Novel protein capture and detection reagents are required to understand comprehensively the interplay of the proteome in basic biological processes and in human health and disease. These reagents need to have high affinity and specificity for particular protein targets, and they need to be easily created and produced, and be amenable to modification and immobilization for high throughput analysis of proteins. Antibodies, the most commonly used protein capture reagents, have high affinity and specificity;however, they are difficult to mass produce and to implement in high throughput protein capture/detection assays due to their protein nature. Single-stranded oligonucleotides (aptamers) have emerged as alternative protein capture reagents. Aptamers that specifically bind to a target protein can be selected from large random-sequence oligonucleotide pools containing as many as 1013-1015 individual molecules by an iterative in vitro process called SELEX. The overarching hypothesis behind this project is that, the SELEX process can be automated and multiplexed to enable simultaneous selection of aptamers to many proteins, and the selected aptamers can be employed in high throughput assays that allow analysis of the target proteins in biological and medical samples. To this end, over 100 target proteins have been chosen as the initial target protein set to test the protocols with proteins having different biochemical properties and subcellular localizations, as wells as with different splicing variants and post-translational modifications. Additionally, this set represents a spectrum of medically relevant proteins. Two complementary SELEX strategies, where a microfluidic device that holds proteins in microarrayed liquid glass (sol-gel) droplets or intact yeast cells that display expressed human proteins on their surface, will be utilized in aptamer selections. Pools of selected aptamers will be sequenced using a massively parallel sequencing technology and templates of individual aptamers will be cloned after synthesis. After validation of non-competitive binding of a pair of aptamer to individual target proteins, these aptamer pairs will be utilized in high throughput sandwich assays. These assays will be tested and optimized earlier in the project using existing protein-specific aptamers and fusions of their target proteins. Finally, these new protein capture/detection reagents and assays will be compared to other reagents and assays, such as antibodies in ELISA. This project is expected to have a major impact on both basic life sciences research and medical research. The technological development will facilitate the selection of aptamers to other biologically and medically important proteins and the selected aptamers and the assays developed with them may have immediate applications in molecular therapeutics and disease diagnosis.
. This project seeks to transform an existing technology to facilitate and streamline identification of novel capture reagents for human proteins and to incorporate these new reagents in assays that can accommodate simultaneous analysis of many proteins. This project will not only develop new technology and assays that enable analysis of human proteins critical in human health and disease, but also generate the novel reagents that can be used for therapy.
|Meng, Hsien-Wei; Pagano, John M; White, Brian S et al. (2014) Discovering aptamers by cell-SELEX against human soluble growth factors ectopically expressed on yeast cell surface. PLoS One 9:e93052|
|Tome, Jacob M; Ozer, Abdullah; Pagano, John M et al. (2014) Comprehensive analysis of RNA-protein interactions by high-throughput sequencing-RNA affinity profiling. Nat Methods 11:683-8|
|Szeto, Kylan; Reinholt, Sarah J; Duarte, Fabiana M et al. (2014) High-throughput binding characterization of RNA aptamer selections using a microplate-based multiplex microcolumn device. Anal Bioanal Chem 406:2727-32|
|Pagano, John M; Kwak, Hojoong; Waters, Colin T et al. (2014) Defining NELF-E RNA binding in HIV-1 and promoter-proximal pause regions. PLoS Genet 10:e1004090|
|Byrnes, Laura J; Singh, Avtar; Szeto, Kylan et al. (2013) Structural basis for conformational switching and GTP loading of the large G protein atlastin. EMBO J 32:369-84|
|Ozer, Abdullah; White, Brian S; Lis, John T et al. (2013) Density-dependent cooperative non-specific binding in solid-phase SELEX affinity selection. Nucleic Acids Res 41:7167-75|
|Gu, Xiaoling; Vedvyas, Yogindra; Chen, Xiaoyue et al. (2012) Novel strategy for selection of monoclonal antibodies against highly conserved antigens: phage library panning against ephrin-B2 displayed on yeast. PLoS One 7:e30680|
|Chen, Xiaoyue; Wong, Richard; Khalidov, Ildar et al. (2011) Inflamed leukocyte-mimetic nanoparticles for molecular imaging of inflammation. Biomaterials 32:7651-61|
|Park, Spencer; Kang, Sungkwon; Veach, Alexander J et al. (2010) Self-assembled nanoplatform for targeted delivery of chemotherapy agents via affinity-regulated molecular interactions. Biomaterials 31:7766-75|