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oai:www.ideals.illinois.edu:2142/31924 2023-07-10 col_2142_14795 col_2142_5131 com_2142_14794 com_2142_14793 com_2142_8903 com_2142_5130

Ha, Taekjip Srinivasan, Divya 2012-06-27T21:19:38Z 2014-06-28T10:00:19Z 2012-05 2012-06-27T21:19:38Z 2012-05 Protein and RNA molecules interact with multiple protein partners to perform essential cellular processes such as post-transcriptional regulation of mRNA. Recently, a single molecule pull-down (SiMPull) assay was developed to isolate and study single protein complexes directly from cell lysates. Unlike ensemble measurements, SiMPull is a powerful tool that allows detection of diverse proteins present in a single complex and quantitation of the number of interacting partners when the proteins are stoichiometrically labeled. Using a similar principle, the objective of this study is to extend the SiMPull assay to isolate and study single cellular RNA-protein complexes. Utilizing a biological system of virally infected mammalian cells, the substrate targeted in the study are viral RNA-protein complexes. Specifically, the highly replicative sendai virus sub-genomic defective interfering (DI) RNA is targeted. The viral DI RNA associates with multiple viral proteins during replication, and is therefore expected to form heterogeneous RNA-protein complexes. Using the RNA sequence information of DI RNA, we designed several short complementary DNA probes to capture single DI RNA molecules for detection with single molecule fluorescence microscopy. We demonstrate specific capture of viral DI RNA molecules using SiMPull, and could quantitatively measure the presence of interacting viral proteins. Therefore, this study provides evidence for the applicability of SiMPull to isolate and study single cellular RNA-protein interactions. Item withdrawn by Mark Zulauf (zulauf@illinois.edu) on 2012-04-24T14:29:38Z Item was in collections: University of Illinois Theses & Dissertations (ID: 1) No. of bitstreams: 1 Srinivasan_Divya.pdf: 5926559 bytes, checksum: 417da5620b02262acfbc1e917cab2817 (MD5) Made available in DSpace on 2012-06-27T21:19:38Z (GMT). No. of bitstreams: 2 Srinivasan_Divya.pdf: 5572485 bytes, checksum: ef4f81be945ffb68a85a494dc97033f2 (MD5) license.txt: 4066 bytes, checksum: 964bdb94e3dd901707860518e8d7cd28 (MD5) Item marked as restricted to the 'UIUC Users [automated]' Group (id=2) by William Ingram (wingram2@illinois.edu) on 2012-06-27T21:24:32Z Item is restricted until 2014-06-27T21:24:27Z Item reinstated by Sarah Shreeves (sshreeve@illinois.edu) on 2014-06-28T10:00:19Z Item was in collections: Dissertations and Theses - Biochemistry (ID: 679) Graduate Theses and Dissertations at Illinois (ID: 204) No. of bitstreams: 2 Srinivasan_Divya.pdf: 5572485 bytes, checksum: ef4f81be945ffb68a85a494dc97033f2 (MD5) license.txt: 4066 bytes, checksum: 964bdb94e3dd901707860518e8d7cd28 (MD5) Item released from any restrictions by Sarah Shreeves (sshreeve@illinois.edu) on 2014-06-28T10:00:19Z http://hdl.handle.net/2142/31924 en Copyright 2012 Divya Srinivasan single molecule viral RNA total internal reflection fluorescence microscopy Ribonucleic acid (RNA) Single molecule pull-down of viral RNA-protein complexes text Biochemistry 1438 Biochemistry 0318 University of Illinois at Urbana-Champaign Thesis M.S. MS:Biochemistry -UIUC 10KS0318MS