Peptide Nucleic Acid Oligomers Research Articles

Iron uptake pathway of Escherichia coli as an entry route for peptide nucleic acids conjugated with a siderophore mimic.

Bacteria secrete various iron-chelators (siderophores), which scavenge Fe3+ from the environment, bind it with high affinity, and retrieve it inside the cell. After the Fe3+ uptake, bacteria extract the soluble iron(II) from the siderophore. Ferric siderophores are transported inside the cell via the TonB-dependent receptor system. Importantly, siderophore uptake paths have been also used by sideromycins, natural antibiotics. Our goal is to hijack the transport system for hydroxamate-type siderophores to deliver peptide nucleic acid oligomers into Escherichia coli cells. As siderophore mimics we designed and synthesized linear and cyclic Nδ-acetyl-Nδ-hydroxy-l-ornithine based peptides. Using circular dichroism spectroscopy, we found that iron(III) is coordinated by the linear trimer with hydroxamate groups but not by the cyclic peptide. The internal flexibility of the linear siderophore oxygen atoms and their interactions with Fe3+ were confirmed by all-atom molecular dynamics simulations. Using flow cytometry we found that the designed hydroxamate trimer transports PNA oligomers inside the E. coli cells. Growth recovery assays on various E. coli mutants suggest the pathway of this transport through the FhuE outer-membrane receptor, which is responsible for the uptake of the natural iron chelator, ferric-coprogen. This pathway also involves the FhuD periplasmic binding protein. Docking of the siderophores to the FhuE and FhuD receptor structures showed that binding of the hydroxamate trimer is energetically favorable corroborating the experimentally suggested uptake path. Therefore, this siderophore mimic, as well as its conjugate with PNA, is most probably internalized through the hydroxamate pathway.

The Application of Peptide Nucleic Acids (PNA) in the Inhibition of Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) Gene Expression in a Cell-Free Transcription/Translation System.

Proprotein convertase subtilisin/kexin 9 (PCSK9) is a protein that plays a key role in the metabolism of low-density lipoprotein (LDL) cholesterol. The gain-of-function mutations of the PCSK9 gene lead to a reduced number of surface LDL receptors by binding to them, eventually leading to endosomal degradation. This, in turn, is the culprit of hypercholesterolemia, resulting in accelerated atherogenesis. The modern treatment for hypercholesterolemia encompasses the use of biological drugs against PCSK9, like monoclonal antibodies and gene expression modulators such as inclisiran-a short, interfering RNA (siRNA). Peptide nucleic acid (PNA) is a synthetic analog of nucleic acid that possesses a synthetic peptide skeleton instead of a phosphate-sugar one. This different structure determines the unique properties of PNA (e.g., neutral charge, enzymatic resistance, and an enormously high affinity with complementary DNA and RNA). Therefore, it might be possible to use PNA against PCSK9 in the treatment of hypercholesterolemia. We sought to explore the impact of three selected PNA oligomers on PCSK9 gene expression. Using a cell-free transcription/translation system, we showed that one of the tested PNA strands was able to reduce the PCSK9 gene expression down to 74%, 64%, and 68%, as measured by RT-real-time PCR, Western blot, and HPLC, respectively. This preliminary study shows the high applicability of a cell-free enzymatic environment as an efficient tool in the initial evaluation of biologically active PNA molecules in the field of hypercholesterolemia research. This cell-free approach allows for the omission of the hurdles associated with transmembrane PNA transportation at the early stage of PNA selection.

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

AbstractThe 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).

Cell-permeable peptide nucleic acid antisense oligonucleotide platform targeting human betacoronaviruses.

Antisense oligonucleotides (ASOs) with therapeutic potential have recently been reported to target the SARS-CoV-2 genome. Peptide nucleic acids (PNAs)-based ASOs have been regarded as promising drug candidates, but intracellular delivery has been a significant obstacle. Here, we present novel modified PNAs, termed OPNAs, with excellent cell permeability that disrupt the RNA genome of SARS-CoV-2 and HCoV-OC43 by introducing cationic lipid moiety onto the nucleobase of PNA oligomer backbone. HCT-8 cells and Caco-2 cells were treated with 1 μM antisense OPNAs at the time of viral challenge and the Viral RNA levels were measured by RT-qPCR three days post infection. NSP 14 targeting OPNA 5 and 11, reduced the viral titer to a half and OPNA 530, 531 and 533 lowered viral gene expression levels to less than 50% of control by targeting the 5' UTR region. Several modifications (oligo size and position, etc.) were introduced to enhance the efficacy of selected OPNAs. Improved OPNAs exhibited a dose-dependent reduction in viral replication and nucleoprotein (NP) protein. When a mixture of oligomers was applied to infected cells, viral titer and NP levels decreased by more than eightfold. In this study, we have developed a modified PNA ASO platform with exceptional chemical stability, high binding affinity, and cellular permeability. These findings indicate that OPNAs are a promising platform for the development of antivirals to combat future pandemic viral infections that do not require a carrier.

TFOFinder: Python program for identifying purine-only double-stranded stretches in the predicted secondary structure(s) of RNA targets.

Nucleic acid probes are valuable tools in biology and chemistry and are indispensable for PCR amplification of DNA, RNA quantification and visualization, and downregulation of gene expression. Recently, triplex-forming oligonucleotides (TFO) have received increased attention due to their improved selectivity and sensitivity in recognizing purine-rich double-stranded RNA regions at physiological pH by incorporating backbone and base modifications. For example, triplex-forming peptide nucleic acid (PNA) oligomers have been used for imaging a structured RNA in cells and inhibiting influenza A replication. Although a handful of programs are available to identify triplex target sites (TTS) in DNA, none are available that find such regions in structured RNAs. Here, we describe TFOFinder, a Python program that facilitates the identification of intramolecular purine-only RNA duplexes that are amenable to forming parallel triple helices (pyrimidine/purine/pyrimidine) and the design of the corresponding TFO(s). We performed genome- and transcriptome-wide analyses of TTS in Drosophila melanogaster and found that only 0.3% (123) of total unique transcripts (35,642) show the potential of forming 12-purine long triplex forming sites that contain at least one guanine. Using minimization algorithms, we predicted the secondary structure(s) of these transcripts, and using TFOFinder, we found that 97 (79%) of the identified 123 transcripts are predicted to fold to form at least one TTS for parallel triple helix formation. The number of transcripts with potential purine TTS increases when the strict search conditions are relaxed by decreasing the length of the probe or by allowing up to two pyrimidine inversions or 1-nucleotide bulge in the target site. These results are encouraging for the use of modified triplex forming probes for live imaging of endogenous structured RNA targets, such as pre-miRNAs, and inhibition of target-specific translation and viral replication.

Large-scale expansions of Friedreich's ataxia GAA•TTC repeats in an experimental human system: role of DNA replication and prevention by LNA-DNA oligonucleotides and PNA oligomers.

Friedreich's ataxia (FRDA) is caused by expansions of GAA•TTC repeats in the first intron of the human FXN gene that occurduring both intergenerational transmissions and in somatic cells. Here we describe an experimental system to analyze large-scale repeat expansions in cultured human cells. It employs a shuttle plasmid that can replicate from the SV40 origin in human cells or be stably maintained in S. cerevisiae utilizing ARS4-CEN6. It also contains a selectable cassette allowing us to detect repeat expansions that accumulated in human cells upon plasmid transformation into yeast. We indeed observed massive expansions of GAA•TTC repeats, making it the first genetically tractable experimental system to study large-scale repeat expansions in human cells. Further, GAA•TTC repeats stall replication fork progression, while the frequency of repeat expansions appears to depend on proteins implicated in replication fork stalling, reversal, and restart. Locked nucleic acid (LNA)-DNA mixmer oligonucleotides and peptide nucleic acid (PNA) oligomers, which interfere with triplex formation at GAA•TTC repeats in vitro, prevented the expansion of these repeats in human cells. We hypothesize, therefore, that triplex formation by GAA•TTC repeats stall replication fork progression, ultimately leading to repeat expansions during replication fork restart.

Synthesis and properties of PNA containing a dicationic nucleobase based on N4-benzoylated cytosine

We report the synthesis of a peptide nucleic acid (PNA) monomer containing N4-bis(aminomethyl)benzoylated cytosine (BzC2+ base). The BzC2+ monomer was incorporated into PNA oligomers using Fmoc-based solid-phase synthesis. The BzC2+ base in PNA had two positive charges and exhibited greater affinity for DNA G base than the natural C base. The BzC2+ base stabilized PNA-DNA heteroduplexes through electrostatic attractions, even in high salt conditions. The two positive charges on the BzC2+ residue did not compromise the sequence specificity of PNA oligomers. These insights will aid the future design of cationic nucleobases.

Charge migration of ferrocene-labeled peptide self-assembled monolayers at various interfaces: The roles of peptide composition

Understanding charge migration across peptide chain of proteins is important for both biology and biomolecular electronics. Despite experimental and theoretical researches showing that the charge migration in wet electrochemistry and on solid state are interrelated, there are still limited insights about this interrelation. Here, taken ferrocene-labeled peptide self-assembled monolayers on the Au substrate as a model, the relation between the charge migration on solid state (charge transport, CTp) and in wet electrochemistry (charge transfer, CT) was investigated. By characterizing the current density from solid state molecular junctions and the electron transfer rate (kET) from electrochemical measurement in solution, we found that the ability of peptides to transmit charge was more sensitive to peptide's length on solid state than that in wet electrochemistry, comparing to the reported alkane and peptide nucleic acid oligomers. In addition, changing peptide compositions, such as doping phenylalanine (F) or aspartic acid (D) into the initial glycine (G) peptide backbone, made a different consequence for charge migration: the kET in wet electrochemistry was F > G ≈ D, while the CTp rate on solid state was G ≈ F > D. Our findings provide further insights into the comprehension of relations between charge migration and peptide structures.

Ultrasound-assisted Peptide Nucleic Acids synthesis (US-PNAS)

Herein, we developed an innovative and easily accessible solid-phase synthetic protocol for Peptide Nucleic Acid (PNA) oligomers by systematically investigating the ultrasonication effects in all steps of the PNA synthesis (US-PNAS). When compared with standard protocols, the application of the so-obtained US-PNAS approach succeeded in improving the crude product purities and the isolated yields of different PNA, including small or medium-sized oligomers (5-mer and 9-mer), complex purine-rich sequences (like a 5-mer Guanine homoligomer and the telomeric sequence TEL-13) and longer oligomers (such as the 18-mer anti-IVS2-654 PNA and the 23-mer anti-mRNA 155 PNA). Noteworthy, our ultrasound-assisted strategy is compatible with the commercially available PNA monomers and well-established coupling reagents and only requires the use of an ultrasonic bath, which is a simple equipment generally available in most synthetic laboratories.

Targeting a KRAS i-motif forming sequence by unmodified and gamma-modified peptide nucleic acid oligomers.

Growing interest in i-motif DNA as a transcriptional regulatory element motivates development of synthetic molecules capable of targeting these structures. In this study, we designed unmodified peptide nucleic acid (PNA) and gamma-modified PNA (γPNA) oligomers complementary to an i-motif forming sequence derived from the promoter of the KRAS oncogene. Biophysical techniques such as circular dichroism (CD) spectroscopy, CD melting, and fluorescence spectroscopy demonstrated the successful invasion of the i-motif by PNA and γPNA. Both PNA and γPNA showed very strong binding to the target sequence with high thermal stability of the resulting heteroduplexes. Interestingly fluorescence and CD experiments indicated formation of an intermolecular i-motif structure via the overhangs of target-probe heteroduplexes formed by PNA/γPNA invasion of the intramolecular i-motif. Targeting promoter i-motif forming sequences with high-affinity oligonucleotide mimics like γPNAs may represent a new approach for inhibiting KRAS transcription, thereby representing a potentially useful anti-cancer strategy.

From Peptide Nucleic Acids to Supramolecular Structures of Nucleic Acid Derivatives.

Nucleic acids play a pivotal role in life processes. The endeavours to shed light on the essential properties of these intriguing building blocks led us to the synthesis of different analogues and the investigation of their properties. First various peptide nucleic acid monomers and oligomers have been synthesized, using an Fmoc/acyl protecting group strategy, and their properties studied. The serendipitous discovery of a side reaction of coupling agents led us to the elaboration of a peptide sequencing method. The capricious behaviour of guanine derivatives spurred the determination of their substitution pattern using 13 C, 15 N NMR, and mass spectrometric methods. The properties of guanines initiated the logical transition to the study of supramolecular systems composed of purine analogues. Thus, xanthine and uracil derivatives have been obtained and their supramolecular self-assembly properties scrutinized in gas, solid, and liquid states and at solid-liquid interfaces.

A novel synthetic method of peptide nucleic acid (PNA) oligomers using Boc/Cbz‐protected PNA trimer blocks in the solution phase

AbstractIn order to use peptide nucleic acid (PNA) oligomers for therapeutic purposes, a high purity level is essential and, in particular, large‐scale production should be easy. However, in the traditional solid‐phase synthesis method, the generation of n − 1 or n − 2 deleted oligomers cannot be completely excluded, and purification of crude oligomers is difficult because the target and impurities overlap. Also, for these reasons, scale‐up is also limited. Here, we report the efficient synthesis of PNA oligomers in the solid phase using fully protected PNA trimers instead of monomers. Fully protected PNA trimer blocks were successfully synthesized via two different routes in the solution phase. When PNA oligomers were synthesized in the solid phase using trimer blocks as monomers, a higher purity level of PNA could be synthesized compared to when oligomers were synthesized using monomers. When using this method, n − 1 or n − 2 truncated products cannot be systematically produced. Furthermore, the trimer blocks coupling method is successful even for sequences containing purine‐rich and/or gamma PNAs, which are difficult to synthesize via the conventional solid‐phase peptide synthesis approach. Finally, two hexameric and one dodecameric PNA oligomers were completely synthesized in the solution phase, with the former showing more than 90% crude purity and the latter showing 70%. We propose that this method may be able to solve several problems related to solid‐phase synthesis at once.

Highly sensitive detection of driver mutations from cytological samples and cfDNA in lung cancer.

BackgroundBronchoscopy is a minimally invasive procedure for establishing the diagnosis of lung cancer. It sometimes fails to obtain tissue samples but readily collects cytological samples.MethodsWe developed PNA‐LNA dual‐PCR (PLDP), which amplified mutant sequences by a high‐fidelity DNA polymerase in the presence of a peptide nucleic acid (PNA) oligomer having a wild‐type sequence. Mutations are detected either by locked nucleic acid (LNA) probes for quick detection of a limited number of mutations, which are EGFR, KRAS, and BRAF mutations in the current study, or by direct sequencing for a comprehensive screening. In a total of 233 lung cancer samples, the results for cytological samples by PLDP were compared with those for tissue samples by cobas® EGFR mutation test (cobas) or by the PNA‐LNA PCR clamp method (P‐LPC). Moreover, the performance of PLDP using cell‐free DNA (cfDNA) was investigated.ResultsPeptide nucleic acid‐LNA dual‐PCR was able to detect each synthesized mutant sequence with high sensitivity. PLDP detected EGFR mutations in 80 out of 149 clinical samples, while the cobas or the P‐LPC detected in 66 matched. The correctness of PLDP was confirmed both by clinical response and by the results of sequencing using a next‐generation sequencer. PLDP detected mutations from cfDNA in approximately 70% of patients who harbors mutations in the tumor.ConclusionsPeptide nucleic acid‐LNA dual‐PCR exhibited an excellent performance, even using cytological samples. PLDP is applicable for the investigation of cfDNA. The combination of bronchoscopy and PLDP is attractive and will expand the utility of bronchoscopy in clinical practice.

Peptide nucleic acid as a template for Taq DNA polymerase

Peptide nucleic acid (PNA), an artificial DNA analog, comprises a purine or pyrimidine base and a pseudo-peptide backbone instead of deoxyribose-phosphate. PNA has been found to have stronger adhesion and higher stability in binding to its complementary DNA than deoxyribose-phosphate. Thus, it could serve as an agent for gene modulation, demonstrating potential in antisense therapy, molecular diagnostics, and nanotechnology. However, the applications of PNA remain limited because its biological activities are not fully known. Here, I demonstrate that a thermostable DNA polymerase, Thermus aquaticus (Taq) polymerase, exhibits transcriptase activity when a PNA oligomer is used as a template and that genetic information of the oligomer can be amplified by PCR using DNA primers. Furthermore, the insertion of a glutamine peptide stretch in the middle part of the PNA template did not interfere with transcription; it was transcribed into a guanosine or adenosine stretch. Intriguingly, this amino acid-to-DNA transcription did not occur when glycine residues were inserted. A synthetic PNA oligomer can, therefore, function as a template for a DNA polymerase, and polyglutamine peptides can be transcribed into guanosine or adenosine. These findings provide a cornerstone to reveal all amino acid genetic codes and transcription activity in the future.

Molecular Assembly of Triplex of Duplexes from Homothyminyl-Homocytosinyl Cγ(S/R)-Bimodal Peptide Nucleic Acids with dA8/dG6 and the Cell Permeability of Bimodal Peptide Nucleic Acids.

Peptide nucleic acids (PNAs) are analogues of DNA with a neutral acyclic polyamide backbone containing nucleobases attached through a t-amide link on repeating units of aminoethylglycine (aeg). They bind to complementary DNA or RNA in a sequence-specific manner to form duplexes with higher stablity than DNA:DNA and DNA:RNA hybrids. We have recently explored a new type of PNA termed bimodal PNA (bm-PNA) designed with two nucleobases per aeg repeating unit of PNA oligomer and attached at Cα or Cγ of each aeg unit through a spacer sidechain. We demonstrated that Cγ-bimodal PNA oligomers with mixed nucleobase sequences bind concurrently two different complementary DNAs, forming double duplexes, one from each t-amide and Cγ face, sharing a common PNA backbone. In such bm-PNA:DNA ternary complexes, the two duplexes show higher thermal stability than individual duplexes. Herein, we show that Cγ(S/R)-bimodal PNAs with homothymines (T8) on a t-amide face and homocytosine (C6) on a Cγ-face form a conjoined pentameric complex consisting of a triplex (bm-PNA-T8)2:dA8 and two duplexes of bm-PNA-C6:dG6. The pentameric complex [dG6:Cγ(S/R)-bm-PNA:dA8:Cγ(S/R)-bm-PNA:dG6] exhibits higher thermal stability than the individual triplex and duplex, with Cγ(S)-bm-PNA complexes being more stable than Cγ(R)-bm-PNA complexes. The conjoined duplexes of Cγ-bimodal PNAs can be used to generate novel higher-order assemblies with DNA and RNA. The Cγ(S/R)-bimodal PNAs are shown to enter MCF7 and NIH 3T3 cells and exhibit low toxicity to cells.

Optimized Synthesis of Fmoc/Boc‐Protected PNA Monomers and their Assembly into PNA Oligomers.

AbstractContinuous advancement of application of peptide nucleic acid (PNA) oligomers encouraged exploration of rapid and efficient synthesis of PNA monomers and oligomers. Among the PNA monomers developed, only a few are commonly used in automated PNA synthesis. Herein, we report short and efficient protocols suitable for large‐scale synthesis of Fmoc/Boc‐protected PNA monomers with advantageous solubility properties; these also facilitate purification due to the traceless nature of the Boc protecting group. Initially, several coupling reagents were screened for assembly of a pentamer containing all four nucleobases, and then the most promising reagents were tested in the synthesis of a decamer. The Fmoc/Boc‐protected monomers proved compatible with both manual synthesis and assembly on an automated peptide synthesizer at room temperature or at 40 °C. As compared to the commonly used coupling agent, 1‐[bis(dimethylamino)methylene]‐1H‐1,2,3‐triazolo[4,5‐b]pyridinium 3‐oxide hexafluorophosphate (HATU), both 2‐(1H‐benzotriazol‐1‐yl)‐1,1,3,3‐tetramethyluronium hexafluorophosphate (HBTU) and [ethyl cyano(hydroxyimino)acetato−O2]tri‐1‐pyrrolidinylphosphonium hexafluorophosphate (PyOxim) proved more favorable, with the latter being superior. A previously reported side reaction of guanine bases in the presence of benzotriazol‐1‐yl‐oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) was not observed with the phosphonium salts.

Targeting a Potential G-Quadruplex Forming Sequence Found in the West Nile Virus Genome by Complementary Gamma-Peptide Nucleic Acid Oligomers.

In the United States, West Nile virus (WNV) infects approximately 2500 people per year, of which 100-200 cases are fatal. No antiviral drug or vaccine is currently available for WNV. In this study, we designed gamma-modified peptide nucleic acid (γPNA) oligomers to target a newly identified guanine-rich gene sequence in the WNV genome. The target is found in the NS5 protein-coding region and was previously predicted to fold into a G-quadruplex (GQ) structure. Biophysical techniques such as UV melting analysis, circular dichroism spectroscopy, and fluorescence spectroscopy demonstrated that the target RNA indeed folds into a moderately stable GQ structure at physiological temperature and potassium concentration. Successful invasion of the GQ by three complementary γPNAs was also characterized by the above-mentioned biophysical techniques. The γPNAs showed very strong binding to the target with low femtomolar affinity at physiological temperature. Targeting this potential guanine quadruplex forming sequence (PQS) and other related sequences with γPNA may represent a new approach for inhibiting both WNV replication and transcription, thereby representing a generally useful antiviral strategy.

Synthesis and characterization of PNA oligomers containing preQ1 as a positively charged guanine analogue

We report the synthesis of a peptide nucleic acid (PNA) monomer containing preQ1, a positively charged guanine analogue. The new monomer was incorporated into PNA oligomers using standard Fmoc-chemistry-based solid-phase synthesis. The preQ1 unit-containing PNA oligomers exhibited improved affinity for their complementary DNA through electrostatic attraction, and their sequence specificity was not compromised. It could be beneficial to incorporate preQ1 into PNA oligomers instead of guanine when creating antisense/antigene agents or research tools.

Vitamin B12- Peptide Nucleic Acid Conjugates.

Vitamin B12 (cobalamin, Cbl) is an essential nutrient for all mammals and some bacteria. From a chemical point of view, it is a highly functionalized molecule, which enables conjugation at multiple positions and attachment of various cargoes. Both mammalian and bacterial cells have developed a specific transport pathway for the uptake of vitamin B12, and as a consequence, cobalamin is an attractive candidate for the delivery of biologically relevant molecules into cells. Indeed, hybrid molecules containing vitamin B12 in their structure have found various applications in medicinal chemistry, diagnostics, and biological sciences.Herein, we describe synthetic strategies toward the synthesis of vitamin B12 conjugates with peptide nucleic acid (PNA ) oligomers. Such short-modified oligonucleotides targeted at bacterial DNA or RNA can act as antibacterial agents if efficiently taken up by bacterial cells. The uptake of such oligonucleotides is hindered by the bacterial cell envelope, but vitamin B12 was found to efficiently deliver antisense PNA into Escherichia coli and Salmonella Typhimurium cells. This paves the way to the use of vitamin B12-PNA conjugates in antibacterial and diagnostic applications.Vitamin B12-PNA conjugates can be prepared via copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) that gives access to covalently linked hybrids or via connecting both building blocks by reduction-sensitive disulfide bridge. Both approaches require prior modification of vitamin B12 by incorporation of the azide moiety or via transformation of the native functional group into a moiety reactive toward thiols. Conjugation of vitamin B12 with PNA-tagged substrates efficiently furnishes designed conjugates.

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.