DNA sequencing technologies have transformed our ability to define genomes and transcriptomes. Techniques to study the proteins they encode, and thus our ability to define new protein functions, discover biologic medicines, and determine disease mechanisms, however, have not similarly advanced. This focused technology development project will provide new methodologies to enable inexpensive, high throughput functional studies of expressed protein libraries. Aim 1: Develop methodology for LASSO-based ORFeome cloning from RNA We propose to adapt our previously established Long Adapter Single Stranded Oligonucleotide (?LASSO?) probe-based DNA sequence capture platform to the cloning of expressed ORFeomes directly from tissue RNA. A reverse transcriptase-mediated gap filling reaction will be defined and combined with an RNA-templated DNA- DNA ligation reaction, to enable LASSO cloning of ORF sequences while maintaining their correct translational reading frame. Captured ORFeome libraries may be shuttled into any expression vector of choice, including in vitro display vectors, like that developed in Aim 2. As validation, a complete human transcriptomic LASSO probe library will be designed, constructed, tested and characterized, prior to being shared with the research community. Aim 2. Develop a self-assembling, barcoded-RNA display format for full length proteins In vitro display technologies permit affinity-based screening of complex protein libraries. High throughput DNA sequencing, when applied to library screening, provides a powerful means to quantify binding interactions. We have previously developed a system that combines DNA sequencing with ribosome display for the ParalleL Analysis of Translated ORFeomes (?PLATO?). The ribosome display format imposes significant limitations, however, which we propose to overcome by developing a novel self-assembling barcoded-RNA version of PLATO (?sabr-PLATO?). Rigorous validation experiments will be used to extensively characterize the performance of the sabr-PLATO protein screening platform. Rapid production of ORFeome libraries using LASSO cloning from tissue-derived RNA, combined with robust screening of the encoded protein libraries using sabr-PLATO, will provide a means for any laboratory to functionally mine the proteome of any tissue from any organism. The synergistic, yet independent Aims of this project utilize the interdisciplinary expertise of the co-PIs, and build upon previous studies successfully carried out in their laboratories.
|Effective start/end date||8/15/18 → 7/31/22|
- National Institute of General Medical Sciences: $364,338.00
- Molecular Biology
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