In-vitro Selektion von DNA-Molekülen für Schimmelpilz-Biosensoren

Schimmelpilze in Innenräumen können bei den Bewohnern verschiedene Gesundheitsprobleme auslösen, wie beispielsweise Mykosen, Mykotoxikosen und Allergien. Standardmethoden für die Quantifizierung und Identifizierung der Pilze sind oft zeitaufwändig und nur durch qualifiziertes Personal durchführbar. Deshalb entwickeln wir unter Nutzung des SELEX-Verfahrens ssDNA – Aptamere für die Sporen von Aspergillus- und Penicillium- Stämmen, die in Biosensoren für die Detektion von Schimmelpilzen in Innenräumen eingesetzt werden sollen. Beide Stämme gehören zu den häufig in Innenräumen auftretenden Schimmelpilzen, von denen bekannt ist, daß sie verschiedene Mykotoxine freisetzen. Die Gewinnung von Aptameren erfolgt unter Verwendung einer evolutionären Methode, dem SELEX-Verfahren (Systematic Evolution of Ligands by Exponential Enrichment). Dabei werden in einem Bindungsschritt aus einem Pool verschiedenster einzelsträngiger DNA-Moleküle (ss DNA) zunächst diejenigen gewonnen, die aufgrund ihrer individuellen Struktur bevorzugt an das Zielmolekül binden. Nach ihrer Ablösung von den Zielmolekülen (intakte Sporen) werden sie mittels PCR vervielfältigt. Dabei enstehen Doppelstränge, von denen die für die Bindungsreaktion an die Zielmoleküle überflüssigen Gegenstränge abgetrennt werden müssen. Dies geschieht über die Spaltung einer Ribose-Bindung im Gegenstrang, die durch modifizierter Primer eingeführt wurde. In dem auf diese Weise entstandenen Pool sind nun besser bindende ssDNA Moleküle angereichert, die für die Bindungsreaktion der nächsten Selektionsrunde zur Verfügung stehen. Dieser Prozeß wird so lange wiederholt, bis keine weitere Verbesserung der Affinität erfolgt. Die Affinität der auf diesem Wege hergestellten Aptamere wird unter Verwendung verschiedener Methoden getestet. Dazu gehören ein Standard-Filter-Bindungs-Test, eine fluoreszenz-mikroskopische Methode, die Messung der Fluoreszenzdepolarisation und die Anwendung der Resonant Mirror Spektroskopie

Refining the Results of a Classical SELEX Experiment by Expanding the Sequence Data Set of an Aptamer Pool Selected for Protein A

New, as yet undiscovered aptamers for Protein A were identified by applying next generation sequencing (NGS) to a previously selected aptamer pool. This pool was obtained in a classical SELEX (Systematic Evolution of Ligands by EXponential enrichment) experiment using the FluMag-SELEX procedure followed by cloning and Sanger sequencing. PA#2/8 was identified as the only Protein A-binding aptamer from the Sanger sequence pool, and was shown to be able to bind intact cells of Staphylococcus aureus. In this study, we show the extension of the SELEX results by re-sequencing of the same aptamer pool using a medium throughput NGS approach and data analysis. Both data pools were compared. They confirm the selection of a highly complex and heterogeneous oligonucleotide pool and show consistently a high content of orphans as well as a similar relative frequency of certain sequence groups. But in contrast to the Sanger data pool, the NGS pool was clearly dominated by one sequence group containing the known Protein A-binding aptamer PA#2/8 as the most frequent sequence in this group. In addition, we found two new sequence groups in the NGS pool represented by PA-C10 and PA-C8, respectively, which also have high specificity for Protein A. Comparative affinity studies reveal differences between the aptamers and confirm that PA#2/8 remains the most potent sequence within the selected aptamer pool reaching affinities in the low nanomolar range of KD = 20 ± 1 nM

Capture-SELEX: Selection of DNA Aptamers for Aminoglycoside Antibiotics

Small organic molecules are challenging targets for an aptamer selection using the SELEX technology (SELEX—Systematic Evolution of Ligans by EXponential enrichment). Often they are not suitable for immobilization on solid surfaces, which is a common procedure in known aptamer selection methods. The Capture-SELEX procedure allows the selection of DNA aptamers for solute targets. A special SELEX library was constructed with the aim to immobilize this library on magnetic beads or other surfaces. For this purpose a docking sequence was incorporated into the random region of the library enabling hybridization to a complementary oligo fixed on magnetic beads. Oligonucleotides of the library which exhibit high affinity to the target and a secondary structure fitting to the target are released from the beads for binding to the target during the aptamer selection process. The oligonucleotides of these binding complexes were amplified, purified, and immobilized via the docking sequence to the magnetic beads as the starting point of the following selection round. Based on this Capture-SELEX procedure, the successful DNA aptamer selection for the aminoglycoside antibiotic kanamycin A as a small molecule target is described

Protein Detection with Aptamer Biosensors

Aptamers have been developed for different applications. Their use as new biological recognition elements in biosensors promises progress for fast and easy detection of proteins. This new generation of biosensor (aptasensors) will be more stable and well adapted to the conditions of real samples because of the specific properties of aptamers

SPR interaction analyses concerning the immobilization orientation of aptamer PA#2/8.

<p>Biacore X100 / sensor chip CAP / ligand: 5’-biotinylated aptamer PA#2/8 (A) or 3'-biotinylated aptamer PA#2/8 (B) / analyte: recombinant Protein A with different concentrations (50–2500 nM, 1000 nM in duplicate). Double-referenced sensorgrams are shown (reference surface modified with unselected SELEX library, buffer injection). Black lines represent the fit to bivalent analyte binding model.</p

Most abundant aptamer sequences in the selected aptamer pool.

<p>Seven groups with 3–8 homologous sequences (#: number of homologous sequences) were identified among the sequenced aptamer clones. The representative aptamer clone of each group is shown. The specific primer binding sites at the 5’- and 3’-end of the aptamer clones are colored in red and blue, respectively.</p