performed. Melting temperature (UV-Tm) of the modified PNA against the complementary DNA was only slightly lower than unmodified PNA.     

Charge dependent behavior of PNA/DNA/PNA triplexes in the gas phase

Intact noncovalent complexes were studied in the gas phase using negative ion nano-ESI mass spectrometry. Among various noncovalent systems studied in the gas phase, the interaction of DNA strands with peptide nucleic acids (PNAs) presents a strong interest as biologically relevant systems. PNAs originally described by Nielsen are used as DNA mimics as possible medical agents by imprisoning DNA single strands into stable noncovalent complexes. Two types of PNAs were investigated in the PNA/DNA multiplex: the original Nielsen's PNA and a modified backbone PNA by the introduction of syn- and anti-(aminoethyl)thiazolidine rings. We first investigated the stoichiometry of PNA/DNA multiplexes formed in solution and observed them in the gas phase via qualitative kinetics of complementary strand associations. It resulted in observing PNA2/DNA triplexes (ts) as the multiply deprotonated species, most stable in both the solution and gas phase. Second, charge-dependant decompositions of these species were undertaken under low-energy collision conditions. It appears that covalent bond cleavages (base releasing or skeleton cleavage) occur from lower ts charge states rather than ts unzipping, which takes place from higher charge states. This behavior can be explained by considering the presence of zwitterions depending on the charge state. They result in strong salt-bridge interactions between the positively charged PNA side chain and the negatively charged DNA backbone. We propose a general model to clearly display the involved patterns in the noncovalent triplex decompositions. Third, the relative stability of three PNA2/DNA complexes was scrutinized in the gas phase by acquiring the breakdown curves of their ts(6-) form, corresponding to the ts unzipping. The chemical structures of the studied PNAs were chosen in order to evidence the possible influence of backbone stereochemistry on the rigidity of PNA2/DNA complexes. It provided significantly different stabilities via V(m) measurements. The relative gas-phase stability order obtained was compared to that found in solution by Chassaing et al., and shows qualitative agreement.     

New aspects in fragmentation of peptide nucleic acids: comparison of positive and negative ions by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

The use of peptide nucleic acids (PNAs) is steadily increasing in biochemistry and diagnostics. So far, PNAs have mostly been investigated using cationic conditions in mass spectrometry. Furthermore, the use of fragmentation techniques developed for peptides and proteins like infrared multiphoton dissociation (IRMPD) and electron capture dissociation (ECD) has barely been examined. However, especially the fragmentation behavior of PNA oligomers in negative ion mode is of high importance, due to the ability to interact with nucleic acids which are almost exclusively analyzed in the negatively charged state. In the current study PNA fragmentations under cationic and anionic conditions were investigated and different fragmentation techniques like collision‐induced dissociation (CID), IRMPD and ECD were applied. Especially when using CID and IRMPD, amide bonds were broken, whereas ECD resulted in the elimination of nucleobases. Differences were also observed between positive and negative ionization, while the sequence coverage for the negative ions was superior to positive ions. The fragmentation behavior using IRMPD led to almost complete sequence coverage. Additionally, in anions the interesting effect of multiple eliminations of HNCO was found. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

Sequence-specific binding of DNA to liposomes containing di-alkyl peptide nucleic acid (PNA) amphiphiles

We present a method to covalently attach peptide nucleic acid (PNA) to liposomes by conjugation of PNA peptide to charged amino acids and synthetic di-alkyl lipids ("PNA amphiphile," PNAA) followed by co-extrusion with disteroylphosphatidylcholine (DSPC) and cholesterol. Attachment of four Glu residues and two ethylene oxide spacers to the PNAA was required to confer proper hydration for extrusion and presentation for DNA hybridization. The extent of DNA oligomer binding to 10-mer PNAA liposomes was assessed using capillary zone electrophoresis. Nearly all PNAs on the liposome surface are complexed with a stoichiometric amount of complementary DNA 10-mers after 3-h incubation in pH 8.0 Tris buffer. No binding to PNAA liposomes was observed using DNA 10-mers with a single mismatch. Longer DNA showed a greatly attenuated binding efficiency, likely because of electrostatic repulsion between the PNAA liposome double layer and the DNA backbone. Langmuir isotherms of PNAA:DSPC:chol monolayers indicate miscibility of these components at the compositions used for liposome preparation. PNAA liposomes preserve the high sequence-selectivity of PNAs and emerge as a useful sequence tag for highly sensitive bioanalytical devices.     

(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.     

Rapid Synthesis of Propargyl-γ-Modified Peptide Nucleic Acid Monomers for Late-Stage Functionalization of Oligomers

The exceptional hybridization properties of peptide nucleic acids (PNAs) coupled with the ease of their synthesis has made this artificial nucleic acid mimetic a desirable platform for diagnostics, therapeutics and supramolecular engineering. PNA backbone modifications have been extensively explored to finetune physicochemical properties and for conjugation of functional molecules. Here, we detail the synthesis of a universal γ-propargyl-PNA backbone from serine, and its acylation with the four DNA canonical nucleobases. The availability of serine as d or l enantiomer provide simple accesses to PNA oligomers for hybridization with natural oligonucleotides or for orthogonal hybridization circuitry. We show that late-stage conjugation enables optimization of the physicochemical properties. This approach is appealing due to its orthogonality to Fmoc-SPPS, its flexibility and ease for introducing diversity by on-resin copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). We exemplified the utility of these novel monomers with PNA based hybridization chain reactions (HCRs).     

The alpha-helical peptide nucleic acid concept: merger of peptide secondary structure and codified nucleic acid recognition

A novel platform for nucleic acid recognition that integrates the alpha-helix secondary structure of peptides with the codified base-pairing capability of nucleic acids is reported. The resulting alpha-helical peptide nucleic acids (alpha PNAs) are composed of a repeating tetrapeptidyl unit, aa(1)-aa(2)-aa(3)-Ser(B), where aa(1) through aa(3) represent generic ancillary amino acids and B = nucleobases linked to Ser via a methylene bridge. Effective syntheses of constituent Fmoc-protected nucleoamino acids (Fmoc-Ser(B)-OH, where B = thymine, cytosine, and uracil) are described along with a protocol for the solid-phase synthesis of 21mer alpha PNAs containing five such nucleobases. By varying the ancillary amino acids, two distinct classes of alpha PNAs were constructed, having a net charge of -1 or +6, respectively, at physiological pH. The modular nature of the alpha PNA platform was illustrated by the synthesis of symmetrical disulfide-bridged alpha PNA dimers containing 10 nucleobases. Hybridization of these alpha PNAs with ssDNA has been examined by thermal denaturation, gel electrophoresis, and circular dichroism (CD) and the data indicated that alpha PNA binds to ssDNA in a cooperative manner with high affinity and sequence specificity. In general, b2 alpha PNAs bind faster and more strongly with ssDNA than do the corresponding b1 alpha PNAs. Parallel alpha PNA-DNA complexes are more stable than their antiparallel counterparts. CD studies also revealed that the hybridization event involves the folding of both species into their helical conformations. Finally, NMR experiments provided conclusive evidence of Watson-Crick base pairing in alpha PNA-ssDNA hybrids.     

High sensitivity detection of 16s rRNA using peptide nucleic acid probes and a surface plasmon resonance biosensor

A signal enhancing method allowing highly sensitive detection of E. coli 16s rRNA was developed using peptide nucleic acid (PNA) as a capture probe and a surface plasmon resonance (SPR) sensor as a detector. 16s rRNA has been used as a genetic marker for identification of organisms, and can be analyzed directly without PCR amplification due to the relatively high number of copies. PNA has a neutral backbone structure, therefore hybridization with 16s rRNA results in the ionic condition being changed from neutral to negative. A cationic Au nanoparticle was synthesized and used for signal amplification by ionic interaction with 16s rRNA hybridized on the PNA probe-immobilized SPR sensor chip. This method resulted in a detection limit of E. coli rRNA of 58.2 ± 1.37 pg mL⁻¹. Using this analytical method, Staphylococcus aureus was detected without purification of rRNA.     

Nucleobase‐Modified PNA Suppresses Translation by Forming a Triple Helix with a Hairpin Structure in mRNA In Vitro and in Cells

Compounds that bind specifically to double‐stranded regions of RNA have potential as regulators of structure‐based RNA function; however, sequence‐selective recognition of double‐stranded RNA is challenging. The modification of peptide nucleic acid (PNA) with unnatural nucleobases enables the formation of PNA–RNA triplexes. Herein, we demonstrate that a 9‐mer PNA forms a sequence‐specific PNA–RNA triplex with a dissociation constant of less than 1 nm at physiological pH. The triplex formed within the 5′ untranslated region of an mRNA reduces the protein expression levels both in vitro and in cells. A single triplet mismatch destabilizes the complex, and in this case, no translation suppression is observed. The triplex‐forming PNAs are unique and potent compounds that hold promise as inhibitors of cellular functions that are controlled by double‐stranded RNAs, such as RNA interference, RNA editing, and RNA localization mediated by protein–RNA interactions.     

Gene sensors based on peptide nucleic acid (PNA) probes: relationship between sensor sensitivity and probe/target duplex stability

Gene sensors based on peptide nucleic acid (PNA) probes were prepared and the relationship between sensor sensitivity and the duplex stability of the probe PNAs and target complementary DNAs was studied using five synthesized PNAs (10-, 15-, 17-, 20-, and 22-mers). It was found that the association constants for the probe PNA/target DNA pairs depend not only on the length but also on the base pair sequence, and that the trend in the sensor responses was the same as that in the association constants for the corresponding pairs. In addition, by using two kinds of probe PNAs with different lengths, it was demonstrated that fabrication of sensors based on probe PNAs with comparable association constants yielded similar response curves and sensor sensitivities.     

Synthesis and evaluation of (1S,2R/1R,2S)-aminocyclohexylglycyl PNAs as conformationally preorganized PNA analogues for DNA/RNA recognition

Conformationally constrained cis-aminocyclohexylglycyl PNAs have been designed on the basis of stereospecific imposition of 1,2-cis-cyclohexyl moieties on the aminoethyl segment of aminoethylglycyl PNA (aegPNA). The introduction of the cis-cyclohexyl ring may allow the restriction of the torsion angle beta in the ethylenediamine segment to 60-70 degrees that is prevalent in PNA(2):DNA and PNA:RNA complexes. The synthesis of the optically pure monomers (10a and 10b) is achieved by stereoselective enzymatic hydrolysis of an intermediate ester 2. The chiral PNA oligomers were synthesized with (1S,2R/1R,2S)-aminocyclohexylglycyl thymine monomers in the center and N-terminus of aegPNA. Differential gel shift retardation with one or more units of modified monomer units was observed as a result of hybridization of PNA sequences with complementary DNA sequences. Hybridization studies with complementary DNA and RNA sequences using UV-T(m) measurements indicate that PNA with (1S,2R)-cyclohexyl stereochemistry enhances selective binding with RNA over DNA as compared to control aegPNA and PNA with the other (1R,2S) isomer.     

The ‘fully protected backbone’ approach as a versatile tool for a new solid-phase PNA synthesis strategy

The ‘fully protected backbone’ (FPB) strategy has been efficiently adapted to the solid-phase synthesis of homothymine, homocytosine and ‘mixed’ pyrimidine PNAs. This versatile and simple method avoids the preparation of PNA monomers and relies on easy available starting materials, highly efficient backbone elongations and effective nucleobase units condensations.