PNA-DNA Duplexes Research Articles

Dynamics of terminal fraying-peeling and hydrogen bonds dictates the sequential vs. cooperative melting pathways of nanoscale DNA and PNA triplexes.

Peptide nucleic acids (PNAs) are charge-neutral synthetic DNA/RNA analogues. In many aspects of biology and biotechnology, the details of DNA and PNA melting reaction coordinates are crucial, and their associative/dissociative details remain inadequately understood. In the current study, we have attempted to gain insights into comparative melting pathways and binding affinity of iso-sequences of an 18-mer PNA-DNA-PNA triplex and the analogous DNA-DNA-DNA triplex, and DNA-DNA and PNA-DNA duplexes. It is intriguing that while the DNA-DNA-DNA triplex melts in two sequential steps, the PNA-DNA-PNA triplex melts in a single step and the mechanistic aspects for this difference are still not clear. We report an all-atom molecular dynamics simulation of both complexes in the temperature range of 300 to 500 K with 20 K intervals. Based on the trajectory analysis, we provide evidence that the association and dissociation are dictated by the differences in fraying-peeling effects from either terminus to the center in a zipper pattern among the PNA-DNA-PNA triplex and DNA-DNA-DNA triplexes. These are shown to be governed by the different characteristics of H-bonding, RMSD, and Free Energy Landscape (FEL) as analyzed by PCA, leading to the DNA-DNA-DNA triplex exhibiting sequential melting, while the PNA-DNA-PNA triplex shows cooperative melting of the whole fragment in a single-step. The PNA-DNA-PNA triplex base pairs are thermodynamically more stable than the DNA-DNA-DNA triplex, with the binding affinity of PNA-TFO to the PNA : DNA duplex being higher than that of DNA-TFO to the DNA : DNA duplex. The investigation of the association/dissociation of PNA-TFO to the PNA-DNA duplex has relevance and importance in the emerging effective applications of oligonucleotide therapy.

Nanopore-Assisted, Sequence-Specific Detection, and Single-Molecule Hybridization Analysis of Short, Single-Stranded DNAs.

We report here on the ability of the α-hemolysin (α-HL) nanopore to achieve label-free, selective, and real-time detection of 15 nt long ssDNA fragments in solution, by exploiting their hybridization with freely added, polycationic peptides-functionalized PNAs. At the core of our work lies the paradigm that when PNAs and ssDNA are mixed together, the bulk concentration of free PNA decreases, depending upon the (mis)match degree between complementary strands and their relative concentrations. We demonstrate that the ssDNA sensing principle and throughput of the method are determined by the rate at which nonhybridized, polycationic peptides-functionalized PNA molecules arrive at the α-HL's vestibule entrance and thread into the nanopore. We found that with the application of a 30-fold salt gradient across the nanopore, the method enhances single-molecule detection sensitivity in the nanomolar range of ssDNA concentrations. This study demonstrates that the transmembrane potential-dependent unzip of single PNA-DNA duplexes at the α-HL's β-barrel entry permits discrimination between sequences that differ by one base pair.

Nonfunctionalized PNAs as Beacons for Nucleic Acid Detection in a Nanopore System.

In this work, single-channel current recordings were used to selectively detect individual ssDNA strands in the vestibule of the α-hemolysin (α-HL) protein nanopore. The sensing mechanism was based on the detection of the intrinsic topological change of target ssDNA molecules after the hybridization with complementary PNA fragments. The readily distinguishable current signatures of PNA-DNA duplexes reversible association with the α-HL's vestibule, in terms of blockade amplitudes and kinetic features, allows specific detection of nucleic acid hybridization.

Nanoscale Probing of Informational Polymers with Nanopores. Applications to Amyloidogenic Fragments, Peptides, and DNA-PNA Hybrids.

The decades long advances in nanotechnology, biomolecular sciences, and protein engineering ushered the introduction of groundbreaking technologies devoted to understanding how matter behaves at single particle level. Arguably, one of the simplest in concept is the nanopore-based paradigm, with deep roots in what is originally known as the Coulter counter, resistive-pulse technique. Historically, a nanopore system comprising the oligomeric protein generated by Staphylococcus aureus toxin α-hemolysin (α-HL) was first applied to detecting polynucleotides, as revealed in 1996 by John J. Kasianowicz, Eric Brandin, Daniel Branton, and David W. Deamer, in the Proceedings of the National Academy of Sciences. Nowadays, a wide variety of other solid-state or protein-based nanopores have emerged as efficient tools for stochastic sensing of analytes as small as single metal ions, handling single molecules, or real-time, label-free probing of chemical reactions at single-molecule level. In this Account, we demonstrate the usefulness of the α-HL nanopore on probing metal-induced folding of peptides, and to investigating the reversible binding of various metals to physiologically relevant amyloid fragments. The widely recognized Achilles heel of the approach, is the relatively short dwell time of the analytes inside the nanopore. This hinders the collection of sufficient data required to infer statistically meaningful conclusions about the physical or chemical state of the studied analyte. To mitigate this, various approaches were successfully applied in particular experiments, including but not restricted to altering physical parameters of the aqueous solution, downsizing the nanopore geometry, the controlled tuning of the balance between the electrostatic and electro-osmotic forces, coating nanopores with a fluid lipid bilayer, employing a pressure-voltage biased pore. From our perspective, in this Account, we will present two strategies aimed at controlling the analyte passage across the α-HL. First, we will reveal how the electroosmotic flow can be harnessed to control residence time, direction, and the sequence of spatiotemporal dynamics of a single peptide along the nanopore. This also allows one to identify the mesoscopic trajectory of a peptide exiting the nanopore through either the vestibule or β-barrel moiety. Second, we lay out the principles of an approach dubbed "nanopore tweezing", enabling simultaneous capture rate increase and escape rate decrease of a peptide from the α-HL, with the applied voltage. At its core, this method requires the creation of an electrical dipole on the peptide under study, via engineering positive and negative amino acid residues at the two ends of the peptide. Concise applications of this approach are being demonstrated, as in proof-of-concept experiments we probed the primary structure exploration of polypeptides, via discrimination between selected neutral amino acid residues. Another useful venue provided by the nanopores is represented by single-molecule force experiments on captured analytes inside the nanopore, which proved useful in exploring force-induced rupture of nucleic acids duplexes, hairpins, or various nucleic acids-ligand conjugates. We will show that when applied to oppositely charged, polypeptide-functionalized PNA-DNA duplexes, the nanopore tweezing introduces a new generation of force-spectroscopy nanopore-based platforms, facilitating unzipping of a captured duplex and enabling the duplex hybridization energy estimation.

Conformational and energetic properties of pyrrolidinyl PNA-DNA duplexes: A molecular dynamics simulation

The binding capability of pyrrolidinyl peptide nucleic acids (PNAs) to DNA have been studied experimentally, also the high affinity and sequence specificity were reported. However, to date a detailed understanding of these properties is unclear due to a lack of crystallographic data. In this work, molecular dynamics (MD) simulations of pyrrolidinyl PNA with different stereochemistry, (2′R,4′R)- and (2′R,4′S)-prolyl-(1S,2S)-2-aminocyclopentanecarboxylic acid (namely acpcPNA and epi-acpcPNA, respectively), binding to DNA were performed. MD simulations of three different forms (A-, B- and P-forms) were conducted to investigate the probable duplex conformation, which revealed that the MD structures were close to B-type duplex. It was found that acpcPNA and epi-acpcPNA backbones play a minor role in the hydrogen bond geometry of base pair, however, these greatly influence the interactions between PNA and DNA strands, leading to the differences in duplex stability. The calculated binding free energy revealed the higher thermodynamic stability of acpcPNA-DNA compared to epi-acpcPNA-DNA system, which is in good agreement with published experimental data. This work offers new insights into the conformational and energetic properties of pyrrolidinyl PNA-DNA duplexes at a molecular level.

Design of Tail-Clamp Peptide Nucleic Acid Tethered with Azobenzene Linker for Sequence-Specific Detection of Homopurine DNA

DNA carries genetic information in its sequence of bases. Synthetic oligonucleotides that can sequence-specifically recognize a target gene sequence are a useful tool for regulating gene expression or detecting target genes. Among the many synthetic oligonucleotides, tail-clamp peptide nucleic acid (TC-PNA) offers advantages since it has two homopyrimidine PNA strands connected via a flexible ethylene glycol-type linker that can recognize complementary homopurine sequences via Watson-Crick and Hoogsteen base pairings and form thermally-stable PNA/PNA/DNA triplex structures. Here, we synthesized a series of TC-PNAs that can possess different lengths of azobenzene-containing linkers and studied their binding behaviours to homopurine single-stranded DNA. Introduction of azobenzene at the N-terminus amine of PNA increased the thermal stability of PNA-DNA duplexes. Further extension of the homopyrimidine PNA strand at the N-terminus of PNA-AZO further increased the binding stability of the PNA/DNA/PNA triplex to the target homopurine sequence; however, it induced TC-PNA/DNA/TC-PNA complex formation. Among these TC-PNAs, 9W5H-C4-AZO consisting of nine Watson-Crick bases and five Hoogsteen bases tethered with a beta-alanine conjugated azobenzene linker gave a stable 1:1 TC-PNA/ssDNA complex and exhibited good mismatch recognition. Our design for TC-PNA-AZO can be utilized for detecting homopurine sequences in various genes.

2-Methoxypyridine as a Thymidine Mimic in Watson-Crick Base Pairs of DNA and PNA: Synthesis, Thermal Stability, and NMR Structural Studies.

The development of nucleic acid base-pair analogues that use new modes of molecular recognition is important both for fundamental research and practical applications. The goal of this study was to evaluate 2-methoxypyridine as a cationic thymidine mimic in the A-T base pair. The hypothesis was that including protonation in the Watson-Crick base pairing scheme would enhance the thermal stability of the DNA double helix without compromising the sequence selectivity. DNA and peptide nucleic acid (PNA) sequences containing the new 2-methoxypyridine nucleobase (P) were synthesized and studied by using UV thermal melting and NMR spectroscopy. Introduction of P nucleobase caused a loss of thermal stability of ≈10 °C in DNA-DNA duplexes and ≈20 °C in PNA-DNA duplexes over a range of mildly acidic to neutral pH. Despite the decrease in thermal stability, the NMR structural studies showed that P-A formed the expected protonated base pair at pH 4.3. Our study demonstrates the feasibility of cationic unnatural base pairs; however, future optimization of such analogues will be required.

Electrochemical paper-based peptide nucleic acid biosensor for detecting human papillomavirus

A novel paper-based electrochemical biosensor was developed using an anthraquinone-labeled pyrrolidinyl peptide nucleic acid (acpcPNA) probe (AQ-PNA) and graphene-polyaniline (G-PANI) modified electrode to detect human papillomavirus (HPV). An inkjet printing technique was employed to prepare the paper-based G-PANI-modified working electrode. The AQ-PNA probe baring a negatively charged amino acid at the N-terminus was immobilized onto the electrode surface through electrostatic attraction. Electrochemical impedance spectroscopy (EIS) was used to verify the AQ-PNA immobilization. The paper-based electrochemical DNA biosensor was used to detect a synthetic 14-base oligonucleotide target with a sequence corresponding to human papillomavirus (HPV) type 16 DNA by measuring the electrochemical signal response of the AQ label using square-wave voltammetry before and after hybridization. It was determined that the current signal significantly decreased after the addition of target DNA. This phenomenon is explained by the rigidity of PNA-DNA duplexes, which obstructs the accessibility of electron transfer from the AQ label to the electrode surface. Under optimal conditions, the detection limit of HPV type 16 DNA was found to be 2.3 nM with a linear range of 10–200 nM. The performance of this biosensor on real DNA samples was tested with the detection of PCR-amplified DNA samples from the SiHa cell line. The new method employs an inexpensive and disposable device, which easily incinerated after use and is promising for the screening and monitoring of the amount of HPV-DNA type 16 to identify the primary stages of cervical cancer.

Is it possible to study the kinetic parameters of interaction between PNA and parallel and antiparallel DNA by stopped-flow fluorescence?

Peptide nucleic acids (PNAs) are among the most interesting and versatile artificial structural mimics of nucleic acids and exhibit peculiar and important properties (i.e. high chemical stability, and a high resistance to cellular enzymes and nucleases). Despite their unnatural structure, they are able to recognize and bind DNA and RNA in a very high, specific and selective manner. One of the most popular, easy and reliable method to measure the stability of PNA-DNA hybrid systems is the melting temperature but the thermodynamic data are obtained using a big quantity of materials failing to provide information on the kinetics of the interaction.In the present work, the PNA decamer 6, with the TCACTAGATG sequence of nucleobases, and the corresponding fluorescent PNA-FITU (fluorescein isothiourea) decamer 8 were synthesized with standard manual Boc-based chemistry. The interaction of the PNA-FITU with parallel and antiparallel DNA has been studied by stopped-flow fluorescence, which is proposed as an alternative technique to obtain the kinetic parameters of the binding.The great advantage of using the stopped-flow technique is the possibility of studying the kinetics of the PNA-DNA duplex formation in a physiological environment. In particular, fluorescence stopped-flow technique has been exploited to compare the affinity of two PNA-DNA duplexes since it can discriminate between parallel and antiparallel DNA binding.

Peptide Nucleic Acid with a Lysine Side Chain at the β-Position: Synthesis and Application for DNA Cleavage

This paper reports the synthesis of new β-Lys peptide nucleic acid (PNA) monomers and their incorporation into a 10-residue PNA sequence. PNA containing β-Lys PNA units formed a stable hybrid duplex with DNA. However, incorporation of β-Lys PNA units caused destabilization of PNA-DNA duplexes to some extent. Electrostatic attractions between β-PNA and DNA could reduce this destabilization effect. Subsequently, bipyridine-conjugated β-Lys PNA was prepared and exhibited sequence selective cleavage of DNA. Based on the structures of the cleavage products and molecular modeling, we reasoned that bipyridine moiety locates within the minor groove of the PNA-DNA duplexes. The lysine side chain of β-PNA is a versatile handle for attaching various functional molecules.

UV-Vis Spectroscopy and Dynamic Light Scattering Study of Gold Nanorods Aggregation

Gold nanorods (AuNRs) were used as spectroscopic sensing elements to detect specific DNA sequences with a single-base mismatch sensitivity. The assay was based on the observation that the stabilizing repulsive forces between CTA(+)-coated AuNRs can be removed by citrate ions, which causes aggregation among AuNRs; whereas nucleic acids of different structures[ i.e., peptide nucleic acid (PNA), single-stranded DNA (ssDNA), PNA-DNA complex, and double-stranded DNA (dsDNA)] can retard the aggregation. Moreover, the dsDNA PNA-DNA duplexes provide larger retardation than that by unhybridized ssDNA and PNA probe. This assay can differentiate single-base mismatched targets with base substitution at different locations (center and end) with AuNRs of a larger aspect ratio. Besides ultraviolet-visable spectroscopy measurement of particle assembly-induced plasmonic coupling that in turn provides a spectroscopic detection of the specific DNA, dynamic light scattering and transmission electron microscope (TEM) were used to measure smaller degree of aggregation that can reveal sodium citrate- and dsDNA-AuNRs interactions in fine detail.

Facilitating Mismatch Discrimination by Surface-Affixed PNA Probes via Ionic Regulation

There has been a search for alternative nucleic acids that can be more effectively used in nucleic acid detection technologies compared to the DNA probes. Peptide nucleic acid (PNA), which contains a non-ionic peptidic backbone, offers such possibilities since it is nuclease-resistant, it binds to DNA with high affinity, and it can be readily self-assembled onto solid substrates, e.g., gold(111), with a molecular backbone orientation away from the substrate. Although application of PNA as a sensor probe has been exemplified, so far there is little or no account of the ionic modulation of single base mismatch discrimination capacity of surface-tethered PNA probes. Herein, we report "on-surface" melting temperatures of PNA-DNA duplexes formed on gold(111) surface, as obtained from fluorescence measurements. We show that surface-tethered PNA forms a stabler duplex than DNA, and is more effective in single base mismatch discrimination than DNA. Importantly, although PNA backbone is non-ionic, variation in the ionic components in hybridization buffer, i.e., varying concentration of monovalent sodium ion, and the nature of anion and the cation, exhibits clear effects on the mismatch discrimination capacity of PNA probes. In general, with decreasing cation concentration, PNA-DNA duplexes are stabilized and mismatch discrimination capacity of the PNA probes is enhanced. The stabilizing/destabilizing effects of anions are found to follow the Hofmeister series, emphasizing the importance of hydrophobic interaction between nucleobases for stability of the PNA-DNA duplexes. Interestingly, the nature of ionic dependence of "on-surface" mismatch detection ability of PNA probes differs significantly from the "solution" behavior of these probes.

Development of PNA-Array Platforms for Detection of Genetic Polymorphism of Cytochrome P450 2C19

The selective and sensitive detection of target DNA is an important issue for clinical diagnostics, including identification of single nucleotide polymorphism (SNP), as well as for fundamental studies in molecular biology. The commonly used DNA-detection techniques are mainly based on the DNA microarrays, which present probe-DNA molecules immobilized onto a solid surface in an array format for the bio-specific hybridization with the unknown target-DNA fragments. Although the DNA microarrays have been utilized as a basic platform for the clinical detection, selectivity and sensitivity are still limited toward the singlenucleotide discrimination, which precludes the accurate analysis of SNP. In addition, duplexes between the target and probe DNAs exist in a stable form only in the presence of salt because of the electrostatic repulsion between negatively charged ribose-phosphate backbones of DNA; these salt conditions also induce the secondary and tertiary structures of DNA oligomers themselves, which could disturb the hybridization between the target and probe DNAs. On the other hand, peptide nucleic acid (PNA), an artificial nucleic acid analog, has been introduced to overcome the drawbacks of natural DNA in the aspects of specificity, sensitivity, and stability, and been as a molecular tool for the identification of SNP. The neutral backbone of PNA enhances the stability of PNA-DNA duplexes due to the absence of interstrand repulsion between the negatively charged phosphate groups of DNA. In addition, the unnatural structure makes PNA not degraded by enzymatic reactions. In this work, we generated a PNA-array platform to detect the SNP of cytochrome P450 2C19 (CYP2C19) gene. The CYP2C19 gene is known to play an important role in the metabolism of therapeutic drugs, including diazepam, proguanil, lansoprazole, and omeprazole. It is noteworthy that 15-20% of the Asians are genetically deficient in CYP2C19, failing to produce enzymes for the metabolism, compared with the occurrence of 3-5% of Caucasian populations. Specifically, there exist two variant alleles leading to the mutant types. The first varient is CYP2C19*2, which has a G → A nucleotide substitution at position 681, resulting in an aberrant splicing site; the second one, CYP2C19*3, which does a G → A substitution at position 636, resulting in a premature termination codon. According to the previous report, the frequency of CYP2C19*2 and CYP2C19*3 for Koreans is 0.21 and 0.12, respectively. Onto the silicon oxide surface were aldehyde groups introduced via imine formation between amine-terminated self-assembled monolayers (SAMs) and glutaldehyde. Another imine linkage was used for immobilizing the PNA probes, and unreacted aldehyde groups were passivated with 2(2aminoethoxy)ethanol. We designed four probe-PNAs based on the clinically important SNPs of CYP2C19, and denoted them 636W and 636M for the wild and mutant types of CYP2C19*3, and 681W and 681M for CYP2C19*2, respectively (Fig. 1). The linker of the PNA probes contained repetitive ethylene glycol groups and ended with several lysine groups. The immobilization of the probe PNAs was confirmed by hybridization of target DNAs. The target DNAs (T-636W, T-636M, T-681W, and T-681M), complementary to each of the PNA probes, were tagged with Cy3 (green) for wild type and Cy5 (red) for mutant type, respectively. The hybridization was performed for 1 h at 40 C with the DNA solution containing both T-636W and T636M (100 nM), and the same conditions were used for hybridization of T-681W/M. As shown in Fig. 2a, each DNA was selectively hybridized with its complementary probe-

Abstract 3068: Detection of K-RAS mutations in Korean patients using PNAClampTM KRAS

Abstract Mutation of the K-RAS gene located in codons 12 and 13 may play an important role in prognosis of related diseases and responses to certain treatments targeting the pathway. Recent studies suggest that in colon cancer patients, K-RAS mutation status is a strong predictor of success in therapeutics of several tyrosine kinase inhibitors. Therefore, it is important to detect K-RAS mutations fast and accurately. To detect a minority of mutant K-RAS among abundant wild-type alleles, we have developed a highly sensitive and simple method for the detection of K-RAS mutations using PNA-mediated real-time PCR clamping, PNAClampTM. PNAClampTM relies on the following two unique properties of PNA probes: 1) PNA-DNA duplexes generally have greater thermal stability than the corresponding DNA-DNA duplex; 2) PNA oligomers are not recognized by DNA polymerases and consequently can serve as a sequence-selective clamp during PCR amplification. This method is fast and accurate with a detection limit of approximately 0.1% mutant alleles using 10 ng to 25 ng of normal DNA as a template. The turnaround time for performance of this assay is only 2 hrs. The PNAClampTM method facilitates the detection of mutations in more than 1000-fold excess of wild-type DNA samples. K-RAS mutations were detected in 28.3% (36/127) of patients. In detail, 29 cases had mutations in codon 12 and 7 cases had mutations in codon 13. To confirm those results, the same clinical samples were analyzed with a direct sequencing assay. Concordant results were obtained from 124 (97.6%) of the 127 clinical samples in the two assays. The discrepancy was observed in three cases. For comparison, the three clinical samples were also analyzed using DxS K-RAS Mutation Kit (DxS Ltd., UK) to give the same outcome. In this study, we will discuss development of PNAClampTM KRAS and its optimization for higher sensitivity. The PNAClampTM KRAS may serve a rapid and reliable alternative for K-RAS mutation detection in the clinical field. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3068. doi:10.1158/1538-7445.AM2011-3068

DNA-programmed spatial screening of carbohydrate–lectin interactions

A wide range of multivalent scaffolds was assembled by using only five different PNA oligomers and various DNA templates. The flexibility of the PNA–DNA duplexes could be increased by introducing nick-sites and partially unpaired regions, as confirmed by MD simulations. The self-organized glyco-assemblies were used in a spatial screening of accessible carbohydrate binding sites in the Erythrina cristagalli lectin (ECL). This systematic investigation revealed a distance dependence which is in agreement with the crystal structure analysis.

A pyrenyl-PNA probe for DNA and RNA recognition

The design and the synthesis of a PNA oligomer containing a pyrenyl residue in the backbone were performed. PNA sequence was chosen complementary to a "G rich" target sequence involved in G-quadruplex formation. The pyrenyl unit replaced a nucleobase in the middle of the PNA through covalent linkage to the backbone by a carboxymethyl unit. A systematic study on the binding properties of this probe towards DNA and RNA complementary strands was carried out by UV and fluorescence spectroscopy. UV melting curves indicated that the PNA probe binds more tightly to RNA rather than to DNA. Thermodynamic data obtained by Van't Hoff fitting of the melting curves indicated that, in the case of RNA, a more favorable interaction occurs between the pyrenyl unit and the RNA nucleobases, leading to a very favorable enthalpic contribution.The fluorescence analysis showed specific quenching of the pyrene emission associated to the formation of the full-match PNA-DNA or PNA-RNA duplexes. Again, this behavior was more evident in the case of RNA, consistently with the stronger interaction of the pyrenyl unit with the complementary strand. In order to study the sequence specificity of the pyrenyl-PNA probe (pyr-PNA), recognition experiments on mismatched DNA and RNA sequences were also performed.

Patterning of Peptide Nucleic Acids Using Reactive Microcontact Printing

PNAs (peptide nucleic acids) have been immobilized onto surfaces in a fast, accurate way by employing reactive microcontact printing. Surfaces have been first modified with aldehyde groups to react with the amino end of the synthesized PNAs. When patterning fluorescein-labeled PNAs by reactive microcontact printing using oxygen-oxidized polydimethylsiloxane stamps, homogeneous arrays were fabricated and characterized using optical methods. PNA-patterned surfaces were hybridized with complementary and mismatched dye-labeled oligonucleotides to test their ability to recognize DNA sequences. The stability and selectivity of the PNA-DNA duplexes on surfaces have been verified by fluorescence microscopy, and the melting curves have been recorded. Finally, the technique has been applied to the fabrication of chips by spotting a PNA microarray onto a flat PDMS stamp and reproducing the same features onto many slides. The chips were finally applied to single nucleotide polymorphism detection on oligonucleotides.

Abstract 5059: Detection of K-RAS mutation in clinical samples by one-step PNA-mediated real-time PCR clamping techniques

Abstract Mutations in the K-RAS oncogene are frequently found in human cancers. K-RAS mutations may indicate prognosis and drug response, and many new cancer therapies are being targeted to the K-RAS pathway. Many researchers suggest that in colon cancer patients the K-RAS mutation status is strong predictor of resistance to therapy with tyrosine kinase inhibitors such as Erbitux® (cetuximab/ Imclone system Inc) or Vectibix® (panitumumab/ Amgen Inc). Therefore, it is important that detection of K-RAS mutations is fast and simple with high accuracy and high sensitivity for predict drug response and prognosis. To detect a minority of mutant K-RAS among abundant wild-type alleles, we developed a high sensitivity and simple method for detection of K-RAS mutations using PNA-mediated real-time PCR clamping. PNA mediated real-time PCR clamping relies on the following two unique properties of PNA probes. 1) PNA-DNA duplexes generally have greater thermal stability than the corresponding DNA-DNA duplex and 2) PNA oligomers are not recognized by DNA polymerases and consequently can serve as sequence selective clamp during PCR amplification. One-step PNA-mediated real-time PCR clamping method was optimized for detection of K-RAS mutations in exon 12 and 13 with high sensitivity. This method was simple and correct with detection limit approximately 0.1% mutant alleles using 10ng to 50ng of normal DNA as the template. The turnaround time for performing this assay was only 2.5hrs. K-RAS mutations were detected in 24.4% (22/90) of clinical samples, of which 18 had mutations in codon 12 and 4 had these in codon 13. To confirm these results, same clinical samples were analyzed with sequencing assay. The concordant results were obtained from 88 (98%) of the 90 clinical samples by the two assays. The PNA-mediated real-time PCR clamping is a rapid and reliable tool for K-RAS mutation detection and allowed a minority of mutant in clinical field. Keywords: K-RAS, Mutation, PNA, cancer, PCR clamping This work was supported be the Small and Medium Business Administration funded by the Korean Government. (S1060400) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5059.

Special Series: Advances in the investigation of the structure‐function relationship in nucleic acids and their assemblies

Special Series: Advances in the investigation of the structure‐function relationship in nucleic acids and their assemblies

A new concept in double duplex DNA invasion by chiral PNAs which simultaneously depress PNA-PNA and improve PNA-DNA duplex stability.

By using suitably designed chiral, lysine-based PNAs (chPNAs), the stability of complementary chPNAchPNA duplexes can be depressed, maintaining or improving at the same time the stability of the corresponding PNA-DNA duplexes. This approach could be used in order to develop chPNAs able to perform double duplex DNA invasion.