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To discover DNA ligands against a predetermined receptor protein complex, we introduce a comprehensive version of ligand-guided selection (LIGS). LIGS is, itself, a variant of systematic evolution of ligands by exponential enrichment (SELEX). Herein, we have optimized LIGS to identify higher affinity aptamers with high specificity. In addition, we demonstrate the expandability of LIGS 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 obtained through Illumina high-throughput (HT) DNA sequencing 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 in this report 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.

We recently introduced a screening technology termed ligand-guided selection, (LIGS), to selectively identify target-specific aptamers from an evolved cell-SELEX library. Cell-SELEX utilizes a large combinatorial single-stranded oligonucleotide library and progressively selects DNA ligands against whole cells with variable DNA-binding affinities and specificities by repeated rounds of partition and amplification. LIGS exploits the partition step and introduces a secondary, pre-existing high-affinity monoclonal antibody (mAb) ligand to outcompete and elute specific aptamers towards the binding target of the antibody, not the cell. Here, using 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. In sum, we 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.

Background:The present study has highlighted the effects of seasonal variation and socio-economic status on generation and composition of municipal solid waste (MSW) in Shalimar Town (ST). The total amount of MSW generated in ST is estimated to be 927 tons per day per year. The average per capita rate of MSW collected in ST is 0.69 kg per day in all four seasons.Results: No significant difference was found in overall waste generation; however, statistical analyses show the significant difference for food waste, paper and plastic (p < 0.01) among socio-economic groups and seasons. The results show that the lowest income group produces 0.39 kg per capita per day during winter months which is the minimum of MSW generated as compared to high (1.1 kg per capita per day) and middle (0.56 kg per capita per day) income groups in the same season. Conclusions:It is also concluded that the low-income group produces the minimum of waste in each of the four seasons. In terms of breakdown of the MSW, organic waste is in the highest percentage (81%) followed by paper (5%), plastic (6%), glass (2%) and others (5%). Food waste is 84% of the entire generated MSW as well as having very low heating value of 5,642 J per g. Elemental and proximate analyses of mixed food waste had carbon 48.72%, nitrogen 2.41%, hydrogen 6.37%, sulphur 0.29 and oxygen 40.15% respectively. © 2015 Kamran et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

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