Peptide Nucleic Acid Molecules Research Articles

Effects of PNA Sequence and Target Site Selection on Function of a 4.5S Non-Coding RNA.

Peptide nucleic acid (PNA) based antisense strategy is a promising therapeutic approach to specifically inhibit target gene expression. However, unlike protein coding genes, identification of an ideal PNA binding site for non-coding RNA is not straightforward. Here, we compare the inhibitory activities of PNA molecules that bind a non-coding 4.5S RNA called SRP RNA, a key component of the bacterial signal recognition particle (SRP). A 9-mer PNA (PNA9) complementary to the tetraloop region of the RNA was more potent in inhibiting its interaction with the SRP protein, compared to an 8-mer PNA (PNA8) targeting a stem-loop. PNA9, which contained a homo-pyrimidine sequence could form a triplex with the complementary stretch of RNA in vitro as confirmed using a fluorescent derivative of PNA9 (F-PNA13). The RNA-PNA complex formation resulted in inhibition of SRP function with PNA9 and F-PNA13, but not PNA8 highlighting the importance of target site selection. Surprisingly, F-PNA13 which was more potent in inhibiting SRP function in vitro, showed weaker antibacterial activity compared to PNA9 likely due to poor cell penetration of the longer PNA. Our results underscore the importance of suitable target site selection and optimum PNA length to develop better antisense molecules against non-coding RNA.

Harnessing Peptide Nucleic Acids and the Eukaryotic Resolvase MOC1 for Programmable, Precise Generation of Double-Strand DNA Breaks.

Programmable site-specific nucleases (SSNs) hold extraordinary promise to unlock myriad gene editing applications in medicine and agriculture. However, developing small and specific SSNs is needed to overcome the delivery and specificity translational challenges of current genome engineering technologies. Structure-guided nucleases have been harnessed to generate double-strand DNA breaks but with limited success and translational potential. Here, we harnessed the power of peptide nucleic acids (PNAs) for site-specific DNA invasion and the generation of localized DNA structures that are recognized and cleaved by the eukaryotic resolvase AtMOC1 from Arabidopsis thaliana. We named this technology PNA-assisted Resolvase-mediated (PNR) editing. We tested the PNR editing concept in vitro and demonstrated its precise target specificity, examined the nucleotide requirement around the PNA invasion for the AtMOC1-mediated cleavage, mapped the AtMOC1-mediated cleavage sites, tested the role of different types and lengths of PNA molecules invasion into dsDNA for the AtMOC1-mediated cleavage, optimized the in vitro PNA invasion and AtMOC1 cleavage conditions such as temperature, buffer conditions, and cleavage time points, and demonstrated the multiplex cleavage for precise fragment release. We discuss the best design parameters for efficient, site-specific in vitro cleavage using PNR editors.

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.

Peptide Nucleic Acids Containing Cationic/Amino-Alkyl Modified Bases Promote Enhanced Hybridization Kinetics and Thermodynamics with Single-Strand DNA.

Peptide nucleic acids (PNAs) are antisense molecules with excellent polynucleotide hybridization properties; they are resistant to nuclease degradation but often have poor cell permeability leading to moderate cellular activity and limited clinical results. The addition of cationic substitutions (positive charges) to PNA molecules greatly increases cell permeability. In this report, we describe the synthesis and polynucleotide hybridization properties of a novel cationic/amino-alkyl nucleotide base-modified PNA (OPNA). This study was designed to quantitate the effect the cationic/amino-alkyl nucleotide base modification had on the kinetic and thermodynamic properties of OPNA-DNA hybridization using surface plasmon resonance and UV thermal melt studies. Kinetic studies reveal a favorable 10-30 fold increase in affinity for a single cationic modification on the base of an adenine, cytosine, or guanidine OPNA sequence compared to the nonmodified PNA strand. The increase in affinity is correlated directly with a favorable decrease in the dissociation rate constant and increase in the association rate constant. Introducing additional amino-alkyl base modifications further favors a decrease in the dissociation rate (3-10-fold per amino-alkyl). The thermodynamics driving the OPNA hybridization is promoted by an additional favorable -80 kJ/mol enthalpy of binding for a single amino-alkyl modification compared to the PNA strand. This increase in enthalpy is consistent with an ion-ion interaction with the DNA strand. These kinetic and thermodynamic hybridization studies reveal for the first time that this type of cationic/amino-alkyl base-modified PNA has favorable hybridization properties suitable for development as an antisense oligomer.

Exploiting the interaction between halloysite and charged PNAs for their controlled release.

The design and development of nanomaterials that could be used in nanomedicine are of fundamental importance to obtain smart nanosystems for the treatment of several diseases. Halloysite, because of its interesting features, represents a suitable nanomaterial for the delivery of different biologically active species. Among them, peptide nucleic acids (PNAs) have attracted considerable attention in recent decades for their potential applications in both molecular antisense diagnosis and as therapeutic agents, although up to now, the actual clinical applications have been very limited. Herein we report a systematic study on the supramolecular interaction of three differently charged PNAs with halloysite. Understanding the interaction mode of charged molecules with the clay surfaces represents a key factor for the future design and development of halloysite based materials which could be used for the delivery and subsequent intracellular release of PNA molecules. Thus, three different PNA tetramers, chosen as models, were synthesized and loaded onto the clay. The obtained nanomaterials were characterized using spectroscopic studies and thermogravimetric analysis, and their morphologies were studied using high angle annular dark field transmission electron microscopy (HAADF/STEM) coupled with Energy Dispersive X-ray spectroscopy (EDX). The aqueous mobility of the three different nanomaterials was investigated by dynamic light scattering (DLS) and ζ-potential measurements. The release of PNA tetramers from the nanomaterials was investigated at two different pH values, mimicking physiological conditions. Finally, to better understand the stability of the synthesized PNAs and their interactions with HNTs, molecular modelling calculations were also performed. The obtained results showed that PNA tetramers interact in different ways with HNT surfaces according to their charge which influences their kinetic release in media mimicking physiological conditions.

Novel Small Multilamellar Liposomes Containing Large Quantities of Peptide Nucleic Acid Selectively Kill Breast Cancer Cells.

Simple SummaryWe present, for the first time, the preparation of small (60–90 nm in diameter) liposomes containing extremely large amounts (~8000 molecules per vesicle) of short, cytosine-rich peptide nucleic acid. The outer surface of liposomes wasfunctionalized with scaffold molecules specific to tumor-associated antigen overexpressing in breast cancer. We have shown that targeted liposomesspecifically interact with cancer cells and reduce their viability in sub-nanomolar concentrations. The results presented here can be widely used in cancer therapy based on cytosine-rich PNA oligonucleotides.Peptide nucleic acid (PNA) may be used in various biomedical applications; however, these are currently limited, due to its low solubility in aqueous solutions. In this study, a methodology to overcome this limitation is demonstrated, as well as the effect of PNA on cell viability. We show that extruding a mixture of natural phospholipids and short (6–22 bases), cytosine-rich PNA through a 100 nm pore size membrane under mild acidic conditions resulted in the formation of small (60–90 nm in diameter) multilamellar vesicles (SMVs) comprising several (3–5) concentric lipid membranes. The PNA molecules, being positively charged under acidic conditions (due to protonation of cytosine bases in the sequence), bind electrostatically to negatively charged phospholipid membranes. The large membrane surface area allowed the encapsulation of thousands of PNA molecules in the vesicle. SMVs were conjugated with the designed ankyrin repeat protein (DARPin_9-29), which interacts with human epidermal growth factor receptor 2 (HER2), overexpressed in human breast cancer. The conjugate was shown to enter HER2-overexpressing cells by receptor-mediated endocytosis. PNA molecules, released from lysosomes, aggregate in the cytoplasm into micron-sized particles, which interfere with normal cell functioning, causing cell death. The ability of DARPin-functionalized SMVs to specifically deliver large quantities of PNA to cancer cells opens a new promising avenue for cancer therapy.

AntimiR-155 Cyclic Peptide-PNA Conjugate: Synthesis, Cellular Uptake, and Biological Activity.

Efficient delivery of nucleic acids into cells still remains a great challenge. Peptide nucleic acids (PNAs) are DNA analogues with a neutral backbone and are synthesized by solid phase peptide chemistry. This allows a straightforward synthetic route to introduce a linear short peptide (a.k.a. cell-penetrating peptide) to the PNA molecule as a means of facilitating cellular uptake of PNAs. Herein, we have devised a synthetic route in which a cyclic peptide is prepared on a solid support and is extended with the PNA molecule, where all syntheses are accomplished on the solid phase. This allows the conjugation of the cyclic peptide to the PNA molecule with the need of only one purification step after the cyclic peptide–PNA conjugate (C9–PNA) is cleaved from the solid support. The PNA sequence chosen is an antimiR-155 molecule that is complementary to mature miR-155, a well-established oncogenic miRNA. By labeling C9–PNA with fluorescein isothiocyanate, we observe efficient cellular uptake into glioblastoma cells (U87MG) at a low concentration (0.5 μM), as corroborated by fluorescence-activated cell sorting (FACS) analysis and confocal microscopy. FACS analysis also suggests an uptake mechanism that is energy-dependent. Finally, the antimiR activity of C9–PNA was shown by analyzing miR155 levels by quantitative reverse transcription polymerase chain reaction and by observing a reduction in cell viability and proliferation in U87MG cells, as corroborated by XTT and colony formation assays. Given the added biological stability of cyclic versus linear peptides, this synthetic approach may be a useful and straightforward approach to synthesize cyclic peptide–PNA conjugates.

Electrochemically mediated in situ growth of electroactive polymers for highly sensitive detection of double-stranded DNA without sequence-preference

The ability to directly detect double-stranded DNA (dsDNA) without sequence-preference continues to be a major challenge. Herein, we report an electrochemical method for the direct, highly sensitive detection of dsDNA based on the strand replacement of dsDNA by peptide nucleic acid (PNA) and the in situ growth of electroactive polymers through the surface-initiated electrochemically mediated atom transfer radical polymerization (SI-eATRP). Thiolated PNA molecules are firstly self-assembled onto gold electrode surface for the specific recognition of target dsDNA (dsDNA-T), which in turn leads to the formation of a high density of PNA/DNA heteroduplexes on the electrode surface for the subsequent attachment of ATRP initiators via the phosphate-Zr4+-carboxylate chemistry. By applying a negative potential to the electrode, the air-stable CuII deactivators can be reduced into the CuI activators so as to trigger the surface-initiated polymerization for the in situ growth of electroactive polymers. Due to the strand replacement of dsDNA by PNA, dsDNA can be directly detected without sequence-preference. Besides, the growth of polymers enables the modification of numerous electroactive probes, thereby greatly improving the electrochemical signal. Under optimal conditions, a good linearity between the electrochemical signal and the logarithm of dsDNA-T concentration over the range from 1.0 fM to 1.0nM, with a detection limit of 0.47 fM, can be obtained. Results indicate that it is highly selective, and holds high anti-interference capability in the presence of human serum samples. Therefore, this method offers great promises in providing a universal and efficient solution for the direct detection of dsDNA.

A new toolbox for genome editing

A new approach, based on the use of peptide nucleic acid molecules to form triple helices and promote locus-specific recombination, demonstrates potential for in vivo gene correction at clinically significant levels and may provide a promising avenue for gene therapy.

Pyrrolidinyl peptide nucleic acid terminally labeled with fluorophore and end-stacking quencher as a probe for highly specific DNA sequence discrimination

Peptide nucleic acid (PNA) labeled with a dye/quencher pair is useful as a high-performance fluorescence probe for DNA sequence determination. Previous work has mainly focused on placing the dye/quencher at different ends of the PNA strand. In this work, we propose a new pyrrolidinyl PNA probe bearing a fluorescent dye and an anthraquinone quencher at the same end of the PNA molecule. The single-stranded PNA probe exhibits a weak fluorescence as a result of close contact between the dye and the quencher. End-stacking of the anthraquinone to the terminal base pair of the PNA·DNA duplex disrupts the fluorophore-quencher interaction, resulting in a large fluorescence increase in the presence of complementary DNA. The high specificity of the present PNA probe allows multicolor, multiplex detection of single mismatched DNA targets. In addition, direct detection of double stranded DNA target by double duplex invasion was demonstrated.

A Novel, Highly Selective RT-QPCR Method for Quantification of MSRV Using PNA Clamping Syncytin-1 (ERVWE1).

HERV-W is a multi-locus family of human endogenous retroviruses (HERVs) that has been found to play an important role in human physiology and pathology. Two particular members of HERV-W family are of special interests: ERVWE1 (coding syncytin-1, which is a glycoprotein essential in the formation of the placenta) and MSRV (multiple sclerosis-associated retrovirus that is thought to play a significant role in human pathology as a result of its increased expression in the brain tissue and blood cells derived from patients with multiple sclerosis (MS)). Both ERVWE1 and MSRV mRNA share high level of similarity and hence a method that allows to exclusively quantify the MSRV expression in clinical samples would be desirable. We developed a quantitative polymerase chain reaction (QPCR) technique for the detection and quantification of the multiple sclerosis-associated retrovirus. The assay utilises fluorescently labelled oligonucleotide probe, which is complementary to the conservative fragment of MSRV env gene and a peptide nucleic acid (PNA) probe, fully complementary to the ERVWE1 sequence fragment that efficiently blocks the polymerase action on ERVWE1 templates. The PNA molecule, if used parallel with hydrolysis probe in QPCR analysis, greatly facilitates the detection efficiency of MSRV even if ERVWE1 is present abundantly in respect to MSRV in the analysed sample. We achieved a wide and measurable range from 1 × 10 e2 to 1 × 10 e8 copies/reaction; the linearity of the technique was maintained even at the low MSRV level of 1 % in respect to ERVWE1. Using our newly developed method we confirmed that the expression of MSRV takes place in normal human astrocytes and in human umbilical vein endothelial cells in vitro. We also found that the stimulation of human monocytes did not influence the specific expression of MSRV but it caused changes in mRNA level of distinct HERV-W templates.

Nanoparticles that deliver triplex-forming peptide nucleic acid molecules correct F508del CFTR in airway epithelium.

Cystic fibrosis (CF) is a lethal genetic disorder most commonly caused by the F508del mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. It is not readily amenable to gene therapy because of its systemic nature and challenges including in vivo gene delivery and transient gene expression. Here, we use triplex-forming PNA molecules and donor DNA in biodegradable polymer nanoparticles to correct F508del. We confirm modification with sequencing and a functional chloride efflux assay. In vitro correction of chloride efflux occurs in up to 25% of human cells. Deep sequencing reveals negligible off-target effects in partially homologous sites. Intranasal application of nanoparticles in CF mice produces changes in nasal epithelium potential differences consistent with corrected CFTR, with gene correction also detected in lung tissue. This work represents facile genome engineering in vivo with oligonucleotides using a nanoparticle system to achieve clinically relevant levels of gene editing without off-target effects.

Antisense long noncoding RNAs regulate var gene activation in the malaria parasite Plasmodium falciparum

The virulence of Plasmodium falciparum, the causative agent of the deadliest form of human malaria, is attributed to its ability to evade human immunity through antigenic variation. These parasites alternate between expression of variable antigens, encoded by members of a multicopy gene family named var. Immune evasion through antigenic variation depends on tight regulation of var gene expression, ensuring that only a single var gene is expressed at a time while the rest of the family is maintained transcriptionally silent. Understanding how a single gene is chosen for activation is critical for understanding mutually exclusive expression but remains a mystery. Here, we show that antisense long noncoding RNAs (lncRNAs) initiating from var introns are associated with the single active var gene at the time in the cell cycle when the single var upstream promoter is active. We demonstrate that these antisense transcripts are incorporated into chromatin, and that expression of these antisense lncRNAs in trans triggers activation of a silent var gene in a sequence- and dose-dependent manner. On the other hand, interference with these lncRNAs using complement peptide nucleic acid molecules down-regulated the active var gene, erased the epigenetic memory, and induced expression switching. Altogether, our data provide evidence that these antisense lncRNAs play a key role in regulating var gene activation and mutually exclusive expression.

In situ synthesis of peptide nucleic acids in porous silicon for drug delivery and biosensing.

Peptide nucleic acids (PNA) are a unique class of synthetic molecules that have a peptide backbone and can hybridize with nucleic acids. Here, a versatile method has been developed for the automated, in situ synthesis of PNA from a porous silicon (PSi) substrate for applications in gene therapy and biosensing. Nondestructive optical measurements were performed to monitor single base additions of PNA initiated from (3-aminopropyl)triethoxysilane attached to the surface of PSi films, and mass spectrometry was conducted to verify synthesis of the desired sequence. Comparison of in situ synthesis to postsynthesis surface conjugation of the full PNA molecules showed that surface mediated, in situ PNA synthesis increased loading 8-fold. For therapeutic proof-of-concept, controlled PNA release from PSi films was characterized in phosphate buffered saline, and PSi nanoparticles fabricated from PSi films containing in situ grown PNA complementary to micro-RNA (miR) 122 generated significant anti-miR activity in a Huh7 psiCHECK-miR122 cell line. The applicability of this platform for biosensing was also demonstrated using optical measurements that indicated selective hybridization of complementary DNA target molecules to PNA synthesized in situ on PSi films. These collective data confirm that we have established a novel PNA–PSi platform with broad utility in drug delivery and biosensing.

Molecular dynamics simulations of PNA–PNA and PNA–DNA duplexes by the use of new parameters implemented in the GROMACS package: a conformational and dynamics study

Peptide Nucleic Acids (PNAs) still represent a growing research area thanks to their potential applications in many fields of science from chemistry and biology to medicine. In these years, structural investigations by means of either experimental or computational techniques have proved to be very useful for the understanding of the structural organization and the binding properties of PNA. In this context, we here report an all-atoms Molecular Dynamics (MD) study of a PNA-PNA duplex and a PNA-DNA hetero-duplex with the well known GROMACS simulation package, by using new force field parameters properly derived for PNA molecules. The good agreement of our results with the crystallographic and NMR data, available for both the systems under investigation, confirms the validity of our approach. Moreover, our simulations reveal new interesting features related to the conformational-dynamic behavior of the studied systems, thus demonstrating the ability of MD simulations to gain insights into the dynamic properties of biologically relevant systems. This force field parametrization represents a good starting point for the implementation of a computational platform, based on the GROMACS package, useful for the rational design of modified PNA molecules with improved conformational features for selective binding toward DNA or RNA.

The Capture of Low-Energy Electrons by PNA versus DNA

This study provides insight into the mechanism of capturing low energy electrons by peptide nucleic acid (PNA) and the role of the oligonucleotide backbone in the capture of low energy electrons. We studied by photoemission self-assembled monolayers of two types of oligonucleo- tides, DNA and PNA. PNA is a synthetic analogue of DNA that has a pseudopeptide backbone and which may have important medical and biotechological applications. We found that in both PNA and DNA, the guanine nucleobases capture the electrons more efficiently than thymines. In PNA, once the electrons are captured, their state is at least partially localized on the nucleobases, and the PNA molecule undergoes structural changes that stabilize the electron. This situation is in contrast to DNA, in which the captured electrons are transferred very efficiently to the backbone, and the final state of captured electron is base independent.

Composites of peptide nucleic acids with ttitanium dioxide nanoparticles. I. Construction of nanocomposites containing DNA/PNA duplexes and their delivery into HeLa cells

In order to investigate the possibility of using titanium dioxide (TiO2) nanoparticles to transport peptide nucleic acids (PNA) in eukaryotic cells, a PNA oligomer has been synthesized, and method of PNA immobilization in the form of hybrid DNA/PNA duplexes on the surface of TiO2 nanoparticles covered with polylysine (PL) has been designed. Attaching of DNA/PNA duplex on TiO2 x PL nanoparticles occurred due to electrostatic interactions between the negatively charged DNA chain and the positively charged amino groups of PL. Binding of the PNA with the nanocomposite achieved through noncovalent Watson-Crick interactions between the PNA and complementary DNA. The capacity of obtained TiO2 x PL x DNA/PNA nanocomposites depending on immobilization conditions was 10-30 nmol PNA per 1 mg of TiO2 particles, which corresponds to -1-3 PNA molecules per one TiO2 particle with size of 4-6 nm. By method ofconfocal laser scanning microscopy on the example of the fluorescein labeled PNA oligomer (Flu)PNA it has been shown that the PNA molecules in composition of TiO2 x PL x DNA/(Flu)PNA nanocomposites effectively penetrate and accumulate in HeLa cells without the use oftransfection agents, electroporation, or other auxiliary procedures has been shown.

Side chain modified peptide nucleic acids (PNA) for knock-down of six3in medaka embryos

BackgroundSynthetic antisense molecules have an enormous potential for therapeutic applications in humans. The major aim of such strategies is to specifically interfere with gene function, thus modulating cellular pathways according to the therapeutic demands. Among the molecules which can block mRNA function in a sequence specific manner are peptide nucleic acids (PNA). They are highly stable and efficiently and selectively interact with RNA. However, some properties of non-modified aminoethyl glycine PNAs (aegPNA) hamper their in vivo applications.ResultsWe generated new backbone modifications of PNAs, which exhibit more hydrophilic properties. When we examined the activity and specificity of these novel phosphonic ester PNAs (pePNA) molecules in medaka (Oryzias latipes) embryos, high solubility and selective binding to mRNA was observed. In particular, mixing of the novel components with aegPNA components resulted in mixed PNAs with superior properties. Injection of mixed PNAs directed against the medaka six3 gene, which is important for eye and brain development, resulted in specific six3 phenotypes.ConclusionsPNAs are well established as powerful antisense molecules. Modification of the backbone with phosphonic ester side chains further improves their properties and allows the efficient knock down of a single gene in fish embryos.

Targeted Gene Modification of Hematopoietic Progenitor Cells in Mice Following Systemic Administration of a PNA-peptide Conjugate

Hematopoietic stem cell (HSC) gene therapy offers promise for the development of new treatments for a variety of hematologic disorders. However, efficient in vivo modification of HSCs has proved challenging, thus imposing constraints on the therapeutic potential of this approach. Herein, we provide a gene-targeting strategy that allows site-specific in vivo gene modification in the HSCs of mice. Through conjugation of a triplex-forming peptide nucleic acid (PNA) to the transport peptide, antennapedia (Antp), we achieved successful in vivo chromosomal genomic modification of hematopoietic progenitor cells, while still retaining intact differentiation capabilities. Following systemic administration of PNA-Antp conjugates, sequence-specific gene modification was observed in multiple somatic tissues as well as within multiple compartments of the hematopoietic system, including erythroid, myeloid, and lymphoid cell lineages. As a true functional measure of gene targeting in a long-term renewable HSC, we also demonstrate preserved genomic modification in the bone marrow and spleen of primary recipient mice following transplantation of bone marrow from PNA-Antp-treated donor mice. Our approach offers a minimally invasive alternative to ex vivo gene therapy, by eliminating the need for the complex steps of stem cell mobilization and harvesting, ex vivo manipulation, and transplantation of stem cells. Therefore, our approach may provide new options for individualized therapies in the treatment of monogenic hematologic diseases such as sickle cell anemia and thalassemia.

An atomic force microscopy investigation on self-assembled peptide nucleic acid structures on gold(111) surface

Peptide Nucleic Acid (PNA) is an important alternative nucleic acid, which exhibits more effective DNA/RNA detection capabilities compared to DNA. Its potential utility in nucleic acid based detection technologies warrants detail understanding of it’s self-assembly behavior on solid substrates, e.g., gold. In the present study, we have applied high-resolution Atomic Force Microscopy (AFM) for obtaining direct visual information on PNA adsorption and formation of a self-assembled monolayer of PNA on gold(111) surface. We show from the molecularly resolved AFM data that PNA molecules form a well-defined 1-dimensional molecule-by-molecule ordering, over a considerable length scale (few hundred nm), as well as 2-dimensional ordering over a wide area of about 10μm×10μm, due to parallel positioning of the 1-dimensional ordered arrangements. The way the parameters like PNA concentration, incubation time, incubation temperature and PNA deposition methods can affect the formation of such ordered self-assembled PNA structures has been investigated. Some of the primary observations are that the minimum PNA concentration and incubation time for large scale (10μm×10μm) 2-dimensional order formation are 0.5μM and 4h, respectively. Furthermore, a dense and well-ordered layer over a large area could be better formed in case of immersion method compared to the droplet contact and droplet deposition methods. From the Reflection Absorption Infra Red Spectroscopy (RAIRS) data, indications for PNA concentration-driven reorientation of the PNA backbone towards more upright configuration on gold(111) surface, were obtained.