Zip Nucleic Acids: new high affinity oligonucleotides as potent primers for PCR and reverse transcription

Most nucleic acid-based technologies rely upon sequence recognition between an oligonucleotide and its nucleic acid target. With the aim of improving hybridization by decreasing electrostatic repulsions between the negatively charged strands, novel modified oligonucleotides named Zip nucleic acids (ZNAs) were recently developed. ZNAs are oligonucleotide–oligocation conjugates whose global charge is modulated by the number of cationic spermine moieties grafted on the oligonucleotide. It was demonstrated that the melting temperature of a hybridized ZNA is easily predictable and increases linearly with the length of the oligocation. Furthermore, ZNAs retain the ability to discriminate between a perfect match and a single base-pair-mismatched complementary sequence. Using quantitative PCR, we show here that ZNAs are specific and efficient primers displaying an outstanding affinity toward their genomic target. ZNAs are particularly efficient at low magnesium concentration, low primer concentrations and high annealing temperatures, allowing to improve the amplification in AT-rich sequences and potentially multiplex PCR applications. In reverse transcription experiments, ZNA gene-specific primers improve the yield of cDNA synthesis, thus increasing the accuracy of detection, especially for genes expressed at low levels. Our data suggest that ZNAs exhibit faster binding kinetics than standard and locked nucleic acid-containing primers, which could explain why their target recognition is better for rare targets

Proximity Dependent Ligation Selection: A New Approach to Generating DNA Aptamers

Nucleic acid aptamers are a group of molecules which emerge from large random sequence pools through in vitro selection, a process called Systematic Evolution of Ligands by EXponential enrichment (SELEX). These recognition molecules are a potential alternative to antibodies and offer a greater thermal stability, robustness and chemical versatility. The first part of this thesis describes the selection of an aptamer against acetylcholinesterase (AChE) using conventional in vitro selection process. The pool enrichment was observed during 15 rounds of selection and a chosen aptamer, R15/19, was characterized. The R15/19 binds with dissociation constant of 55±8pM but does not inhibit the enzyme. This aptamer can be used as a part of immobilization mediator. However, this manual in vitro selection procedure is time consuming as it needs multiple rounds of selection. Therefore, a novel aptamer selection method has been developed that is aimed at shortening selection times. Taking an advantage of multiple interactions, it is possible to generate a single sequence that contains two binding sites through a single selection process. The approach is adopted from proximity dependent DNA ligation assay (PLA) and called ‘proximity ligation selection’ (PLS). Instead of using one random pool for selection, with PLS, we use two. These two starting pools were designed to contain different additional sequences, apart from the primer and random regions, at either the 5’ or the 3’ end. The basis of this method is only in the presence of target protein molecules, is there a close proximity of pairs of bound sequences. And only if those pairs are in an appropriate orientation in which the 3’ hydroxyl terminal of one sequence is sufficiently close to the 5’-phosphate terminal of the other sequence, are the extended free ends of each member of the proximity oligonucleotide pairs were brought closely by base-pairing to subsequently added connector oligonucleotide. The hybridized complexes are then joined using a DNA ligase, allowing the formation of a ligated product that contains a dual binding site on the target. Thrombin and lysozyme were used as model targets. For thrombin, none of the PLS products showed a G-quadruplex signature in CD spectroscopy. Using the SPR, the Kd of the aptamer dimer 2/19-5 (from F and R pool) and 4/19-7 (from TBA29plus and R pool) are 1.6M and 1.2M, respectively. The ELONA test was used to measure a Kd for each monomer and there was no binding observed from each monomer of the aptamer against thrombin. In the case of lysozyme, the SPR and ELONA revealed the Kd of the aptamer dimer (from two starting pools) of 8.7M and 9nM, respectively. The Kd of each constitute of anti-lysozyme aptamer dimer using ELONA showed the Kd of 35.5 and 51.5nM. The Kd obtained from SPR was high, suggesting a low binding affinity. We assume that the aptamer molecule can freely fold into a favored structure once it meets the target and thus can perform binding better than in the SPR. Although we did not succeed in generating aptamer dimers that had higher affinity than monomers, there are several steps in the procedure that need to be further improved.Open Acces

A novel substitution 1381V in the sterol 14alpha-demethylase (CYP51) of Mycosphaerella graminicola is differentially selected by azole fungicides

The recent reduction in the efficacy of azole fungicides in controlling Septoria leaf blotch of wheat, caused by Mycosphaerella graminicola, has prompted concerns over possible development of resistance, particularly in light of the recent emergence of widespread resistance to quinone outside inhibitors (QoIs). We have recently implicated alterations in the target-encoding sterol 14 alpha-demethylase protein (CYP51), and over-expression of genes encoding efflux pumps, in reducing sensitivity to the azole class of sterol demethylation inhibitors (DMIs) in M. graminicola. Here we report on the prevalence and selection of two CYP51 alterations, substitution I381V and deletion of codons 459 and 460 (Delta Y459/G460), in populations of M. graminicola. Neither alteration has previously been identified in human or plant pathogenic fungi resistant to azoles. The presence of Delta Y459/G460 showed a continuous distribution of EC50 values across isolates with either I381 or V381, and had no measurable effect on azole sensitivity. Data linking fungicide sensitivity with the presence of I381V in M. graminicola show for the first time that a particular CYP51 alteration is differentially selected by different azoles in field populations of a plant pathogen. Substitution I381V although not an absolute requirement for reduced azole sensitivity, is selected by tebuconazole and difenoconazole treatment, suggesting an adaptive advantage in the presence of these two compounds. Prochloraz treatments appeared to select negatively for I381V, whereas other azole treatments did not or only weakly impacted on the prevalence of this substitution. These findings suggest treatments with different members of the azole class of fungicides could offer a resistance management strategy

High-Throughput Engineering and Analysis of Class II Mhc/Peptide Binding by Yeast Co-Display

Polymorphisms of major histocompatibility complex (MHC) and molecular mechanisms of their antigen-presenting specificity and promiscuity have great impact on T cell-mediated immune responses and related diseases. Challenges in elucidating the characteristics of antigenic peptide binding by MHC motivate the development of high throughput experimental tools to quantitatively analyze interactions between hundreds of MHC allelic proteins and various peptide sequences. We demonstrated such a method by co-displaying target peptides and class II MHC (MHC-II) on the yeast surface in an intracellular association-dependent manner. The optimized yeast co-display system enabled quantitative mapping of side-chain preferences and general motifs for peptides binding to MHC-II by site-directed mutagenesis or peptide library screening, and also allowed rapid tailoring of MHC-II peptide binding specificity by directed evolution approaches, which derived MHC-II allelic mutants with altered peptide binding specificity or hyper-promiscuity. Comparison of these experimentally engineered mutants with naturally discovered MHC-II proteins recovered valuable information about structure-function relationship in the evolutionary mechanisms for polymorphic MHC-II molecules, which could direct future immunotherapeutic innovation

Association of AURKA polymorphisms with prostate cancer risk

Die vorliegende Diplomarbeit ist ein Teil des Projekts „Molekulare Epidemiologie von Prostatakrebs“, einer Zusammenarbeit der Abteilung für angewandte und experimentelle Onkologie des Instituts für Krebsforschung sowie der urologischen Abteilungen der Medizinischen Universität Wien, des Sozialmedizinischen Zentrums Süd und des Sozialmedizinischen Zentrums Ost. Ziel dieser Studie ist es, ein polygenetisches Modell zu entwickeln, um Hochrisikopatienten identifizieren zu können und neue Perspektiven in der Prävention und der Therapie des Prostatakrebses zu eröffnen. Prostatakrebs stellt die häufigste bösartige Neubildung bei Männern in Österreich dar. In dieser Diplomarbeit wurden vier Single Nucleotide Polymorphisms (SNPs), welche im AURKA-Gen lokalisiert sind, genotypisiert. Das Gen für AURKA (Aurora Kinase A), eine Serin/Threonin-Proteinkinase, befindet sich am Chromosom 20q13, einem bekannten Prostatakrebs-Suszeptibilitätslokus. AURKA ist an einigen ausschlaggebenden Vorgängen während der Mitose beteiligt. Genetische Polymorphismen im AURKA-Gen tragen zu interindividuellen Unterschieden in der Chromosomenstabilität bei und beeinflussen dadurch das Prostatakrebsrisiko. Die Studienpopulation dieser fortlaufenden Studie besteht aus 1027 Prostatakrebsfällen und 550 Kontrollpatienten mit benigner Prostatahypoplasie (BPH). Vier ausgewählte tagging SNPs (rs2180691, rs8117896, rs1468055, rs1476394) wurden mit Hilfe des T5’ Nuclease TaqMan MGB Assay bestimmt. Zusammenfassend wurde kein signifikanter Zusammenhang zwischen den untersuchten SNPs und dem Prostatakrebsrisiko gefunden. Jedoch zeigte eine Haplotypenanalyse ein reduziertes Prostatakrebsrisiko (OR: 0,7021; 95% CI: 0,49599-0,99384) für Männer unter 64 Lebensjahren mit einem GCAC Haplotyp. Weitere Untersuchungen in größeren Studienpopulationen sind nötig, um den Einfluss von Polymorphismen im AURKA-Gen auf das Prostatakrebsrisiko festzustellen.The present diploma thesis is part of the project “Molecular Epidemiology of Prostate Cancer”, a collaboration of the Department of Applied and Experimental Oncology of the Institute of Cancer Research and the Departments of Urology of the Medical University Vienna, Sozialmedizinisches Zentrum Süd and Sozialmedizinisches Zentrum Ost. Aim of this study is to create a polygenetic model to identify high-risk patients and to open new perspectives in prevention and therapy of prostate cancer. Prostate cancer is the most common malign neoplasm in men in Austria. In this diploma thesis four single nucleotide polymorphisms (SNPs) within the AURKA gene were genotyped. AURKA (Aurora Kinase A), a serin/threonin protein kinase, is located on chromosome 20q13, a known prostate cancer susceptibility locus, and is involved in some crucial events during mitosis. Genetic polymorphisms in AURKA gene may contribute to interindividual differences in chromosomal stability and therefore influence the risk of prostate cancer. The study population of this ongoing study consists of 1027 prostate cancer cases and of 550 benign prostatic hyperplasia (BPH) controls. Four selected tagging SNPs (rs2180691, rs8117896, rs1468055, rs1476394) were determined using -T5’ nuclease TaqMan MGB Assay. Overall, no significant association could be found between the investigated SNPs and prostate cancer risk. However, a haplotype analysis revealed a reduced prostate cancer risk (OR: 0.7021; 95% CI: 0.49599-0.99384) for men below 64 years of age with the GCAC haplotype. Further investigations in larger study populations are required to assess the influence of polymorphisms in the AURKA gene on prostate cancer risk

Fine mapping of susceptibility loci to malaria clinical episodes in a family-based cohort from Senegal

O parasita da malária, P. falciparum, mata na ordem de um milhão de crianças Africanas em cada ano, e esta é uma pequena fracção do número de pessoas infectadas em todo o mundo. A evolução clínica de uma infecção por este parasita depende em certa medida, da constituição genética do indivíduo infectado. O papel dos factores genéticos que regulam a gravidade da infecção da malária tem sido repetidamente demonstrado em humanos e animais. Os estudos de associação são realizados com o objectivo de identificar os genes implicados na causalidade do resultado da infecção. Foi detectado anteriormente, linkage no cromossoma humano 5p15 ao número de ataques de Plasmodium falciparum (PFA) em Dielmo, uma aldeia senegalesa [48]. Posteriormente, e antes deste estudo, um levantamento usando um ensaio "GoldenGate" da Illumina, com cerca de 1.450 SNPs foi realizada na região de Linkage com o fenótipo PFA. A análise foi realizada com três programas estatísticos baseados na família: Merlin, QTDT e FBAT/PBAT. Estes programas identificaram três genes candidatos associados com o fenótipo PFA: três SNPs (rs4867417, rs7714218 e rs11959398), localizados no gene PDZD2, um SNP (rs11134099) no gene ADAMTS16, e outro (rs3777320) localizado no gene SEMA5A. O objectivo deste estudo foi investigar estas associações. Os SNPs das regiões destes genes candidatos foram escolhidos por sequenciação de exões situados na região candidata ou por análise bioinformática utilizando dados do HapMap da população Yoruba. O estudado para genotipagem foi através das análises de pré-design ou “Custom” dos SNPs (Applied Biosystems). Os dados foram incluídos num banco de dados e a verificação dos erros de transmissão mendeliana foi efectuada. As análises estatísticas foram realizadas utilizando dois programas de associação familiar, PBAT e QTDT. Foram utilizados diferentes modelos de transmissão de alelos e foi definido como limite de significância p-value = 10-3. As análises de SNPs dos genes PDZD2 e ADAMTS16 não confirmaram a associação, mas encontrou-se associação significativa com SNPs do gene SEMA5A. Um SNP (rs3777325) foi significativamente associado com o fenótipo PFA usando ambos os programas (p-value= - 6.49x10-4 usando o programa PBAT e p-value = 2.0x10-4 usando o programa QTDT). A análise de haplótipos de dois SNPs adjacentes (rs4541632 e rs1018956), também mostrou uma associação significativa do haplótipo GC (p-value= -6.82x10-5) utilizando o programa PBAT. Este estudo confirma que o locus de susceptibilidade para o fenótipo PFA está localizado no gene SEMA5A. Mais estudos serão necessários para replicar essa associação e identificar o polimorfismo causal.The malaria parasite, P. falciparum, kills on the order of a million African children each year, and this is a small fraction of the number of infected individuals world-wide. The clinical outcome of an infection by this parasite depends to some extent on the genetic makeup of the infected individual. The role of genetic factors that regulate the severity of malaria infection has been repeatedly demonstrated in humans and animals. Association studies are conducted with the aim of identifying the causal genes implicated in the outcome of infection. Linkage was previously detected on human chromosome 5p15 controlling the number of Plasmodium falciparum attacks (PFA) in Dielmo, a Senegalese village [48]. Subsequently, and prior to this present study, a fine mapping study using a "GoldenGate assay” from Illumina, with about 1450 SNPs was performed in this region of linkage with PFA phenotype. Analysis was performed with three statistical family-based programs: Merlin, QTDT, and FBAT/PBAT. These programs identified three candidate genes associated with PFA phenotype: three SNPs (rs4867417, rs7714218, and rs11959398) located in PDZD2, one SNP (rs11134099) in ADAMTS16, and one (rs3777320) in SEMA5A. The aim of this present study was to investigate these associations. Novel SNPs in the candidate regions of these genes were selected either by sequencing exons located in these candidate regions or by bioinformatics analysis using HapMap data from Yoruba population. SNPs were studied using either Pre-design or Custom SNP genotyping assay (Applied Biosystems). Data were included in an Access Database and checked for error of Mendelian transmission. Statistical analyses were performed using two family-based association programs, PBAT and QTDT. We used different models of allele transmission and defined p=10-3 as significance threshold. The analyses did not confirm the association with SNPs of PDZD2 or ADAMTS16, but did find significant association with SNPs of SEMA5A. One SNP (rs3777325) was significantly associated with PFA phenotype using both programs (p-value= -6.49x10-4 using the PBAT program and p-value=2.0x10-4 using the QTDT program). A haplotype analysis of two adjacent SNPs (rs4541632 and rs1018956) also showed a significant association of the haplotype GC (p-value= -6.82x10-5) using the PBAT program. This work confirms that a susceptibility locus to PFA phenotype is located inside SEMA5A. Further studies will be necessary to replicate this association and identify the causal polymorphism

The evaluation and interpretation of controls used in three commercially available quantification kits for forensic DNA analysis

This research presented an idea to aid crime laboratories in the selection of the best quantification method for use in DNA analysis in their laboratory. The goal of this research was to evaluate the suitability of using an external reference standard, the NIST SRM 2372 Human Quantitation Standard, to increase the quality of quantification results of human DNA analysis in commercially available kits. Additionally, the DNA standard included with the commercial kit, used for developing a standard curve, was evaluated for accuracy and stability beyond the recommended time frame. The commercial quantification kits tested were: QuantifilerRTM Trio DNA Quantification Kit (Applied Biosystems(TM), Life Technologies), PlexorRTM HY System (Promega Corporation), and InvestigatorRTM Quantiplex HYres (Qiagen). It was found that generally the concentration of the NIST SRM 2372 was not accurately quantifying in all three quantification kits. Furthermore, it was found that each standard curve dilution series varied in accuracy, as well as stability

Molecular diagnosis of medical viruses.

The diagnosis of infectious diseases has been revolutionized by the development of molecular techniques, foremost with the applications of the polymerase chain reaction (PCR). The achievable high sensitivity and ease with which the method can be used to detect any known genetic sequence have led to its wide application in the life sciences. More recently, real-time PCR assays have provided additional major contributions, with the inclusion of an additional fluorescent probe detection system resulting in an increase in sensitivity over conventional PCR, the ability to confirm the amplification product and to quantitate the target concentration. Further, nucleotide sequence analysis of the amplification products has facilitated epidemiological studies of infectious disease outbreaks, and the monitoring of treatment outcomes for infections, in particular with viruses which mutate at high frequency. This review discusses the applications of qualitative and quantitative real-time PCR, nested PCR, multiplex PCR, nucleotide sequence analysis of amplified products and quality assurance with nucleic acid testing (NAT) in diagnostic laboratories

Study of genetic variants in chromosome 5p15.33 region in non-smoker lung cancer patients

Introduction: Genome-wide association studies have identified that genetic polymorphisms in the telomerase reverse transcrip-tase (TERT) and cleft lip and palate transmembrane 1-like (CLPTM1L) genes may play important roles in the development of lung cancer in never smokers.Material and methods: This study was aiming to evaluate the associations between the risk of lung cancer in never smokers and single nucleotide polymorphisms in these genes by Real-Time Taqman assay, in forty lung cancer patients and forty apparently healthy age-matched controls selected from the chest department, Kasr Al-Ainy hospital from June 2018 to January 2019. Results: Adenocarcinoma was the most common histopathological subtype of lung cancer in the study patients. Also, the prevalence of females having adenocarcinoma was more common than males. The heterozygous form of the CLPTM1L occurred more frequently in the subjects aged above 46 years (P=0.019). There was a significant association between (rs 2730100) (c. 1574-3777C>A) TERT and CLPTM1L (rs 451360) (c.1532+ 1051C>A) genotypes and the incidence of lung cancer in never smokers, especially adenocarcinoma, a subtype of non-small cell lung carcinoma (NSCLC).Conclusions: Polymorphism in the telomerase reverse transcriptase (TERT) and cleft lip and palate transmembrane 1 like (CLPT-M1L) genes may play an important role in the development of NSCLC, especially adenocarcinoma subtype. The two genes are located in the chromosome 5p15.33