OBJECTIVETo investigate the effect of blocking polypyrimidine complex binding to DNA site by using peptide nucleic acid (PNA) on γ-globin gene expression.

METHODSPYR-PNA, β-PNA and RS-PNA (random sequence-PNA) were designed and synthesized, then were transfected into K562 cells with the cationic liposome lipofectamine 2000 used as vector. The expression of γ-globin gene at both the transcriptional and translational level was detected by RT-PCR and the Western blot respectively at 24 h, 48 h and 72 h after transfection with PNAs.

RESULTSCompared with RS-PNA and control groups, the expression of γ-globin gene at mRNA and protein levels in PYR-PNA group was significantly up-regulated(P<0.05), especially at 48 h after tranfection, the levels of mRNA and protein in PYR-PNA group were increased by 2.0 and 2.5 times than those in control group, respectively.

CONCLUSIONPYR-PNA can significantly up-regulate the expression of γ-globin gene in K562 cells, this study may provide a new research idea for gene therapy of β-thalassemia.

The aim of this study was to build a gene chip system with surface plasmon resonance (SPR) technique, for which Gamma-peptide nucleic acid (Gamma-PNA) functioned as a probe, in order to improve sensitivity and its specificity. With the use of self-assembled monolayer (SAM) technology, surface chemistry of two-dimensional structure was used. Gamma-PNA was designed according to the bioinformatics, and was plated on the SPR chip modified by SAM. Subsequently, relevant parameters of the experiment were ensured and optimized. The results showed that the performances of Gamma-PNA probe was little affected by the ion concentration of buffer, and it had a strong light signal in a stable state. As the ion concentration was 0, there were still good hybrid reactions; pH value had less influence upon Gamma-PNA probe, and acid environment of buffer could be better. Gamma-PNA probe combined with sensor technologies achieved made the probe with dispensable labels and real-time detection. It also improved the efficiency of the hybridization and the stability, providing the foundation for clinical application.
Nucleic Acid Hybridization ; Nucleic Acid Probes ; Oligonucleotide Array Sequence Analysis ; methods ; Peptide Nucleic Acids ; genetics ; Sensitivity and Specificity ; Surface Plasmon Resonance

Peptide nucleic acid (PNA) is a DNA surrogate in which the phosphate deoxyribose backbone of DNA is replaced by repeating N-(2-aminoethyl)glycine units. PNA can hybridize to the complementary DNA and RNA with higher affinity than their oligonucleotide counterparts. This character of PNA not only makes it a new tool for the studies of molecular biology but also the potential candidate for gene-targeting drugs. The non-ionic backbone of PNA leads to stable hybrids with the nucleic acids, but at the same time, the neutral backbone results in poor cellular uptake. To address this problem, studies on modified PNA progress rapidly in recent years. We reviewed literature reports combined with our study about the delivery methods, including backbone modified PNA and PNA-ligand conjugates, and the cellular uptake of modified PNA. In addition, we summarized the problems and future prospect of the cellular delivery of modified PNA.
DNA, Complementary ; Drug Delivery Systems ; Glycine ; analogs & derivatives ; Humans ; Nucleic Acid Hybridization ; Oligonucleotides ; Peptide Nucleic Acids ; chemistry ; RNA

Both the primed in situ (PRINS) and the peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) techniques constitute alternatives to the conventional (fluorescence in situ hybridization, FISH) procedure for chromosomal investigations. The PRINS reaction is based on the use of a DNA polymerase and labeled nucleotide in an in situ primer extension reaction. Peptide nucleic acid probes are synthetic DNA analogs with uncharged polyamide backbones. The two procedures present several advantages (specificity, rapidity and discriminating ability) that make them very attractive for cytogenetic purposes. Their adaptation to human spermatozoa has allowed the development of new and fast procedures for the chromosomal screening of male gametes and has provided efficient complements to FISH for in situ assessment of aneuploidy in male gametes.
Aneuploidy ; Humans ; Male ; Peptide Nucleic Acids ; chemistry ; Primed In Situ Labeling ; methods ; Spermatozoa ; metabolism

BACKGROUND/AIMS: Helicobacter pylori eradication rates are decreasing because of increases in clarithromycin resistance. Thus, finding an easy and accurate method of detecting clarithromycin resistance is important. METHODS: We evaluated 70 H. pylori isolates from Korean patients. Dual-labeled peptide nucleic acid (PNA) probes were designed to detect resistance associated with point mutations in 23S ribosomal ribonucleic acid gene domain V (A2142G, A2143G, and T2182C). Data were analyzed by probe-based fluorescence melting curve analysis based on probe-target dissociation temperatures and compared with Sanger sequencing. RESULTS: Among 70 H. pylori isolates, 0, 16, and 58 isolates contained A2142G, A2143G, and T2182C mutations, respectively. PNA probe-based analysis exhibited 100.0% positive predictive values for A2142G and A2143G and a 98.3% positive predictive value for T2182C. PNA probe-based analysis results correlated with 98.6% of Sanger sequencing results (κ-value=0.990; standard error, 0.010). CONCLUSIONS: H. pylori clarithromycin resistance can be easily and accurately assessed by dual-labeled PNA probe-based melting curve analysis if probes are used based on the appropriate resistance-related mutations. This method is fast, simple, accurate, and adaptable for clinical samples. It may help clinicians choose a precise eradication regimen.
Clarithromycin* ; Fluorescence ; Freezing ; Helicobacter pylori* ; Helicobacter* ; Humans ; Methods* ; Peptide Nucleic Acids ; Point Mutation ; RNA

Peptide nucleic acid(PNA) is a kind of artificial DNA mimic. PNA hybridizes with DNA or RNA by means of Watson-Crick's base-pairs complementary with high stability, affinity and selectivity. PNA not only regulates. DNA replication, but also adjusts DNA transcription (or reverse transcription) and translation. Many applications have been explored as a new kind of molecular biological tool and a gene-targeting strategy.
Gene Expression Regulation ; drug effects ; Peptide Nucleic Acids ; pharmacology ; Protein Biosynthesis ; drug effects ; Transcription, Genetic ; drug effects

BACKGROUND: KRAS is one of commonly mutated genetic "drivers" in non-small cell lung cancers (NSCLCs). Recent studies indicate that patients with KRAS-mutated tumors do not benefit from adjuvant chemotherapy, so there is now a focus on targeting KRAS-mutated NSCLCs. A feasible mutation detection method is required in order to accurately test for KRAS status. METHODS: We compared direct Sanger sequencing and the peptide nucleic acid (PNA)-mediated polymerase chain reaction (PCR) clamping method in 134 NSCLCs and explored associations with clinicopathological factors. Next-generation sequencing (NGS) was used to validate the results of discordant cases. To increase the resolution of low-level somatic mutant molecules, PNA-mediated PCR clamping was used for mutant enrichment prior to NGS. RESULTS: Twenty-one (15.7%) cases were found to have the KRAS mutations using direct sequencing, with two additional cases by the PNA-mediated PCR clamping method. The frequencies of KRAS mutant alleles were 2% and 4%, respectively, using conventional NGS, increasing up to 90% and 89%, using mutant-enriched NGS. The KRAS mutation occurs more frequently in the tumors of smokers (p=.012) and in stage IV tumors (p=.032). CONCLUSIONS: Direct sequencing can accurately detect mutations, but, it is not always possible to obtain a tumor sample with sufficient volume. The PNA-mediated PCR clamping can rapidly provide results with sufficient sensitivity.
Alleles ; Carcinoma, Non-Small-Cell Lung* ; Chemotherapy, Adjuvant ; Constriction* ; Humans ; Lung Neoplasms ; Peptide Nucleic Acids ; Polymerase Chain Reaction*

BACKGROUND: Peptide nucleic acid (PNA) probes are artificial DNA analogues with a hydrophobic nature that can penetrate the mycobacterial cell wall. We evaluated a FISH method for simultaneous detection and identification of Mycobacterium tuberculosis (MTB) and non-tuberculous mycobacteria (NTM) in clinical respiratory specimens using differentially labeled PNA probes. METHODS: PNA probes targeting the mycobacterial 16S ribosomal RNA were synthesized. The cross-reactivity of MTB- and NTM-specific probes was examined with reference strains and 10 other frequently isolated bacterial species. A total of 140 sputum specimens were analyzed, comprising 100 MTB-positive specimens, 21 NTM-positive specimens, and 19 MTB/NTM-negative specimens; all of them were previously confirmed by PCR and culture. The PNA FISH test results were graded by using the United States Centers for Disease Control and Prevention-recommended scale and compared with the results from the fluorochrome acid-fast bacterial stain. RESULTS: The MTB- and NTM-specific PNA probes showed no cross-reactivity with other tested bacterial species. The test results demonstrated 82.9% agreement with the culture results with diagnostic sensitivity of 80.2% and diagnostic specificity of 100.0% (kappa=0.52, 95% confidence interval: 0.370-0.676). CONCLUSIONS: Dual-color PNA FISH showed high specificity for detecting and identifying mycobacteria in clinical specimens. However, because of its relatively low sensitivity, this method could be more applicable to culture confirmation. In application to direct specimens, the possibility of false-negative results needs to be considered.
Cell Wall ; Centers for Disease Control and Prevention (U.S.) ; DNA ; Fluorescence* ; In Situ Hybridization* ; Mycobacterium tuberculosis* ; Nucleic Acid Probes* ; Peptide Nucleic Acids ; Polymerase Chain Reaction ; RNA, Ribosomal, 16S ; Sputum

PURPOSE: Direct sequencing (DS) is the standard method for detection of epidermal growth factor receptor (EGFR) gene mutation in non-small cell lung cancer (NSCLC); however, low detection sensitivity is a problem. The aim of this study is to demonstrate higher detection rate of EGFR gene mutation with peptide nucleic acid (PNA) clamping compared with DS. MATERIALS AND METHODS: This is a single arm, prospective study for patients with stage IIIB/IV or relapsed NSCLC. Using tumor DNA from 138 patients, both DS and PNA clamping for EGFR gene in exon 18, 19, 20, and 21 were performed. Discrepant results between the two methods were verified using Cobas and a mutant enrichment based next generation sequencing (NGS). Patients with activating mutations were treated with EGFR tyrosine kinase inhibitor (EGFR-TKI, gefitinib, or erlotinib) as first line treatment. RESULTS: Of 138 paired test sets, 24 (17.4%) and 45 (32.6%) cases with activating mutations were detected by DS and PNA clamping, respectively. The difference of detection rate between the two methods was 15.2% (95% confidence interval, 8.7% to 17.8%; p < 0.001). Between the two methods, 25 cases showed discrepant results (n=23, PNA+/DS-; n=2, PNA-/DS+). Mutations were confirmed by Cobas or NGS in 22 of 23 PNA+/DS- cases. The response rates to EGFR-TKI were 72.2% in the PNA+/DS+ group and 85.0% in the PNA+/DS- group. CONCLUSION: PNA clamping showed a significantly higher detection rate of EGFR gene mutation compared with DS. Higher sensitivity of PNA clamping was not compromised by the loss of predictive power of response to EGFR-TKI.
Arm ; Carcinoma, Non-Small-Cell Lung* ; Constriction* ; DNA ; Exons ; Genes, erbB-1* ; Humans ; Peptide Nucleic Acids ; Prospective Studies ; Protein-Tyrosine Kinases ; Receptor, Epidermal Growth Factor

BACKGROUND: The aims of this study were to evaluate the abilities of direct sequencing (DS), peptide nucleic acid (PNA) clamping, and pyrosequencing methods to detect epidermal growth factor receptor (EGFR) mutations in formalin-fixed paraffin-embedded (FFPE) non-small cell lung carcinoma (NSCLC) samples and to correlate EGFR mutational status as determined by each method with the clinical response to EGFR tyrosine kinase inhibitors (TKIs). METHODS: Sixty-one NSCLC patients treated with EGFR TKIs were identified to investigate somatic mutations in the EGFR gene (exons 18-21). RESULTS: Mutations in the EGFR gene were detected in 38 of the 61 patients (62%) by DS, 35 (57%) by PNA clamping and 37 (61%) by pyrosequencing. A total of 44 mutations (72%) were found by at least one of the three methods, and the concordances among the results were relatively high (82-85%; kappa coefficient, 0.713 to 0.736). There were 15 discordant cases (25%) among the three different methods. CONCLUSIONS: All three EGFR mutation tests had good concordance rates (over 82%) for FFPE samples. These results suggest that if the DNA quality and enrichment of tumor cells are assured, then DS, PNA clamping, and pyrosequencing are appropriate methods for the detection of EGFR mutations.
Constriction ; DNA ; Genes, erbB-1 ; Humans ; Lung ; Lung Neoplasms ; Peptide Nucleic Acids ; Polymerase Chain Reaction ; Protein-Tyrosine Kinases ; Receptor, Epidermal Growth Factor ; Tyrosine