Identifizierung und Charakterisierung von Metallchelat-bindenden Peptiden mittels Phage-Display

Rekombinante Proteine können verändert werden um Metallionen zu binden. Dieses Prinzip wird zur Aufreinigung mittels immobilisierter Metallchelat Chromatographie (IMAC) verwendet. Eine Fusion mit einer kurzen Sequenz von 6 Histidinresten zur Bindung von Übergangsmetallionen wie Cu2+, Ni2+, Co2+ and Zn2+ bewirkt dies. Eine Phagen-Bibliothek mit einer 15-mer Zufallssequenz wurde verwendet, um Peptide mit Affinität zu Übergangsmetallionen sowie harten Lewissäuren wie Fe3+ and Al3+ zu selektieren. Verschiedene Metallchelate sowie Stringenzen wurden bei der Selektion verwendet. Vier Selektionsrunden reichten zur Anreicherung für Metallchelat spezifischer Phagenpopulationen aus. Übergangsmetall bindende Peptide mit der größten Affinität hatten 4 Histidinreste, flankiert von hydrophoben Aminosäuren und Prolin. Die Spezifität für den Chelatbildner korrelierte mit der relativen Position des Bindungsmotifs im Peptid sowie der Hydrophobizität.Recombinant proteins can be engineered for affinity to metal ions. This principle is exploited for the purification by immobilised affinity chromatography (IMAC). Fusion of a short sequence of 6 histidine residues to a protein is sufficient for binding to transition metal ions like Cu2+, Ni2+, Co2+ and Zn2+. A phage-displayed random 15-mer library was used to select peptides binding to transition metal ions as well as to hard lewis acids such as Fe3+ and Al3+. Different formats for the immobilisation of metal ions and stringency of selection were employed. Four rounds of panning were sufficient to enrich a phage population with specific binding to the given metal chelates. Peptides binding to transition metals with highest affinity contained 4 consecutive histidine residues, flanked with hydrophobic amino acids and proline. Specificity for the chelating support material correlated with relative position of the binding motif in the sequence and hydrophobicity. As an example, a helper phage carrying a transition metal binding peptide was constructed and shown to purify by IMAC. Panning on Fe3+ yielded positively charged peptides. The best binder contained 4 lysine and two histidine residues and was shown to bind to Ni2+ in absence of imidazole. Binding to Fe3+- IDA complexes could be efficiently competed with either primary amines, phosphate or pH 8 and higher. This bispecific peptide may serve for a cascade purification on Ni2+ and Fe3+ columns, yielding higher homogeneity. Furthermore, affinity purification with iron abrogates the tedious removal of contaminating toxic heavy metal ions in traditional IMAC

Detektion des Kartoffelspindelknollen Viroids mit Hilfe der Loop Mediated Isothermal Amplification

PSTV ist ein hoch infektiöses Viroid, das in Kartoffeln verkleinerte und spindelähnliche Knollen verursacht. Um Ernteverlusten vorzubeugen, ist eine Detektion in frühen Infektionsstadien von großer Bedeutung. Aufgrund ihrer hohen Sensitivität und Spezifität wird die PCR als Standardnachweisverfahren für PSTV verwendet. Nachteilig an dieser Methode sind der apparative Aufwand und die zeitaufwändige Durchführung. Als viel versprechende Alternative konnte der Nachweis von PSTV mit Hilfe der Reverse Transcription Loop Mediated Isothermal Amplification (RT-LAMP) gezeigt werden. Hierbei handelt es sich um eine einfache und schnelle Methode, für die wenig aufwändige Laborausrüstung benötigt wird. Dabei ermöglichte eine an den Amplifikationsprozess gekoppelte Fluoreszenzreaktion die Detektion von Produkten direkt nach der Nachweisreaktion mit dem bloßen Auge (bzw. unter UV-Licht). Die Ergebnisse konnten mit Hilfe einer Real Time Detektion des auftretenden Fluoreszenzsignals bestätigt werden.PSTV is a highly infectious viroid which leads to small and spindle shaped tubers in potatoes. The detection at an early stage of infection is important to minimize loss of harvest. Normally PCR is used for the detection of PSTV, because of its high sensitivity and specificity. Disadvantages of this method are the requirement of sophisticated equipment and the time consuming process. As a promising alternative, the detection of PSTV with the reverse transcription loop mediated isothermal amplification (RT-LAMP) was shown. LAMP is a very fast and simple detection method requiring only standard laboratory equipment. A fluorescence reaction, coupled to the amplification process, allowed the detection of amplification products directly after the reaction with the naked eye (or under UV light, respectively). Real time detection of the occurring fluorescence signal was possible and confirmed the obtained results

A calibrated diversity assay for nucleic acid libraries using DiStRO—a Diversity Standard of Random Oligonucleotides

We have determined diversities exceeding 1012 different sequences in an annealing and melting assay using synthetic randomized oligonucleotides as a standard. For such high diversities, the annealing kinetics differ from those observed for low diversities, favouring the remelting curve after annealing as the best indicator of complexity. Direct comparisons of nucleic acid pools obtained from an aptamer selection demonstrate that even highly complex populations can be evaluated by using DiStRO, without the need of complicated calculations

Probing the SELEX Process with Next-Generation Sequencing

Background SELEX is an iterative process in which highly diverse synthetic nucleic acid libraries are selected over many rounds to finally identify aptamers with desired properties. However, little is understood as how binders are enriched during the selection course. Next-generation sequencing offers the opportunity to open the black box and observe a large part of the population dynamics during the selection process. Methodology We have performed a semi-automated SELEX procedure on the model target streptavidin starting with a synthetic DNA oligonucleotide library and compared results obtained by the conventional analysis via cloning and Sanger sequencing with next-generation sequencing. In order to follow the population dynamics during the selection, pools from all selection rounds were barcoded and sequenced in parallel. Conclusions High affinity aptamers can be readily identified simply by copy number enrichment in the first selection rounds. Based on our results, we suggest a new selection scheme that avoids a high number of iterative selection rounds while reducing time, PCR bias, and artifacts

Hinge-initiated Primer-dependent Amplification of Nucleic Acids (HIP) – A New Versatile Isothermal Amplification Method

The growing demand for cost-effective nucleic acid detection assays leads to an increasing number of different isothermal amplification reaction methods. However, all of the most efficient methods suffer from highly complex assay conditions due to the use of complicated primer sets and/or auxiliary enzymes. The present study describes the application of a new linker moiety that can be incorporated between a primer and a secondary target binding site which can act both as a block to polymerase extension as well as a hinge for refolding. This novel “hinge-primer” approach results in an efficient regeneration of the primer binding site and thus improves the strand-displacement and amplification process under isothermal conditions. Our investigations revealed that the reaction with forward and reverse hinge-primer including an abasic site is very efficient. The assay complexity can be reduced by combining the hinge-primer with a corresponding linear primer. Furthermore, the reaction speed can be increased by reducing the length of the amplified target sequence. We tested the sensitivity down to 104 copies and found a linear correlation between reaction time and input copy number. Our approach overcomes the usually cumbersome primer-design and extends the range of isothermal amplification methods using a polymerase with strand-displacement activity

Standardization and quality management in next-generation sequencing

DNA sequencing continues to evolve quickly even after >30 years. Many new platforms suddenly appeared and former established systems have vanished in almost the same manner. Since establishment of next-generation sequencing devices, this progress gains momentum due to the continually growing demand for higher throughput, lower costs and better quality of data. In consequence of this rapid development, standardized procedures and data formats as well as comprehensive quality management considerations are still scarce. Here, we listed and summarized current standardization efforts and quality management initiatives from companies, organizations and societies in form of published studies and ongoing projects. These comprise on the one hand quality documentation issues like technical notes, accreditation checklists and guidelines for validation of sequencing workflows. On the other hand, general standard proposals and quality metrics are developed and applied to the sequencing workflow steps with the main focus on upstream processes. Finally, certain standard developments for downstream pipeline data handling, processing and storage are discussed in brief. These standardization approaches represent a first basis for continuing work in order to prospectively implement next-generation sequencing in important areas such as clinical diagnostics, where reliable results and fast processing is crucial. Additionally, these efforts will exert a decisive influence on traceability and reproducibility of sequence data

High-throughput protein arrays: prospects for molecular diagnostics

High-throughput protein arrays allow the miniaturized and parallel analysis of large numbers of diagnostic markers in complex samples. Using automated colony picking and gridding, cDNA or antibody libraries can be expressed and screened as clone arrays. Protein microarrays are constructed from recombinantly expressed, purified, and yet functional proteins, entailing a range of optimized expression systems. Antibody microarrays are becoming a robust format for expression profiling of whole genomes. Alternative systems, such as aptamer, PROfusion™, nano- and microfluidic arrays are all at proof-of-concept stage. Differential protein profiles have been used as molecular diagnostics for cancer and autoimmune diseases and might ultimately be applied to screening of high-risk and general populations. Abstract 2 : High-throughput protein arrays are still largely experimental but have taken the first steps towards becoming diagnostic tools, which will eventually arrive at the doctor's practice and as over-the-counter devices

A Novel Optical Method To Reversibly Control Enzymatic Activity Based On Photoacids

Most biochemical reactions depend on the pH value of the aqueous environment and some are strongly favoured to occur in an acidic environment. A non-invasive control of pH to tightly regulate such reactions with defined start and end points is a highly desirable feature in certain applications, but has proven difficult to achieve so far. We report a novel optical approach to reversibly control a typical biochemical reaction by changing the pH and using acid phosphatase as a model enzyme. The reversible photoacid G-acid functions as a proton donor, changing the pH rapidly and reversibly by using high power UV LEDs as an illumination source in our experimental setup. The reaction can be tightly controlled by simply switching the light on and off and should be applicable to a wide range of other enzymatic reactions, thus enabling miniaturization and parallelization through non-invasive optical means