Development of Ligand Guided Selection (LIGS) to Identify Specific DNA Aptamers Against Cell Surface Proteins

Oligonucleotide aptamers (nucleic acid-based affinity reagents) are an emerging class of synthetic molecules that display high affinity and specificity towards their targets. Aptamer molecules for a target of interest are obtained using a combinatorial chemistry-based method termed systematic evolution of ligands by exponential enrichment (SELEX). SELEX is an in vitro selection process in which a random oligonucleotide library is subjected to repeated cycles of target incubation, separation, and amplification until target-specific evolved sequences become prevalent in the library. Typically, SELEX is used against target molecules such as small molecules and proteins, in their purified state. However, aptamers selected against purified cell surface proteins show limited scope, due to aptamer’s inability to recognize the cell surface protein expressed in its natural environment. To address this issue, a modified SELEX method, named cell-SELEX, was developed, which uses the whole live cells as targets. Cell-SELEX enabled the identification of aptamers against cell surface proteins in their native environment without the need for target purification. A major limitation of cell-SELEX, however, is that the targeting epitope on the cell surface cannot be predefined. In this dissertation, we developed a novel variant of the SELEX method named ligand-guided selection (LIGS) to successfully identify specific aptamers against predetermined cell surface proteins in their native, functional state. This method is designed to uniquely exploit the selection step, which is the core of the SELEX process. In chapter 2, the LIGS method is introduced as a new biochemical-screening platform that employs the binding of naturally occurring, stronger and highly specific secondary molecular entity to its target as a partition step, to identify highly specific artificial nucleic acid ligands. Here, we used an antibody (Ab) that binds to the membrane-bound Immunoglobulin M (mIgM) to selectively elute aptamers that are specific for mIgM from a SELEX pool that is partially enriched toward mIgM expressing Ramos cells. Three aptamers that were selected showed high specificity toward Ramos cells. Furthermore, our results show that the aptamers identified by LIGS could be outcompeted by mIgM Ab, demonstrating that LIGS can be successfully applied to select aptamers from a partially evolved cell-SELEX library, against predetermined receptor proteins using a cognate ligand. In Chapter 3, we explored the applicability of LIGS towards a multi-domain receptor complex, using an anti-CD3ε mAb against the cluster of differentiation 3 (CD3ε), as the guiding ligand against one of the domains of the T-cell Receptor (TCR) complex expressed on Jurkat.E6 cells. We discovered three specific aptamers against TCR complex expressed on an immortalized line of human T lymphocyte cells. These findings demonstrate that specific aptamers can be identified utilizing an antibody against a single domain of a multidomain protein complex in their endogenous state with neither post- nor pre-SELEX protein manipulation. Chapter 4 outlines, the systematic truncation of an aptamer named R1, which was selected against mIgM in the study concluded in chapter one, to design shorter variants with enhanced affinity. Importantly, herein, we succeeded to show that the specificity of the most optimized variant of R1 aptamer (R1.2) is similar to that of the anti-IgM antibody, indicating that the specificity of the ligand utilized in selective elution of the aptamer determines the specificity of the LIGS-generated aptamer. Furthermore, the results demonstrated that truncated variants of R1 are able to recognize mIgM-positive human B lymphoma BJAB cells at physiological temperature indicating that low-affinity aptamers generated in LIGS could be enhanced by post-SELEX modifications without compromising their specificity. Since the aptamers obtained from initial LIGS experiments possessed only moderate binding affinities to their target receptors, we developed a comprehensive version of ligand-guided selection (LIGS), by optimizing LIGS to identify higher affinity aptamers with high specificity. In addition, we expanded the LIGS method by performing specific aptamer elution at 25 °C, utilizing multiple monoclonal antibodies (mAbs) against cultured cells and primary cells obtained from human donors expressing the same receptor. Eluted LIGS libraries were subjected to Illumina high-throughput (HT) DNA sequencing and were analyzed by bioinformatics tools to discover five DNA aptamers with apparent affinities ranging from 3.06 ± 0.485 nM to 325 ± 62.7 nM against the target, T-cell receptor-cluster of differentiation epsilon (TCR-CD3ε) expressed on human T-cells. The specificity of the aptamers was validated utilizing multiple strategies, including competitive binding analysis and a double-knockout Jurkat cell line generated by CRISPR technology. The cross-competition experiments using labeled and unlabeled aptamers revealed that all five aptamers compete for the same binding site. Collectively, the data presented in Chapter 5, introduce a modified LIGS strategy as a universal platform to identify highly specific multiple aptamers toward multi-component receptor proteins in their native state without changing the cell-surface landscape. These aptamers can be used to develop therapeutic agents, especially for cancer immunotherapy

DIFFERENCE OF SURFACE DERMATOFAGOID ALLERGEN CONCENTRATION ON 0-2 YEARS AGE GROUP AND BABY AND CHILDREN CLOTHES MANUFACTURED FROM VARIOUS FABRICS

WOS: 000279625200013Allergic illness based on home dust affects not only the life style of the adults as well as the life quality of the children. Mites are found in all kinds of home textile products and clothing. The main reason that triggers the asthma illness in babies and children is these bugs called mites which cannot be seen with naked eye. In this study, the factors that cause allergic effects due to textile clothing are aimed to be investigated in the most used baby underwear. With this purpose, underwears produced from six different kinds of knitted fabrics worn to each child for three days were investigated. Under the light of this study, the most appropriate fabric types were determined in order to prevent asthma in children based on mites

Designing a jacket for motorcycle drivers by combination of leather and denim

In the wake of the development of apparel sector and the increase in consumer demands, designing clothes aimed at their intended use has gained importance. Visual and aesthetic designs in line with fashion trends, as well as clothes that can meet technical requirements are used in numerous fields and there are many studies carried out on developing these technical textiles. Many clothes, examples of which could be given as those designed for firemen, swimmers and dancers, should have distinctive properties depending on their field of use and intended use, and should provide the user with protection and convenience in occupation. For motorcycle clothes, leather is usually preferred as material, however leather is an expensive material. In this study, materials of denim fabric and leather have been investigated in terms of cost and air permeability. Designing a reasonable motorcycle driver jacket is aimed. Thus, a leather-denim combination jacket has been designed, consisted of front and shoulders parts which are mostly exposed to wind, made up of leather material and other parts made up of denim fabric. The cost of this jacket has been assessed, comparing it to that of a full leather jacket, and a new product has been developed that could perform a similar function at a much cheaper price.&nbsp

The role of G-quadruplex structures of LIGS-generated aptamers R1.2 and R1.3 in IgM specific recognition

Exploiting a variant of SELEX called "Ligand-Guided Selection" (LI-GS), we recently identified two novel truncated G-rich aptamers, called R1.2 and R1.3, specific for membrane-bound IgM (mIgM), the hallmark of B cells. Herein, the conformational behaviour of these aptamers has been analysed by multiple biophysical methods. In order to investigate their functional secondary structures, these studies have been carried out in pseudo-physiological buffers mimicking different cellular environments. Both aptamers proved to be highly polymorphic, folding into stable, unimolecular G-quadruplex structures in K+-rich buffers. In turn, in buffered solutions containing Na+/Mg2+ ions, R1.2 and R1.3 formed mainly duplex structures. Remarkably, these aptamers were able to effectively bind mIgM on B-cell lymphoma exclusively in the presence of potassium ions. These findings demonstrate the key role of G-quadruplex folding in the molecular recognition and efficient binding of R1.2 and R1.3 to mIgM expressed in lymphoma and leukemia cells, providing a precious rational basis for the design of effective aptamer-based biosensors potentially useful for the detection of cancer-relevant biomarkers

Effects of mean platelet volume on survival of the patients undergoing below-knee bypass

Background: This study aims to investigate the relationship of the mean platelet volume (MPV) values, risk factors, treatment protocols with the mortality in patients undergoing below-knee bypass

A new approach to liquid penetrant inspection: radiolabeled QDots

WOS: 000406490600002Penetrant technique is a sensitive non-destructive testing (NDT) method for detecting and locating the presence of cracks in sample surface. Today, NDT is used in a wide range of industries including aerospace, biotechnology, defence, marine, oil-gas and energy plants. This work focuses on potential use of radiolabeled ZnS coated CdTe quantum dots (QDots) as a penetrant for liquid penetrant testing. The synthesized QDots as a precursor were tested for surface defects detection in welded joints. The experimental results show the highest activities were found in defects on the sample surface. These finding are consistent with the sample NDT inspection test report

Investigation of therapeutic efficiency of phenytoin (PHT) labeled with radioactive I-131 in the cancer cell lines

WOS: 000372264900019The aim of this study is to determine the incorporations of PHT radiolabeled with I-131 (I-131-PHT) on U-87 MG, Daoy and A549 cancerous cell lines. For this, cold and radio-labeling studies were carried out. The radio-labeling yield of I-131-PHT was obtained about 95 %. Subsequently, cell culture studies were carried out and radio-labeling yields of I-131, I-131-PHT on U-87 MG, Daoy and A549 cancerous cells were investigated. Cell culture studies demonstrated that the incorporation values of (IPHT)-I-131 on the three cell lines decreased with increasing radioactivity. Consequently, I-131-PHT may be a good radiopharmaceutical for targeting radionuclide therapy of Central Nervous System Tumors.Celal Bayar University Research FundCelal Bayar University [2012-089]The authors are thankful for the financial support from the Celal Bayar University Research Fund (Contract No. 2012-089)