Analytical separation of plasmid DNA isoforms using anion exchanging chromatographic monoliths with 6 µm channels.

High-performance liquid chromatography (HPLC)-based analytical assays are used to effectively monitor purity and quantity of plasmid DNA (pDNA) throughout the purification process. However, the phenomenon of physical entrapment of open circular (OC) isoforms pDNA inside narrow channels of chromatographic support decreases its accuracy and precision and the effect increases with pDNA size. The purpose of the study was to develop a chromatographic method for accurate analytical separation between isoforms of <16 kbp pDNA using weak anion exchanging monolithic column with large (6 µm) convective channels. Purified samples of 4.7 and 15.4 kbp large pDNA with known isoform composition were prepared and their isoforms separated in ascending salt gradient. Both OC and supercoiled (SC) isoforms were baseline separated at a flow rate below 0.5 mL min-1 in a guanidinium chloride (GdnCl) gradient with a ≥95% OC pDNA elution recovery. However, these chromatographic conditions increased 2 times the peak width for linear (LIN) pDNA isoform compared to the results using monoliths with 1.4 µm channel size. If other chaotropic agents, such as urea or thiocyanate (SCN), were added to Gdn ions, the elution volume for LIN isoform decreased. Optimization of combined GdnCl/GdnSCN gradient for pDNA elution resulted in a simple and robust chromatographic method, where OC-LIN and LIN-SC pDNA (up to 15 kbp size) were separated with resolution above 1.0 and above 2.0, respectively. The accessibility and general acceptance of anion exchange chromatography for pDNA analytics give the newly developed method a great potential for in-process control monitoring of pDNA production processes.

In Silico Selection Approach to Develop DNA Aptamers for a Stem-like Cell Subpopulation of Non-small Lung Cancer Adenocarcinoma Cell Line A549.

BACKGROUND: Detection of circulating lung cancer cells with cancer-stem like characteristics would represent an improved tool for disease prognosis. However, current antibodies based methods have some disadvantages and therefore cell SELEX (Systematic Evolution of Ligands by Exponential Enrichment) was used to develop DNA aptamers, recognizing cell surface markers of non-small lung carcinoma (NSLC) cells. MATERIALS AND METHODS: The human adenocarcinoma cell line A549 was used for selection in seven cell SELEX cycles. We used human blood leukocytes for negative selection, and lung stem cell protein marker CD90 antibody binding A549 cells for positive selection. RESULTS: The obtained oligonucleotide sequences after the seventh SELEX cycle were subjected to in silico selection analysis based on three independent types of bioinformatics approaches, selecting two closely related aptamer candidates in terms of consensus sequences, structural motifs, binding affinity (Kd) and stability (ΔG). We selected and identified the aptamer A155_18 with very good binding characteristics to A459 cells, selected for CD90 antibody binding. The calculated phylogenetic tree showed that aptamers A155_18 and the known A549 cell aptamer S6 have a close structural relationship. MEME sequence analysis showed that they share two unique motifs, not present in other sequences. CONCLUSIONS: The novel aptamer A155_18 has strong binding affinity for A549 lung carcinoma cell line subpopulation that is expressing stem cell marker CD90, indicating a possible stemness, characteristic for the A459 line, or a subpopulation present within this cell line. This aptamer can be applied as diagnostic tool, identifying NSLC circulating cells.

Optimization of SELEX: comparison of different methods for monitoring the progress of in vitro selection of aptamers.

Oligonucleotide aptamers are selected from libraries typically comprising up to 10(15) different sequences by an iterative process of binding, separation, amplification and purification, called SELEX. During this process, the diversity of the oligonucleotide pool decreases until, presumably, only sequences with highest binding affinities towards chosen targets remain. This selection technique is time-consuming, labor-intensive and expensive. Though well posed in principles, the SELEX procedure is noise sensitive, due to amplification of unspecific-binding sequences, and it is not surprising that aptamer selection is often not successful in practice. In view of that, a follow-up of the progress of selection during its course with simple yet reliable methods is necessary. In this paper, we describe five independent assays to estimate the sequence complexity of SELEX pools including qualitative restriction fragment length polymorphism analysis, melting curve analysis, quantitative fluorescence intensity measurements of bound ssDNA, real time PCR quantification and pool dissociation constant analysis during the progress of aptamer selection against streptavidin. Properties and features of each method are discussed and compared. Pool dissociation constant analysis and sequencing serve as reference methods.

Quantitative cell wall protein profiling of invasive and non-invasive Saccharomyces cerevisiae strains.

A new, simple approach for the isolation and quantitative analysis of the cell wall (CW) proteins from invasively growing Saccharomyces cerevisiae strains is described in this contribution. The proposed method was proved compatible with agar-invasion assays and was demonstrated to be useful as a screening tool for rapid analysis of CW protein determinants related to yeast adhesion and invasion processes. CW protein isolation was performed enzymatically on viable cells by using mild, isosmotic reaction conditions and pure, proteinase free glucanase, thus avoiding destruction of cells and protein structures, which is a drawback of the existing methods based on hot SDS, DTT or NaOH treatment. Moreover, the method requires as low as 10mg of collected cell biomass for sufficient protein yield, which makes it suitable for the study of yeast invasion at the proteomic level. The extraction protocol was optimized for fast, direct analysis of multiple protein samples by SDS-PAGE, avoiding pre-concentration or purification steps, but still preserving high resolution of protein bands. The developed method was used to compare CW protein profile of i) invasive and non-invasive strains, ii) invasive and non-invasive morphological part of the colony and iii) cells cultivated at optimal and increased growth temperature. Results of quantitative SDS-PAGE analysis of S. cerevisiae CW proteins revealed the presence of up to 20 protein bands with molecular masses in the range 60-220 kDa. In addition, comparative analysis of CW protein profiles resulted in significant changes in the protein profile expression relevant to different cultivation temperature, cell morphology (invasive vs. non-invasive growth) and yeast strain.

Application of chromogenic reagents in surface plasmon resonance (SPR).

In this paper, a new simple approach for sensitivity optimization in surface plasmon resonance (SPR) chemosensors based on colorimetric ligands is presented. A new design of SPR sensor with tunable analytical wavelength (lambda(SPR)) was constructed for this purpose, to perform studies on the ligand absorbance spectra related sensitivity enhancement. Unlike commercial SPR sensors which operate at one lambda(SPR), the new device can be used for sensitivity analysis at selected lambda(SPR) in the range 550-750 nm, offering the possibility to identify the highest sensitivity lambda(SPR) in regard to the spectral changes of the selected ligand. Measurements can be easily done in ligand bulk solutions without immobilization steps. Sensitivity enhancement analysis and optimization of lambda(SPR) on chromogenic reagents with hypsochromic shift in their absorption spectra are demonstrated in this contribution. Optimal selection of analytical wavelength, set at the absorbance peak of chromogenic reagent Eriochrome Black T (EBT) was observed to result in up to two times increased SPR sensitivity to Cd(2+) compared to wavelengths selected in other parts of the ligand absorbance spectra, with a limit of detection (LOD) 0.2 ppm. The sensitivity enhancement at optimal lambda(SPR) was observed to be related to increased refractive index (n), drop in extinction coefficient (alpha) and simultaneous hypsochromic shift of the EBT absorbance spectra causing the lambda(SPR) to match the absorbance peak shoulder.